Two classes of pyrethroid action in the cockroach

Pyrethroids are divided into two classes (Types I and II) based on their effects on the cercal sensory nerves recorded in vivo and in vitro and on the symptomology they produce in dosed cockroaches, Periplaneta americana. Type I compounds include pyrethrins, S-bioallethrin, [1R,cis]resmethrin, kadethrin, the 1R,trans and 1R,cis isomers of tetramethrin, phenothrin, and permethrin, and an oxime O-phenoxybenzyl ether. Electrophysiological recordings from dosed individuals reveal trains of cercal sensory spikes and sometimes also spike trains from the cercal motor nerves and in the CNS. Low concentrations of these pyrethroids act in vitro to induce repetitive firing in a cercal sensory nerve following a single electrical stimulus. This in vitro measurement, standardized for evaluating structure-activity relationships, shows that only 1R, insecticidal isomers are highly effective neurotoxins. The most potent compounds on the isolated nerve are [1R,trans]- and [1R,cis]tetramethrin, each active at 3 × 10^?13M. The poisoning symptoms of Type I compounds are restlessness, incoordination, hyperactivity, prostration, and paralysis. Type II compounds include [1R,cis,αS]- and [1R,trans,αS]cypermethrin, deltamethrin, and [S,S]fenvalerate. These α-cyanophenoxybenzyl pyrethroids do not induce repetitive firing in the cercal sensory nerves either in vivo or in vitro; moreover, they cause different symptoms, including a pronounced convulsive phase. Two other pyrethroids with an α-cyano substituent, i.e., fenpropathrin and an oxime O-α-cyanophenoxybenzyl ether, are classified as Type I based on their action on a cercal sensory nerve but the symptoms with these compounds resemble Type II. The two classes of pyrethroid action evident with the cockroach are discussed relative to their neurophysiological effects and symptomology in other organisms.     

Decreased nerve sensitivity and decreased cuticular penetration as mechanisms of resistance to pyrethroids in a (1R)-trans-permethrin-selected strain of the house fly

A fenthion-resistant strain of the house fly (Musca domestica L.) was selected with bioresmethrin resulting in ca. 90-fold resistance to the selecting agent. This strain was subsequently selected with (1R)-trans-permethrin producing ca. 140-fold resistance to this latter insecticide. The permethrin-resistant (147-R) strain was highly cross-resistant to several other pyrethroids and demonstrated resistance to knockdown by these insecticides as well as by DDT. The sensitivity of the central nervous system to four pyrethroids was investigated. The 147-R strain was 2.6-fold less sensitive to (1R)-trans-ethanoresmethrin than the susceptible (NAIDM-S) strain, and >43-fold and >67-fold less sensitive to (1R,S)-cis, trans-tetramethrin and (1R)-trans-permethrin, respectively. It also displayed decreased penetration of (1R,S)-trans-[¹⁴C]permethrin when compared to the NAIDM-S strain. Lower nerve sensitivity and decreased cuticular penetration are potential mechanisms of resistance to pyrethroids in house flies in the United States.     

Insect spiracle as the main penetration route of pyrethroids

The penetration route of adhered pyrethroids following direct aerosol spraying was studied in the German cockroach (Blattella germanica) by investigating the relationship between the application site of insecticide and knockdown efficacy. In direct spray, KT₅₀ was 26.4 s and the adhered amount of pyrethroid was 0.745 μg. On the contrary, required amount of pyrethroid to obtain the same KT₅₀ was one-eighth in topical application to the mesothoracic spiracle, while 2.6 times to the ventral mesothorax. KT₅₀ of cockroaches with blocked mesothoracic spiracles was greater than those with unblocked spiracles by 1.8-fold. The amount of directly sprayed pyrethroid penetrating through the inner wall of the mesothoracic trachea was significantly higher than the amount penetrating through the body wall of the ventral mesothorax. Therefore, the knockdown effect of the direct spray was believed to be caused by the flow of pyrethroids into the mesothoracic spiracles and its subsequent penetration through the inner wall of the mesothoracic trachea.     

Insecticides and electrophysiological symptoms: A comparative study on a tarsal motor nerve of Locusta migratoria

Spontaneous discharges of a tarsal motor nerve preparation of the jumping leg of Locusta migratoria were recorded extracellularly under the influence of various insecticides applied to the in situ metathoracic ganglion of the insect. Insecticides from different chemical classes were found to exhibit rather specific electrophysiological symptoms: cholinesterase inhibitors produced groups of action potentials with highly increased frequencies, and led to the appearance of new units which were not present in untreated control insects. They also caused transitory phases of conduction blocks, the length of which increased as intoxication proceeded. The DDT analog tested could be characterized by pronounced repetitive dischanges, whereas the neuroactivity of pyrethroids was typically associated with a very regular spike pattern in continuous phases of extremely high frequencies. Also chlordimeform, a formamidine insecticide, was found to increase the spike frequency, but to a much lesser extent than that observed for the other insecticides tested.     

Substrate-specificity and toxicological significance of pyrethroid-hydrolyzing esterases of mouse liver microsomes

An esterase or esterases in acetone powder preparations of mouse liver microsomes hydrolyze the cyclopropanecarboxylate ester linkage of pyrethroid insecticide chemicals derived from primary alcohols. The rate of cleavage of (+)-trans-chrysanthemates with various alcohol moieties decreases in the following order: 5-propargyl-2-furylmethyl; 5-benzyl-3-furylmethyl (bioresmethrin); 3-phenoxybenzyl; tetrahydrophthalimidomethyl esters. The hydrolysis rate of benzylfurylmethyl esters with various acid moieties decreases in the order: (+)- or (?)-trans-chrysanthemate; (+)-trans-ethanochrysanthemate; tetramethylcyclopropanecarboxylate; (+)- or (?)-cis-chrysanthemate or (+)-cis-ethanochrysanthemate. The trans-isomers of chrysanthemates and ethanochrysanthemates are hydrolyzed from 2.6- to more than 50-fold more rapidly than the corresponding cis-isomers. This enzyme system does not hydrolyze secondary alcohol esters, i.e., allethronyl (+)-trans- and (+)-cis-chrysanthemates.On intraperitoneal administration to mice, the (+)-trans-chrysanthemate and -ethanochrysanthemate of benzylfurylmethanol are of very low toxicity relative to the corresponding (+)-cis-isomers and the tetramethylcyclopropanecarboxylate. S,S,S-tributyl phosphorotrithioate (DEF) pretreatment increases the toxicity of these five compounds by 2.6- to more than 188-fold, with the exception of bioresmethrin whose toxicity is not altered. When the toxicity is increased, it is probably the result of esterase inhibition since DEF strongly inhibits the esterase activity of fresh liver microsomes while the mixed-function oxidase system remains active. The oxidase system metabolizes the chrysanthemates more rapidly than the ethanochrysanthemates of benzylfuryl-methanol. Depending upon the pyrethroid involved, the esterase or the mixed-function oxidase system, or both may be responsible for limiting the toxicity of these pyrethroids to mice.     

Symptoms of mirex,dieldrin, and DDT poisoning in the field cricket,Gryllus pennsylvanicus burmeister,and effect on activity of the central nerve cord

In the field cricket, Gryllus pennsylvanicus, the characteristic feature of mirex poisoning was the long latent period of at least 72 hr followed by hyperactivity, ataxia, and convulsions and paralysis. Dieldrin poisoning showed a short latent period followed directly by ataxia and convulsions and paralysis. The stages of DDT poisoning were locomotor instability, ataxia and convulsions and paralysis followed by death.Increased spontaneous activity of the central nerve cord in the initial stages of poisoning was characteristic for all three insecticides. DDT poisoned crickets showed DDT-type trains or groups of spikes in the central nerve cord during ataxia and early convulsions, but no synaptic after-discharges were observed at any stage of poisoning. Dieldrin and mirex poisoned crickets did not show DDT-type trains but a prolonged synaptic after-discharge was evident in the early stages of poisoning. In mirex poisoned crickets the increased spontaneous activity and the synaptic after-discharge was more acute than for dieldrin.     

Insecticide resistance profiles can be misleading in predicting the survival of Myzus persicae genotypes on potato crops following the application of different insecticide classes

BACKGROUND: The accuracy of predicting the survival of insecticide‐resistant aphids following the application of commonly used insecticides from the carbamate, the pyrethroid, a mix of the two or the neonicotinoid chemical classes was evaluated in a potato field in Scotland. Equal proportions of five genotypes of the peach‐potato aphid, Myzus persicae (Sulzer), with none, resistance to dimethyl‐carbamates, resistance to pyrethroids or combinations conferring resistance to both chemical classes were released into potato field plots. The insecticides were sprayed separately onto these plots, the aphid populations were analysed after 6–8 days and the process repeated. RESULTS: For each assessment after the three separate spray events, plots treated with the carbamate had 48, 147 and 28%, those treated with pyrethroid 53, 210 and 89%, those treated with carbamate/pyrethroid 28, 108 and 64% and those treated with neonicotinoid 43, 55 and 11% of the numbers of M. persicae by comparison with untreated controls. Only the proportions of surviving aphids from the genotype containing no insecticide resistance traits and the genotype containing elevated carboxylesterases matched ratios predicted from the selective advantage afforded by the resistance traits alone. Survival of aphids from the other three genotypes that carried 1–3 of the insecticide resistance traits differed from expectations in all cases, possibly owing to physiological differences, including their vulnerability to predators and hymenopterous parasitoids present at the site and/or their carrying unknown insecticide resistance mechanisms. CONCLUSION: Control strategies based on knowledge of the genetically determined insecticide resistance profile of an M. persicae population alone are insufficient. Hence, other important factors contributing to aphid survival under insecticide pressure need to be considered. Copyright © 2012 Society of Chemical Industry     

Symptomatic and neurophysiological activities of new synthetic non-ester pyrethroids,ethofenprox, MTI-800, and related compounds

Ethofenprox (MTI-500), MTI-800, and related compounds, which have a m-phenoxybenzyl moiety but lack ester bonding, were compared with DDT-type compounds and pyrethroid insecticides for their lethal and convulsive activities toward American cockroaches. The most potent among them ranked between phenothrin and cyphenothrin. Neurophysiological effects were also examined by extra- and intracellular recording and voltage clamp techniques. Some derivatives induced repetitive discharges in the excised central nerve cord of the American cockroach. The after-potential recorded intracellularly from the crayfish giant axon was markedly increased by some compounds. Voltage clamp experiments with the crayfish giant axon showed that ethofenprox decreased the peak sodium current and induced a large residual current during a step depolarization. It also induced a large and prolonged tail current after a step repolarization of the membrane. The effects of the test compounds on the action potential and the sodium current were similar to those of DDT-type compounds and the pyrethroids such as allethrin and phenothrin. A shift of sodium channel population from normal to modified was thought to result in modifications of the sodium current which, in turn, cause the increase in after-potential and the induction of repetitive discharges.     

Pyrethroid toxicology: Mouse intracerebral structure-toxicity relationships

Mouse intracerebral (ic) toxicity studies with 29 pyrethroids confirm earlier mouse intraperitoneal (ip) and rat oral and intravenous findings in three respects: α-cyano-3-phenoxybenzyl esters produce choreoathetosis, convulsions, and salivation, whereas compounds lacking the α-cyano group yield tremors and convulsions; high stereospecificity is involved in inducing both poisoning syndromes; large toxicity differences for (1R,trans) vs (1R,cis) resmethrin and permethrin do not extend to ethanomethrin and the cyanophenoxybenzyl esters. The ic investigations further establish that: profuse salivation is not unique for pyrethroids with the α-cyano group; the inactivity of (1R,trans) resmethrin in the brain is not due to detoxification; pyrethroids acting most rapidly in the brain are those with the highest knockdown activity for insects; the cyanophenoxybenzyl esters, in comparison with the non-cyano pyrethroids, have a high ic toxicity relative to their synergized ip toxicity indicating the importance of the brain in the Type II poisoning syndrome.     

Hemolymph carbohydrate and lipid mobilization in Locusta migratoria in relation to the progress of poisoning following bioresmethrin treatment

Effects of bioresmethrin, a pyrethroid insecticide, on the mobilization of metabolic reserves in the hemolymph of Locusta migratoria were studied. Bioresmethrin treatment of intact locusts caused depletion of hemolymph carbohydrate (hypoglycemia) and elevation in blood lipid (hyperlipemia). The mobilization of these metabolic reserves was found to be closely related to the poisoning syndrome of this insecticide. Bioresmethrin-induced hyperlipemia was not detected in neck-ligated locusts, whereas hypoglycemia was still observed. The results of this study suggest that bioresmethrin-induced elevation in hemolymph lipid is due to the release of hyperlipemic hormone(s). The depletion of hemolymph carbohydrate, on the other hand, may be due to their increased utilization in response to the hyperactivity caused by the insecticide treatment.     

Resistance of Pakistani field populations of spotted bollworm Earias vittella (Lepidoptera: Noctuidae) to pyrethroid,organophosphorus and new chemical insecticides

BACKGROUND: The spotted bollworm Earias vittella (Fab.) is a serious pest of cotton and okra in Pakistan. Owing to persistent use of insecticides, this pest has developed resistance, especially to pyrethroids. The present studies aimed at determining the extent of resistance to pyrethroid, organophosphorus and new chemical insecticides in Pakistani populations of E. vittella. RESULTS: Field populations of E. vittella were monitored at Multan, Pakistan, from 1999 to 2007 for their resistance against six pyrethroid, four organophosphorus and six new chemical insecticides using a leaf‐dip bioassay. Of the pyrethroids, resistance was generally low to zeta‐cypermethrin and moderate to high or very high to cypermethrin, deltamethrin, esfenvalerate, bifenthrin and lambda‐cyhalothrin. Resistance to organophosphates chlorpyrifos, profenofos, triazophos and phoxim was recorded at very low to low levels. Among new chemicals, E. vittella had no or a very low resistance to spinosad, emamectin benzoate and methoxyfenozide, a very low to low resistance to abamectin, a very low to moderate resistance to indoxacarb and a moderate resistance to chlorfenapyr. CONCLUSION: The results indicate a lack of cross‐resistance between pyrethroid and organophosphorus insecticides in E. vittella. Rotation of insecticides showing no, very low or low resistance, but belonging to different insecticide classes with unrelated modes of action, may prevent or mitigate insecticide resistance in E. vittella. Copyright © 2009 Society of Chemical Industry     

昆虫对拟除虫菊酯类杀虫剂的代谢抗性机制研究进展

随着拟除虫菊酯类杀虫剂在卫生和农业害虫防治中的广泛应用,昆虫对此类杀虫剂产生抗性的报道越来越多。目前已明确昆虫对拟除虫菊酯类杀虫剂的抗性机制包括表皮穿透率下降、靶标抗性以及代谢抗性,其中代谢抗性机制较为普遍,而且其与昆虫对多种杀虫剂的交互抗性关系密切。目前,随着基因组、转录组以及蛋白质组学等新技术的发展及应用,昆虫对拟除虫菊酯类杀虫剂的代谢抗性机制研究也取得了很多新进展。昆虫体内细胞色素P450酶（P450s）、羧酸酯酶（CarE）及谷胱甘肽S-转移酶（GSTs）等重要解毒酶系的改变均与昆虫对拟除虫菊酯类杀虫剂的代谢抗性有关,其中这3类解毒酶的活性及相关基因表达量的变化是昆虫对此类杀虫剂产生代谢抗性的主要原因。明确昆虫对拟除虫菊酯类杀虫剂的代谢抗性机制,对合理使用此类杀虫剂及延缓抗药性的产生均具有重要意义。本文在总结拟除虫菊酯类杀虫剂代谢路径及相关生物酶研究概况的基础上,综述了近年来有关昆虫对此类杀虫剂代谢抗性机制研究的主要进展。     

A pharmacological study of pyrethroid neurotoxicity in mice

Permethrin (cis-, trans-, and technical grade [tech.]) and deltamethrin, representatives of the non-cyano- and cyano-containing classes of synthetic pyrethroids, produced neurotoxic symptoms when administered to mice. ED₅₀ values for this effect were compared following intracerebroventricular (icv) or peripheral (iv) injection. Both permethrin and deltamethrin showed large increases in potency following icv administration, suggesting a mainly central site of action for both classes of pyrethroid. Pretreatment of mice with drugs affecting central noradrenergic, cholinergic, or serotonergic transmission was demonstrated to potentiate the toxic response to iv injections of tech.-permethrin, while some symptoms of toxicity could be partially alleviated by ip pretreatment with diazepam, aminooxyacetic acid, or cycloheximide.     

Esterase inhibitors as synergists for (+)-trans-chrysanthemate insecticide chemicals

Four synergists are used to evaluate the relative contribution of esterases and oxidases in the metabolism of four pyrethroids, the (+)-trans- and (+)-cis-isomers of resmethrin and tetramethrin, by five insect species and by mice. Three of these compounds are known pyrethroid synergists, S,S,S-tributyl phosphorotrithioate acting as an esterase inhibitor and piperonyl butoxide and O-(2-methylpropyl) O-(2-propynyl) phenylphosphonate acting as oxidase inhibitors. The fourth synergist, 1-naphthyl N-propylcarbamate, is an esterase inhibitor selected by screening 65 candidate esterase and oxidase inhibitors for maximal potency in synergizing the toxicity of trans-resmethrin to milkweed bugs. Naphthyl propylcarbamate synergizes the toxicity of trans-resmethrin and -tetramethrin to milkweed bugs, cockroaches, houseflies, cabbage loopers, and mealworms but not to mice. The persistence of trans-resmethrin in milkweed bugs treated by injection is increased by the esterase inhibitors while that of cis-resmethrin is increased by the oxidase inhibitors. The optimal synergist varies with the species and the pyrethoid, being related to both the nature of the pyrethroid alcohol moiety and the trans- or cis-configuration of the acid moiety. This probably results from species variations in the relative significance of esterases and oxidases in pyrethroid detoxification.     

Why does DDT toxicity change after a blood meal in adult female Culex pipiens?

The toxicity of topically applied DDT to adult female anautogenous mosquitoes (Culex pipiens L.) showed dramatic variations in blood-fed insects. It decreased very rapidly about twofold to a minimum at 24 hr after a blood meal, then increased within 72 hr back to values typical of non-blood fed insects. A comparison of the metabolism of [¹⁴C]DDT in vivo revealed an increase in DDT dehydrochlorination to DDE at 72 hr after a blood meal, but this increase was not responsible for the variations in DDT toxicity at 24 hr. Changes in penetration rates were not observed and changes in the distribution of DDT could likewise not be related to the short period of decreased toxicity of DDT. Fenvalerate and trans-permethrin, two pyrethroid insecticides which are believed to have a mode of action similar to that of DDT, were also significantly less toxic 24 hr after a blood meal. By contrast, the cyclodiene insecticide aldrin and the carbamate insecticide propoxur were not less toxic 24 hr after a blood meal. The results suggest that after a blood meal an unidentified and transient change in C. pipiens specifically decreases DDT/pyrethroid toxicity. A hypothesis concerning this transient change is advanced. The results illustrate the difficulties in explaining physiological changes in insecticide toxicity.     

CNS insensitivity to pyrethroids in the resistant kdr strain of house flies

Solutions of tetramethrin, RU 11679, or cismethrin caused uncoupled convulsions in 30–40 min in exposed thoracic ganglia from S_NAIDM house flies at concentrations down to 10^?10M: whereas these same compounds at 10^?6M concentrations failed to produce poisoning symptoms when perfused onto the exposed ganglia of the kdr strain of house fly. The pyrethroid analogs examined had a negative temperature coefficient of action on the exposed thoracic ganglia from S_NAIDM flies. DDT and GH-74 possessed positive temperature coefficients of action on the exposed thoracic ganglion of susceptible house flies. It is concluded that the central nervous system of the kdr strain of house fly is resistant to pyrethroid action; furthermore, the resistance appears to be widespread throughout the house fly nervous system, involving sensory, motor, and central neural elements.     

Mutated sodium channel genes and elevated monooxygenases are found in pyrethroid resistant populations of Sri Lankan malaria vectors

The present status of pyrethroid resistance in vectors of malaria; Anopheles culicifacies and Anopheles subpictus, was tested in two malarious Districts, Anuradhapura and Trincomalee, of Sri Lanka. Both species were resistant to permethrin and susceptible to cypermethrin and cyfluthrin. An. subpictus were resistant to deltamethrin. λ-Cyhalothrin and etofenprox resistance was shown only by Anuradhapura An. subpictus. Although there were no differences among the populations for esterase and glutathione S-transferase activities, increased monooxygenase levels were found among Trincomalee populations. The voltage-gated sodium channel gene, the target site gene of pyrethroids, was partially sequenced to screen for mutations previously associated with insecticide resistance. The classic leucine to phenylalanine substitution, TTA to TTT, was detected in An. subpictus. It appears that both kdr type and monooxygenase resistance underlie pyrethroid resistance in these two malaria vectors of Sri Lanka.     

Pharmacokinetics of cis- and trans-substituted pyrethroids in the American cockroach

The pharmacokinetic behavior of cis and trans isomers of pyrethroids after topical application to adult male American cockroaches (Periplaneta americana L.) was examined using the insecticidal 1R,cis (NRDC 157; I) and 1R,trans (NRDC 163; III) isomers of 3-phenoxybenzyl 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate and their insecticidally inactive 1S,cis (II) and 1S,trans (IV) enantiomers. III was detected in the hemolymph, nerve cord, and fat body of animals receiving a just-lethal dose (0.6 μg/g) within 2 hr after topical application. The pattern of accumulation of III was similar to that previously determined for I at a just-lethal dose, but quantitative comparisons revealed that the cis isomer I was delivered from the site of application to the nerve cord eightfold more efficiently than III. The inactive enantiomers II and IV were administered at the same dose (0.60 μg/g) to compare the rates of cuticular penetration and in vivo degradation of cis and trans isomers in the absence of intoxication symptoms. II penetrated somewhat more rapidly than IV and achieved higher levels in whole body extracts, but there was no difference between isomers in the rates of overall degradation of applied pyrethroid. These studies demonstrated a twofold difference in internal availability, but they did not reveal sufficient pharmacokinetic selectivity to explain the large difference in the access of I and III to the nerve cord observed in the tissue uptake studies. III was hydrolyzed by nerve cord homogenates in vitro at a rate 5 times greater than that of I, but neither ester underwent detectable oxidative metabolism in this system. Local selective metabolism by the nerve cord is suggested as an important determinant of the levels of parent pyrethroid found in this tissue. These results demonstrate the significance of pharmacokinetic selectivity in determining the relative access of topically applied cis- and trans-substituted pyrethroids to the insect nervous system.     

Structure-related effects of pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis

The effects of seven different pyrethroid insecticides on the lateral-line sense organ and on peripheral nerves of the clawed frog, Xenopus laevis, were investigated by means of electrophysiological methods. The results show that two classes of pyrethroid can be clearly distinguished. (i) Pyrethroids without an α-cyano group (permethrin, cismethrin, and bioresmethrin). These noncyano pyrethroids induce short trains of nerve impulses in the lateral-line sense organ. In peripheral nerve branches they induce a depolarizing afterpotential and repetitive firing. These effects are very similar to those previously reported for allethrin. (ii) Pyrethroids with an α-cyano-3-phenoxybenzyl alcohol (cypermethrin, fenpropathrin, deltamethrin, and fenvalerate). In the lateral-line sense organ these α-cyano pyrethroids induce very long trains of nerve impulses which may last for seconds and may contain hundreds or even thousands of impulses. The α-cyano compounds do not cause repetitive activity in peripheral nerves. Instead they induce a quickly reversible, stimulus frequency-dependent suppression of the action potential. Since the chemical structure of cypermethrin differs from that of permethrin only in the α-cyano group and because all four α-cyano compounds act in a very similar way, it is concluded that the α-cyano substituent is responsible for the large differences in neurotoxic effects. In the lateral-line sense organ the duration of nerve impulse trains induced by the noncyano as well as the α-cyano pyrethroids increases dramatically when the temperature is lowered. Further, in sensory fibers the effects of both classes of pyrethroid on the nerve action potential are more pronounced compared to their effects on motor fibers. It is argued that the different neurotoxic effects reported here originate from a common mechanism of action of pyrethroids, which is a prolongation of the transient increase of sodium permeability of the nerve membrane associated with excitation.It is concluded that the sodium channel in the nerve membrane is the major target site of noncyano and α-cyano pyrethroids.     

The pyrethroid resistance status and mechanisms in Aedes aegypti from the Guerrero state,Mexico

Dengue is one of the most important vector-borne diseases worldwide and is a public health problem in Mexico. Most programs in dengue endemic countries rely on insecticides for Aedes control. In Mexico, pyrethroid insecticides (mainly permethrin and deltamethrin) have been extensively used over a decade as adulticides and represented a strong selection for insecticide resistance for dengue vectors in several parts of the country. We studied the type, frequency and distribution of insecticide resistance mechanisms in Aedes aegypti from six municipalities in the state of Guerrero selected on the basis of historically intense chemical control and a high risk for dengue transmission. Ae. aegypti eggs were collected from October 2009 to January 2010 using ovitraps. F₁ adults, emerged from these collections, were exposed to permethrin, deltamethrin and DDT in WHO diagnostic tests and showed high resistance levels to both pyrethroids and DDT. This was consistent with the presence of increased metabolic enzyme activities and target site insensitivity due to kdr mutations. Biochemical assays showed elevated esterase and glutathione S-transferase activities in the six municipalities. The V1016I kdr mutation on the IIS6 domain of the sodium channel gene was present in an overall frequency of 0.80. A second mutation, F1534C on the IIIS6 domain of the same gene was also detected, being the first report of this mutation in Guerrero. The multiple resistance mechanisms present in Ae. aegypti from Guerrero state represent a warning for the efficacy of the pyrethroid usage and consequently for the success of the dengue control program.