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.     

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.     

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.     

Indoxacarb resistance in the house fly, Musca domestica

Indoxacarb (DPX-MP062) is a recently introduced oxadiazine insecticide with activity against a wide range of pests, including house flies. It is metabolically decarbomethoxylated to DCJW. Selection of field collected house flies with indoxacarb produced a New York indoxacarb-resistant (NYINDR) strain with >118-fold resistance after three generations. Resistance in NYINDR could be partially overcome with the P450 inhibitor piperonyl butoxide (PBO), but the synergists diethyl maleate and S,S,S-tributyl phosphorothioate did not alter expression of the resistance, suggesting P450 monooxygenases, but not esterases or glutathione S-transferases are involved in the indoxacarb resistance. Conversely, the NYINDR strain showed only 3.2-fold resistance to DCJW, and this resistance could be suppressed with PBO. Only limited levels of cross-resistance were detected to pyrethroid, organophosphate, carbamate or chlorinated hydrocarbon insecticides in NYINDR. Indoxacarb resistance in the NYINDR strain was inherited primarily as a completely recessive trait. Analysis of the phenotypes vs. mortality data revealed that the major factor for indoxacarb resistance is located on autosome 4 with a minor factor on autosome 3. It appears these genes have not previously been associated with insecticide resistance.     

Mechanisms of resistance to diflubenzuron in the house fly, Musca domestica (L.)

The mechanisms of resistance to the chitin synthesis inhibitor diflubenzuron were investigated in a diflubenzuron-selected strain of the house fly (Musca domestica L.) with > 1000 × resistance, and in an OMS-12-selected strain [O-ethyl O-(2,4-dichlorophenyl)phosphoramidothioate] with 380 × resistance to diflubenzuron. In agreement with the accepted mode of action of diflubenzuron, chitin synthesis was reduced less in larvae of the resistant (R) than of a susceptible (S) strain. Cuticular penetration of diflubenzuron into larvae of the R strains was about half that of the S. Both piperonyl butoxide and sesamex synergized diflubenzuron markedly in the R strains, indicating that mixed-function oxidase enzymes play a major role in resistance. Limited synergism by DEF (S,S,S-tributyl phosphorotrithioate) and diethylmaleate indicated that esterases and glutathione-dependent transferases play a relatively small role in resistance. Larvae of the S and R strains exhibited a similar pattern of in vivo cleavage of ³H- and ¹⁴C-labeled diflubenzuron at N₁C₂ and N₁C₁ bonds. However, there were marked differences in the amounts of major metabolites produced: R larvae metabolized diflubenzuron at considerably higher rates, resulting in 18-fold lower accumulation of unmetabolized diflubenzuron by comparison with S larvae. Polar metabolites were excreted at a 2-fold higher rate by R larvae. The high levels of resistance to diflubenzuron in R-Diflubenzuron and R-OMS-12 larvae are due to the combined effect of reduced cuticular penetration, increased metabolism, and rapid excretion of the chemical.     

Mechanisms of resistance to the juvenoid methoprene in the house fly Musca domestica L

The mechanisms of resistance and cross resistance to the juvenoids methoprene and R-20458 in the house fly, Musca domestica, were examined. Radiolabeled methoprene was found to be metabolized faster in resistant and cross-resistant house fly larvae than in susceptible larvae, and methoprene and R-20458 penetrated more slowly into larvae of the resistant strain. In vivo and in vitro metabolism of methoprene was largely by oxidative pathways followed by conjugation in all strains examined, and little or no ester change of methoprene was noted in vitro. In vitro oxidative metabolism of methoprene, R-20458, juvenile hormone I, and several model substrates was higher in resistant and cross-resistant larvae than in susceptible larvae. Juvenoid functionalities susceptible to metabolic attack by resistant strains are indicated.     

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.     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae)

A housefly strain, originally collected in 1998 from a dump in Beijing, was selected with beta-cypermethrin to generate a resistant strain (CRR) in order to characterize the resistance and identify the possible mechanisms involved in the pyrethroid resistance. The resistance was increased from 2.56- to 4419.07-fold in the CRR strain after 25 consecutive generations of selection compared to a laboratory susceptible strain (CSS). The CRR strain also developed different levels of cross-resistance to various insecticides within and outside the pyrethroid group such as abamectin. Synergists, piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF), increased beta-cypermethrin toxicity 21.88- and 364.29-fold in the CRR strain as compared to 15.33- and 2.35-fold in the CSS strain, respectively. Results of biochemical assays revealed that carboxylesterase activities and maximal velocities to five naphthyl-substituted substrates in the CRR strain were significantly higher than that in the CSS strain, however, there was no significant difference in glutathione S-transferase activity and the level of total cytochrome P450 between the CRR and CSS strains. Therefore, our studies suggested that carboxylesterase play an important role in beta-cypermethrin resistance in the CRR strain.     

Comparative metabolism of six ethoxychlor analogs and DDT in DDT-resistant and susceptible strains of the house fly Musca domestica L

The metabolic fate of six ³H-ring-substituted ethoxychlor analogs with altered aliphatic moieties and [¹⁴C]p,p′-DDT was investigated in susceptible and DDT-resistant strains of the house fly Musca domestica Linnaeus. The chloroalkane analogs, dichloroethane, chloropropane, and dichloropropane were primarily metabolized to the corresponding dehydrochlorinated products. This pathway was relatively more prominent in the resistant strain than in the susceptible strain. Biotransformation and detoxication of the isobutane, nitropropane, and neopentane derivatives was through microsomal oxidation (O-deethylation) of aryl ethoxy degradophores, and oxidation of the aliphatic moieties to produce the corresponding benzophenones, with no substantial differences between the resistant and susceptible strain. There was a strong correlation between the Taft ($\text{σ}{}^1$) values for the altered aliphatic moieties of chloroalkane analogs and their rate of dehydrochlorination in both the strains. These results suggest the importance of altered aliphatic moieties in developing resistance-proof DDT derivatives.     

Selection of Anopheles stephensi with DDT and dieldrin and cross-resistance spectrum to pyrethroids and fipronil

The field strain of Anopheles stephensi, the main malaria vector in south of Iran, was colonized in laboratory and selected with DDT and dieldrin in two separate lines for 3 generations to a level of 19.5- and 14-fold for DDT and dieldrin resistance, respectively. Synergist tests with chlorofenethol (DMC) and piperonyl butoxide (PBO) on the selected strains indicated that dehydrochlorination and oxidative detoxification might be the underlying mechanisms involved in the resistance to dieldrin and DDT in selected strains. DDT selection decreased susceptibility to DDT and pyrethroids including lambdacyhalothrin, permethrin deltamethrin and cyfluthrin. The result also showed that selection with dieldrin caused negative and positive cross-resistance to pyrethroid and fipronil, respectively. Based on these results, it can be concluded that besides metabolic resistance mechanisms, other factors such as mutation in γ aminobutyric acid (GABA) and voltage-gated sodium channels (Kdr) might be involved.     

Characterization of multiple glutathione transferases from the house fly, Musca domestica (L)

Glutathione transferases have been purified to a high degree of homogeneity from three strains of house fly by a procedure involving affinity chromatography on glutathione-sulfobromophthalein conjugate immobilized on Sepharose 4B, followed by preparative isoelectrofocusing. The affinity chromatography yielded purifications of between about 10- and 100-fold, depending on the strain and the substrate with which activity was measured. Each strain was shown to possess several proteins with glutathione S-transferase activity which fell into two clearly defined groups. The first group, of relatively low isoelectric point, showed activity with CDNB but little with DCNB, p-nitrobenzylchloride, or 1,2-epoxy-3-(p-nitrophenoxy)propane, whereas the second group, of higher isoelectric points, showed substantial activity with all substrates tested. Studies on the subunit structure of these enzymes demonstrated the existence of three different sized subunits of M_r 20,000, 22,000, and 23,500. From the experimental evidence recorded here, the existence of at least three functionally different glutathione transferases is inferred.     

Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.     

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.     

A new resistance to pyrethroids in tribolium castaneum (herbst)

The characteristics of a new high-level, field-derived resistance to pyrethroids in Tribolium castaneum (Herbst) were investigated using impregnated-paper and treated-grain assays. Piperonyl butoxide almost completely suppressed the resistance, suggesting that the major resistance mechanism was microsomal oxidation. Resistance extended to all pyrethroids tested and to carbaryl but not to organophosphorus insecticides or to methoprene. Resistance was strongest against α—CN phenoxybenzyl cyclopropanecarboxylate pyrethroids and was correlated with structural modifications of the pyrethroid molecule, results also consistent with oxidative resistance. This resistance will ultimately result in failures to control T. castaneum if pyrethroids, such as deltamethrin, cypermethrin or cyfluthrin, are used in the field, even if they are synergised with piperonyl butoxide. The resistance does not jeopardise organophosphorus materials (e.g. fenitrothion, chlorpyrifos-methyl, pirimiphos-methyl, methacrifos) or methoprene.     

Toxicokinetics of carbofuran in house fly, Musca domestica L

The kinetics of accumulation and elimination of lethal doses of [¹⁴C]carbofuran in the hemolymph of the house fly suggest a one-compartment open model. Carbofuran in the hemolymph appeared to be in equilibrium with that in the tissues very soon after treatment.Following topical application of carbofuran, the rate of onset of symptoms of poisoning was correlated with the amount of carbofuran in the hemolymph, and the onset of convulsions only occurred after the concentration of carbofuran in the hemolymph reached μM levels. This value correlated well with neurobioassays of known concentrations of carbofuran perfused in saline onto the isolated thoracic ganglion.Following topical doses, carbofuran concentration in the hemolymph reached a peak within an hour and then gradually declined. At an LD₆₀ dose, the initial decline in carbofuran concentration in the hemolymph over time was significantly slower than the decline after an LD₁₀ dose, suggesting saturation kinetics.Hemolymph was collected from house flies for up to 3 hr following topical application of toxic amounts of carbofuran. Thereafter, hemolymph volume decreased and blood samples could not be collected. Curiously, hemolymph samples could be collected for 5 hr from house flies that were injected with toxic doses of carbofuran.     

Structure-activity relationships of 1,2,3-benzothiadiazoles as synergists for carbaryl against the house fly (Musca domestica)

Fifty six 1,2,3-benzothiadiazoles and related compounds were evaluated as carbaryl synergists against the house fly (Musca domestica). Many of these were excellent synergists, the most active being those containing various combinations of halogen, alkyl, or alkoxy substituents in the 5- and/or 6-positions of the ring.Regression analysis on the data from 14 compounds for which substituents constants were available established that synergistic activity can be satisfactorily described by equations in terms of the hydrophobic bonding constant (π) and the homolytic free radical constant (σ ·).The results with compounds related to the 1,2,3-benzothiadiazoles suggest that synergistic activity is associated primarily with the diazosulfide moiety.     

Monitoring of resistance of insecticides in house fly (Musca domestica) populations in hungary

Samples of 24 house fly (Musca domestica L.) populations were collected from animal farms in Hungary in 1990 and kept in the laboratory to determine their susceptibility to different types of insecticide: organochlorines, organophosphates, carbamates, pyrethroids, macrocyclic lactone and insect growth regulators. The adulticides were tested with topical bioassay in all 24 populations, the larvicides were studied with treated larval medium in 16 populations. The data were expressed as LD₅₀ and LC₅₀ values (ng fly ?1 and mg kg ?1 larval medium respectively). The percentages of populations which had resistance ratios > 10 at LD₅₀ or LC₅₀ were: 63% to DDT, 50% to methoxychlor, 13% to lindane, 83% to malathion, 63% to trichlorfon, 4% to propetamphos, 96% to dioxacarb, 46% to propoxur, 4% to methomyl, 13% to pyrethrum, 96% to bioresmethrin, 63% to permethrin, 58% to cypermethrin, 79% to SK-80, 79% to deltamethrin, 38% to invermectin, 0% to diflubenzuron, 0% to cyromazine. Correlation analysis showed a high degree of positive correlation among the adulticides except for ivermectin, bioresmethrin and SK-80. No cross-resistance was found between the larvicides and the conventional adulticides. Differences of insecticide resistance levels among the populations surveyed were studied by principal component and factor analysis. A fairly good relationship between resistance status and control practices used on farms was revealed. The populations originating from those farms where the application of adulticides had been frequent or regular and where high resistance was shown to most chemicals could be separated from the others.