Knockdown resistance to DDT and pyrethroids: from target-site mutations to molecular modelling

Naturally derived insecticides such as pyrethrum and man-made insecticides such as DDT and the synthetic pyrethroids act on the voltage-gated sodium channel proteins found in insect nerve-cell membranes. The correct functioning of these channels is essential for the normal transmission of nerve impulses, and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein that inhibit the binding of the insecticide and result in the insect developing resistance. This perspective outlines the current understanding of the molecular processes underlying target-site resistance to these insecticides (termed kdr and super-kdr), and how this knowledge may in future contribute to the design of novel insecticidal compounds.     

Recent advances in the chemistry of the pyrethroids. Forword

The sequences of compounds, leading from the natural pyrethrins to the present established pyrethroids, are described so as to provide a background for considering the more recent advances discussed in succeeding papers.     

DDT and pyrethroids: Receptor binding and mode of action in the house fly

The mode of action of DDT and pyrethroids was investigated in the house fly, Musca domestica L, using drug:receptor binding techniques. Both in vivo and in vitro binding studies demonstrated the existence of membrane receptors which bind specifically to [¹⁴C]DDT and [¹⁴C]cis-permethrin. The receptors show properties to be expected of a critical target site of these insecticides. These include negative temperature correlation with binding, relatively nonsensitivity to DDE, and sensitivity to Ca²⁺. The receptor sites are readily saturated at 45–90 nM [¹⁴C]DDT and have an apparent disassociation constant (K_d) of 12.2 nM. The maximum number of binding sites was estimated to be 17 pmol DDT/mg membrane protein (0.34 pmol/house fly head). Competition studies showed DDT, cis-permethrin, and cypermethrin bind to the same receptor but not at precisely the same site. The addition of Ca²⁺ to the incubation buffer significantly inhibited the binding of both [¹⁴C]DDT and [¹⁴C]cis-permethrin, suggesting the receptor binding is Ca²⁺ sensitive and may have a role in ion conductance.     

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.     

气相色谱-质谱法测定水-沉积物系统中13种拟除虫菊酯类农药

建立了水-沉积物系统中13种拟除虫菊酯类农药的残留分析方法.样品采用固相萃取和加压流体萃取法分别作为水和沉积物的前处理方法,利用气相色谱-质谱(GC-MS)在选择离子扫描模式下进行检测,内标法定量.结果表明,13种拟除虫菊酯类农药在一定浓度范围内线性关系良好(R2≥0.995);水中添加水平在0.50～50μg/L范围...     

Esterases and resistance to synthetic pyrethroids in the Egyptian cotton leafworm

Esterases hydrolyzing α-naphthyl acetate (α-NA), β-naphthyl acetate (β-NA), and p-nitrophenyl acetate (p-NPA) were investigated colorimetrically in larval homogenates of synthetic pyrethroid susceptible (S) and resistant (R) strains of Spodoptera littoralis (Boised). The hydrolytic activity towards the three substrates in cybolt, decamethrin, and fenvalerate R strains were from 3 to 6.5 times as high as in the S strain. The increase in esterase activity was closely associated with the development of resistance in the R strains. DEF (S,S,S-tributyl phosphorotrithioate) proved to be an inhibitor for all esterases, with a particularly potent action on p-NPA-hydrolyzing enzymes. The inhibitory action was more pronounced in R strains than in the S strain. Pretreatment with DEF increased the toxicity of pyrethroid compounds in the R strains more than in the S strain and hence decreased the levels of resistance in these strains. This is evidence that the esterases contribute to the resistance against synthetic pyrethroids in S. littoralis larvae.     

Metabolism and degradation of the pyrethroids tralomethrin and tralocythrin in insects

Tralomethrin and tralocythrin undergo debromination, forming deltamethrin and cypermethrin, respectively, following topical administration to house flies, feeding to cabbage looper larvae, or incubation with house fly homogenates and cockroach nerve cords. The debromination is probably not an enzymatic process since it occurs rapidly on incubation with glutathione, cysteine, and albumin. Following debromination, an esterase(s) in house fly homogenate hydrolyzes delta-methrin and cypermethrin. The insecticidal activity of tralomethrin and tralocythrin may be due in part to the liberation of deltamethrin and cypermethrin in the insect or its nervous system.     

Depolarization of motor nerve terminals by pyrethroids in susceptible and kdr-resistant house flies

When applied at concentrations of one nM or higher to a house fly larval neuromuscular preparation, deltamethrin (DM) and fenvalerate (FV) greatly increased miniature excitatory postsynaptic potential (mepsp) rate and blocked neuromuscular transmission. The DM-induced mepsp discharge was abolished by tetrodotoxin (TTX), removal of Ca²⁺ from the saline, or by application of hyperpolarizing stimuli to the nerve, indicating that it was due to depolarization of the presynaptic terminals. Also, in the presence of TTX, K⁺ depolarization increased mepsp rate at the same external K⁺ concentration before and after DM treatment, confirming that DM released transmitter by depolarizing the nerve terminals rather than by altering the voltage dependence of transmitter release. The potassium channel blocker tetraethylammonium (TEA) increased mepsp rate somewhat, while aconitine (20 μM), which keeps sodium channels open, increased mepsp rate consistently. Pretreatment of nerves with a subthreshold dose of TEA greatly increased the mepsp rate-increasing activity of DM and aconitine, while a subthreshold level of aconitine did not synergize DM. These observations suggest that DM, like aconitine, depolarized nerves by modifying the sodium channels. Knockdown resistant (kdr) larvae were resistant to the depolarizing action of DM and aconitine but not to that of TEA, indicating that the kdr gene produced a modified sodium channel which was less sensitive to the action of pyrethroids and aconitine. During sustained transmitter release by DM, evoked release gradually declined, resulting in a condition called early block in which spontaneous release was high and release could be evoked by electrotonic depolarization of the nerve terminals, but not by a nerve action potential. Early block was probably due to conduction block in the nerve terminals. Early block eventually gave way to late block, characterized by the decline of spontaneous release to subnormal levels and complete failure of evoked release. After late block, the calcium ionophore X-537A could not release transmitter, suggesting that late block was due to depletion of available transmitter. DM did not have a direct effect upon extrasynaptic muscle membrane. However, after late block, muscles were left insensitive to the putative transmitters glutamate and aspartate when these were bath or iontophoretically applied. A low rate of mepsps persisted after late block, indicating that the muscles were still sensitive to the natural transmitters.     

Promotion of norepinephrine release and inhibition of calcium uptake by pyrethroids in rat brain synaptosomes

A series of 25 pyrethroids were assessed for their effects on Na⁺-dependent norepinephrine release and on Ca²⁺ uptake in vitro using a crude rat brain synaptosomal preparation. The most effective pyrethroids required a concentration of 3–10 μM to promote norepinephrine release. Plotting release data versus lipophilicity (as log P) for each compound resulted in a parabolic curve with log P_opt being 5.4 for maximal release. The release promoted by most of the compounds assessed at 30 μM could not be or was only partially reversed by either tetrodotoxin or substituting choline for Na⁺ conditions which readily reversed the release promoting effects of veratridine. Thus, many pyrethroids, particularly those without the α-cyano group, did not display their expected effects on the Na⁺ channel in rat brain. When assessed at 5 μM, pyrethroids inhibited, had no effect, or caused increases in the amount of Ca²⁺ incorporated in the presence of ATP. The effectiveness of the various pyrethroids to inhibit Ca²⁺ uptake again displayed a parabolic relationship with log P_opt being 6.4. It was concluded that the variations in pyrethroid effects on norepinephrine release and Ca²⁺ uptake are not solely related to their particular chemical structures, but to lipophilicity. The effects of many pyrethroids on Ca²⁺ metabolism, particularly displacement of bound Ca²⁺, better explain the transmitter release promoting properties in vitro rather than a direct effect on the Na⁺ channel. No direct relationship between known toxicity to mammals and Ca²⁺ inhibition by pyrethroids was established.     

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.     

细胞壁降解酶在油茶炭疽病菌致病过程中的作用研究

为明确细胞壁降解酶在油茶炭疽病菌致病过程中的作用,本文研究了活体内外炭疽病菌产生的细胞壁降解酶活性及其对叶片的降解情况。结果表明,活体外以羧甲基纤维素钠(CMCNa)为诱导底物,羧甲基纤维素酶(Cx酶)和漆酶活性最高;以柑橘果胶为诱导底物,果胶酶活性最高;以油茶叶为诱导底物,纤维素酶、果胶酶和漆酶可产生较高活力;并且经5种诱导物诱导的酶液对叶片均有降解作用。发病叶片的各部位,以病健交界处细胞壁降解酶活性最高。接种4d后开始发病,其细胞壁降解酶活性迅速增强;6d后滤纸酶(FPA)、β-葡萄糖苷酶和漆酶活性达最大值,分别为4.53、7.44、1.21U/mg;而Cx酶和果胶酶在第8天时酶活性最高,分别为15.79和25.49U/mg;接种10~16d,酶活性比较稳定。上述结果表明,纤维素酶、果胶酶和漆酶在油茶炭疽病菌致病过程中起重要作用。     

Enhancement of neurotransmitter release from invertebrate synaptosomes by pyrethroids during pulsed-depolarization: A functional assay for effects on repolarization

The release of [³H]neurotransmitters was used as a functional assay to assess the actions of selected neurotoxins on the synaptosomal membranes prepared from the invertebrate nervous systems of squid and house fly. A reproducible release of [³]neurotransmitter was evoked by pulsed-depolarization in the presence of elevated K⁺ or of veratridine. Pretreatment with deltamethrin resulted in a substantial enhancement of [³H]neuro-transmitter release during pulsed-depolarization. This enhanced neurotransmitter release was greatly reduced or absent when synaptosomes of knockdown-resistant house flies were examined. No enhanced neurotransmitter release due to deltamethrin pretreatment was apparent from any synaptosomal preparation under non-depolarizing conditions. Under similar conditions, collaborative experiments demonstrated that deltamethrin causes a significant change in protein phosphorylation activities which follow depolarization. The most significant change caused by deltamethrin was the prolonged elevation of the level of phosphorylation on a number of key synaptic proteins beyond the normal time of their recovery to the dephosphorylated state. The most notable protein reacting to deltamethrin in this manner was calcium-cadlmodulin-dependent protein kinase.     

The actions of pyrethroids upon the peripheral nervous system and associated organs in the locust

The actions of a number of pyrethroids upon the crural nerve, chordotonal organs and skeletal muscles of the desert locust were investigated. The compounds were principally 3-phenoxybenzyl esters (with or without an α substituent), and particular attention was given to fenvalerate and related compounds. Four different effects were observed, both during the early stages of intoxication of intact locusts and during perfusion of physiological preparations with low concentrations of the pyrethroids. The type of effect produced was determined by the molecular structure of the pyrethroid. Knockdown activity was associated with one particular effect, and it is suggested that it is the type of action caused by a pyrethroid, rather than its rate of penetration into the insect, that determines whether or not it will have knockdown activity.     

DDT and pyrethroids: Receptor binding in relation to knockdown resistance (kdr) in the house fly

The nature of target site or knockdown resistance (kdr) to DDT and pyrethroids was studied by investigating specific binding of [14_C] DDT and [14_C] cis-permethrin to the previously established membrane receptors from the heads of susceptible (sbo) and resistant (kdr) strains of the house fly, Musca domestica L. In vivo studies showed the heads from sbo flies bound two to three times more DDT than those from kdr flies at all doses tested. Reduced binding was also observed in kdr flies in in vitro [14_C] DDT binding assays. Scatchard analysis indicated that kdr flies have the same affinity but fewer receptors per milligram protein in the CNS than sbo flies. Assays with [14_C] cis-permethrin also showed binding was much reduced in kdr flies in comparison with sbo flies. Based on these results, the nature of the target site insensitivity of kdr flies may relate to their having a reduced number of receptors for the insecticides.     

Picrotoxinin receptor in the central nervous system of the American cockroach: Its role in the action of cyclodiene-type insecticides

The nature of the picrotoxinin receptor was studied using the central nervous system (CNS) of the American cockroach. It first was confirmed by using an electrophysiological technique that the abdominal nerve cord of the American cockroach was sensitive to picrotoxinin. By using a $\text{[}{}^3\text{H]α-dihydropicrotoxinin}$ binding test it was determined that the picrotoxinin receptor in CNS of this insect had a higher affinity toward picrotoxinin and heptachlor epoxide than the corresponding receptor in the rat brain. Also, the cockroach brain preparation had a higher percentage of specific binding in the total binding, making this material suitable for receptor studies. By using a sucrose density centrifugation technique, it was determined that the fraction sedimented at the interphase of 1.0 to 1.2 M sucrose at 100,000g contained the highest level of specific binding site. The receptor showed a sensitivity to all insecticidal cyclodienes tested, namely photodieldrin, oxychlordane, endrin, heptachlor epoxide, γ-chlordane, dieldrin, aldrin, heptachlor, and isodrin (expressed in the order of potency). Among four BHC isomers, the γ-isomer showed the highest potency to bind with this receptor.     

Surfaces and other factors modifying the effectiveness of pyrethroids against insects in public health

Features of the evolution of synthetic pyrethroid insecticides are reviewed and related to the target function in control. The factors affecting residual activity are discussed and related to the surfaces to which different formulations are applied. Many insecticide/formulation/surface combinations lose effectiveness rapidly. The consequence of this in relation to the recovery of cockroach populations are discussed using mathematical models and field observations.     

海南普通大蓟马对拟除虫菊酯药剂抗性监测及钠通道突变检测

普通大蓟马Megalurothrips usitatus在海南省对豇豆造成严重危害且抗药性逐渐增强。本研究测定了2019年至2021年海南省普通大蓟马对氯菊酯和甲氰菊酯的抗性。结果表明,海口、乐东和三亚3个地理种群对甲氰菊酯处于极高水平抗性,对氯菊酯处于高水平抗性,且抗性逐年增强。对普通大蓟马钠离子通道序列分析发现存在M283R突变,该突变位于钠离子通道同源结构域Ⅰ。突变频率检测显示,2019年至2021年连续3年海口种群该突变位点的突变频率分别为1/30、1/30、3/30,有升高趋势。本研究发现海南省普通大蓟马从2019年到2021年对拟除虫菊酯类药剂的抗药性呈逐年上升趋势。     

Resistance to synthetic pyrethroids in the bollworm,Heliothis armigera from Andhra Pradesh,India

Helio this armigera collected in October 1987 from Juzzuru in the coastal cotton growing district of Krishna in Andhra Pradesh (Eastern India) were highly resistant to cypermethrin and fenvalerate and moderately resistant to endosulfan. Prior to this, in 1986, H. armigera in the Hyderabad area were resistant to DDT but not to pyrethroids or endosulfan. By late 1987 these latter populations were highly resistant to pyrethroids and mildly resistant to endosulfan. Concurrently, the resistance to DDT increased. It is suggested that resistant moths from the east coast migrated downwind in a northwesterly direction with the prevailing winds which occur at that time of year. The level of pyrethroid resistance in H. armigera infesting pigeon-pea and chick-pea fields around Hyderabad increased steadily up to March 1988. These results are discussed with special reference to the resistance mechanisms likely to be involved.     

Differential susceptibilities to pyrethroids in field populations of Chilo suppressalis (Lepidoptera: Pyralidae)

To assess the feasibility of pyrethroids for rice insect control, we examined susceptibilities of six field populations of rice stem borer Chilo suppressalis (Walker) to 10 pyrethroids using the topical application method in laboratory in 2004 and 2005. Our results showed that the seven pyrethroids with high fish-toxicity (i.e., β-cyfluthrin, λ-cyhalothrin, β-cypermethrin, deltamethrin, S-fenvalerate, α-cypermethrin, and fenpropathrin) were more effective against C. suppressalis than the three compounds with low fish-toxicity (i.e., cycloprothrin, etofenprox, and silafluofen). The results also showed that all 10 of the pyrethroids were much more effective than methamidophos and monosultap for C. suppressalis control. In addition, we found that susceptibilities of some field populations of C. suppressalis to some high fish-toxicity pyrethroids were significantly reduced, and our results indicated that a Ruian (RA) field population showed a year-to-year variation in susceptibility to most tested pyrethroids between 2004 and 2005. Our data indicated that the tolerance levels increased dramatically in RA population, especially to β-cyfluthrin and deltamethrin. This study provided the first assessment of resistance to pyrethroids in field populations of C. suppressalis. In addition, a close correlation between resistance ratios to the 10 compounds and differences of the structures of these compounds was established in the RA05 population, which was resistant to most of the pyrethroids tested while it was still very susceptible to fenvalerate with no cross resistance. Finally, the feasibility and precaution were discussed in selecting pyrethroids as alternatives to replace high toxicity organophosphates for C. suppressalis control and insecticide resistance management.