Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies

The mechanisms of resistance to pyrethroids were studied in a permethrin-selected (147-R) strain of the house fly, Musca domestica L. Approximately 12-fold synergism was obtained with a mixture of (1R)-trans-permethrin:piperonyl butoxide (1:5) so that the resistance decreased from 97-fold to 22-fold. Tests with the esterase inhibitor S,S,S-tributyl phosphorotrithioate produced very little synergism in either the resistant (R) strain (1.6-fold) or the susceptible (S) strain (1.9-fold). An investigation of the microsomal components revealed that compared to the S strain, the R strain demonstrated twice as much cytochrome P-450 and cytochrome b₅ and double the rate of NADPH-cytochrome c reductase activity. In addition, the rate of p-nitroanisole O-demethylation was found to be six times greater in the R strain. An in vivo accumulation study showed that the R strain displayed a decreased rate of penetration of trans-[¹⁴C]permethrin. When treated at equitoxic doses the R strain was found to tolerate 50-fold more internal permethrin than the S strain. An in vitro metabolism study indicated that there was no difference between strains in the overall rate of metabolism of trans-[¹⁴C]permethrin. The evidence obtained supports the conclusion that several resistance factors are involved but that decreased sensitivity of the nervous system to the action of pyrethroids is the principal mechanism of resistance in the 147-R strain.     

The residual contact toxicity of bay sir 8514 toSpodoptera littoralis larvae

The contact effect of residues on glass of the chitin synthesis inhibitor BAY SIR 8514 againstSpodoptera littoralis (Boisduval) larvae was investigated and probitlog dosage curves were established. The ED₅₀ for cumulative mortality up to the adult stage was 0.0017 g/m² for 100-mg and 0.004 g/m² for 200-mg larvae. The toxicity of BAY SIR 8514 through this route of administration was considerably higher than that of diflubenzuron found in previous work.     

Laboratory selected resistance to diflubenzuron in larvae of aedes aegypti

The variation in tolerance to diflubenzuron [1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] was examined in fourth instar larvae of seven strains of Aedes aegypti, some of which were resistant to DDT and permethrin. The difference between the least and the most tolerant to diflubenzuron was approximately two-fold. There was no correlation with resistance to the other insecticides. A DDT-resistant strain (T8) was selected 10 times (during 12 generations) with diflubenzuron. The LC₅₀ to diflubenzuron had increased 3.3 times by the S₈ generation but there was no further increase in later generations despite further selection. Associated with this increase, a marked decrease in resistance to DDT was observed but no change in permethrin tolerance. A genetically enriched strain (Hotchpotch) was synthesised from 35 strains of different geographic origin and crossed to the selected T8 strain before subsequent generations were selected five times with diflubenzuron. This procedure resulted in an 8 to 12-fold increase in the LC₅₀ value over that for unselected T8, accompanied by a decrease in the slope of the log dose against probit mortality line.     

The inhibition of chitin synthesis in spodoptera littoralis larvae by flufenoxuron,teflubenzuron and diflubenzuron

The chitin precursor [¹⁴C] N-acetylglucosamine injected into the haemolymph of Spodoptera littoralis (Boisduval) larva was incorporated into the chitin exponentially with time. When caterpillars were injected with precursor at the commencement of feeding on acylurea-treated leaf discs, flufenoxuron, teflubenzuron and diflubenzuron were found to be equally effective inhibitors of chitin synthesis, measured after 21 h. The dose response curves by feeding are not parallel, indicating that the relative potency of the compounds will vary across the dose range. When chitin precursor was injected simultaneously with topically applied diflubenzuron, flufenoxuron or teflubenzuron, all three acylureas were found to be equally effective as inhibitors of chitin synthesis when measured after five hours. The I₅₀values (50% inhibition of chitin synthesis) were not significantly different; average 600 ng, compared with LD₅₀values (50% lethal dose) of ～ 13 ng for flufenoxuron and teflubenzuron but 130 ng for diflubenzuron (topical application). Injection of precursor 24 h after topical application of insecticide gave an I₅₀value which had dropped 670- and 150-fold for flufenoxuron and teflubenzuron respectively but only 20-fold for diflubenzuron. It is postulated that the reason for the low increase in diflubenzuron effectiveness with time was due either to less diflubenzuron than flufenoxuron reaching the site of action, or more probably, a faster rate of metabolism and excretion for diflubenzuron. The lower toxicity of diflubenzuron compared with flufenoxuron and teflubenzuron may not be due to any inherent differences in biochemical effectiveness, but rather to different penetration/metabolism properties.     

The mechanism of resistance to lindane and hexadeuterated lindane in the Third Yumenoshima strain of house fly

The factors which cause lindane resistance in the Third Yumenoshima strain, a strain of house flies highly resistant to insecticides, were studied using hexadeuterated lindane. Hexadeuterated lindane has the same physicochemical properties as lindane, but the former is much less biodegradable than the latter. The LD₅₀ ratio of lindane to hexadeuterated lindane in this strain, deuterium isotope effect on LD₅₀ values, was larger than that in S_NAIDM, a susceptible (nonresistant) strain. The penetration rates of labeled and nonlabeled lindane through the insect cuticle were about the same for both strains. Thus, penetration rate does not cause resistance. The metabolic degradation of lindane in the resistant strain in vivo occurred much faster than in the susceptible strain. This was also the case for lindane degradation processes in vitro such as microsomal oxidation and glutathione conjugation. In both strains, significant isotope effects were observed in the degradation rates in vitro of labeled and nonlabeled lindane. Therefore, principal biodegradation and detoxication pathways should include reactions which cleave the CH bonds. When the much less biodegradable d₆ counterpart of lindane was applied to both strains, the susceptible strain became much more highly intoxicated than the other within 20 to 30 min. This indicates that a combination of both greater degradability and probably lower sensitivity at the action site are the main factors underlying resistance in the Third Yumenoshima strain.     

Inhibition of chitin synthesis by two 1-(2,6-disubstituted benzoyl)-3-phenylurea insecticides. II

The knowledge of the biochemical mode of action of 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea (diflubenzuron) is presented, explaining the insecticidal effect. Like its structural analog, 1-(2,6-dichlorobenzyl)-3-(3,4-dichlorophenyl)urea (Du 19111), it inhibits chitin synthesis in the cuticle of larvae. Virtually complete inhibition was demonstrable 15 min after the application of diflubenzuron. Neither diflubenzuron nor Du 19111 has any effect upon chitinase activity either in vivo or in vitro. The insecticidal effect upon the cuticle, therefore, must be explained as an inhibition of chitin synthesis and not as an activation of chitin degradation. In contrast to the action of Du 19111, no accumulation of N-acetylglucosamine occurs upon treatment of larvae with diflubenzuron. Similarities and differences in the mode of action of both compounds are discussed, together with other effects reported in the literature.     

Multifactorial resistance to transpermethrin in field-collected strains of the tobacco budworm Heliothis virescens F.

The penetration, degradation and excretion of [³H]transpermethrin were examined in susceptible and field-collected pyrethroid-resistant strains of the tobacco budworm Heliothis virescens. No consistent differences in labelled materials excreted or recovered in cuticle rinses were found between the resistant (R) and susceptible (S) larvae. Considerably lower levels of the parent compound were present internally in R compared with S larvae after 24h (P <0.01), clearly identifying a metabolic resistance mechanism in Meloland and Westmorland larvae. Moderate levels of absorbed permethrin accompanied by an absence of poisoning symptoms were observed in certain individuals of both R strains, suggesting a second resistance mechanism. Neurobioassays of R larvae showed a consistently lower sensitivity of the neuromuscular system to pyrethroids when compared with the S larvae, thus confirming the indication from metabolic studies of the additional (site-insensitive) mechanism. Toxicity values suggest a cross-resistance to other pyrethroids.     

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.     

The joint action of mixtures of insecticides,or of insect growth regulators and insecticides,on susceptible and diflubenzuron-resistant strains of Spodoptera littoralis boisd

The joint action of insecticides, or of mixtures of insect growth regulators and insecticides, on the susceptible (S) strain and diflubenzuron-resistant (R_d) strain of the cotton leafworm Spodoptera littoralis Boisd. was investigated. The joint action of the insecticides and/or insect growth regulator mixtures was determined by mixing them in proportion to their activity equivalents at the LD₂₅ or ED₂₅ levels. A total of 15 mixtures of two synthetic pyrethroids, two organophosphorus, one carbamate and one organochlorine insecticides, were applied to the fourth-instar larvae of the S and R_d strains. The insecticide mixtures cypermethrin/methomyl and cypermethrin/endrin exhibited high and moderate levels of synergism on the S strain, respectively. However the mixtures chlorpyrifos/methomyl, phosfolan/methomyl, and phosfolan/endrin produced antagonism, while the other mixtures showed varying levels of additive effects. The response of the fourth-instar larvae of the S strain, to the joint action of diflubenzuron/juvenoid, diflubenzuron/insecticide, or insecticide/juvenoid mixtures, revealed that diflubenzuron produced high levels of synergism when combined with methoprene and progressively less with fenvalerate, methomyl and cypermethrin. On the other hand, the mixture diflubenzuron/triprene was antagonistic. Fenvalerate with the two juvenoids produced synergism while methomyl showed an additive effect with methoprene. However, the mixtures cypermethrin/methoprene, cypermethrin/triprene and methomyl/triprene produced antagonism. The mixtures that produced potentiation on the fourth-instar larvae of the S strain lost their high potency when tested against the R_d strain. The results also indicated that insecticide/juvenoid mixtures, when applied on 2-day-old pupae of the S strain, were synergistic, except in the case of cypermethrin/methoprene and methomyl/triprene mixtures, for which additive effects were observed. When the mixtures that had synergistic effects on the S strain were tested on the R_d strain, the results revealed that their synergistic effects were apparently reduced. This was attributed to the fact that the generalised levels of tolerance in the R_d strain towards various compounds may have influenced the several defence mechanisms to act against the synergistic action of the chemical mixtures.     

The effect of IKI-7899, a new chitin synthesis inhibitor,on larvae ofTribolium castaneum andSpodoptera littoralis

The effect of IKI-7899 and diflubenzuron on larval development and emergence ofTribolium castaneum and ofSpodoptera littoralis was determined. The LC₅₀ of IKI-7899 at adult emergence ofT. castaneum larvae fed the compound in their diet and the ED₅₀ of S.littoralis larvae treated topically were approximately one-sixth those of diflubenzuron.     

The effect of BAY SIR 8514 onspodoptera littoralis (Boisduval) eggs and larvae

The chitin biosynthesis inhibitor diflubenzuron and its analog BAY SIR 8514 are equitoxic for eggs ofSpodoptera littoralis. BAY SIR 8514 residues on alfalfa fed toSpodoptera larvae were more toxic than diflubenzuron residues, whereas BAY SIR 8514 topically applied to these larvae was consistently twice as toxic as diflubenzuron, according to different criteria.     

The effect of CME 134 onSpodoptera Littoralis Eggs and larvae

CME 134, a new benzoylphenyl urea chitin synthesis inhibitor, was less active than diflubenzuron and BAY SIR 8514, when tested againstSpodoptera littoralis eggs by a dipping method. AgainstS. littoralis larvae the compound was tested by feeding treated alfalfa, topical application and contact with crystalline residues on glass, followed by observation until the adult stage. With both 200–250 and 360–440-mg larvae 100% mortality was obtained by one-day feeding of alfalfa treated with 0.15 ppm a.i. Topical application to 100- and 200-mg larvae showed CME 134 to be about five and nine times more active than BAY SIR 8514 and diflubenzuron, respectively. These differences were even much greater in the contact tests. Cotton field plots were sprayed with either CME 134 or diflubenzuron formulations, leaves were collected at different intervals and fed for one day toS. littoralis larvae in the laboratory. 0.0009% a.i. CME 134 residues gave complete kill of 30–50-mg larvae after 5 and 20 days, and 86% kill after 28 days of aging. With 0.003 and 0.009% a.i., complete kill was obtained in 200–250-mg larvae until 50 days after spraying.     

Pyrethroid resistance in a strain of Spodoptera littoralis is correlated with decreased sensitivity of the CNS in vitro

The effects of permethrin and cypermethrin on the isolated abdominal nerve cord of insecticide-resistant [R] and -susceptible [S] strains of Spodoptera littoralis larvae have been studied. Above ca. 19°C, permethrin at 10^?7M caused a prolonged spike train to follow electrical stimulation of the nerve cord. The time of onset of this repetitive firing was significantly greater for the [R] strain. Moreover, cypermethrin, to which this strain shows negligible resistance, did not cause such repetitive discharges. Thus, resistance to permethrin but not to cypermethrin appears to be based on a qualitative difference between the pyrethroids. Nerve blockage by the two pyrethroids was also investigated, with particular reference to temperature. Once again, differences were apparent: when considered relative to untreated controls, permethrin caused quicker nerve blockage as temperature was reduced whereas the blocking action of cypermethrin was not affected by temperature. However, the times taken to cause nerve blockage by permethrin in [R] and [S] larvae were not significantly different, making it unlikely that nerve blockage plays a major role in this resistance. Two methods were employed to reduce the resistance factor in vitro. The synergist dodecyl imidazole failed to significantly reduce the time taken for permethrin to cause either repetitive firing or nerve blockage. However, reducing the calcium concentration in the saline did significantly reduce the latency of repetitive firing caused by permethrin in [R] larvae, thus increasing the nerve sensitivity to approximately the same level as normal calcium, [S] insects.     

Resistance risk assessment of two insect development inhibitors,diflubenzuron and cyromazine,for control of the housefly Musca domestica. part i: Larvicidal tests with insecticide-resistant laboratory and danish field populations

Larvicide tests with diflubenzuron (DFB) and cyromazine (CYR) were carried out against 15 laboratory housefly strains and 89 field strains collected from 87 Danish and 2 Swedish farms, 1975–89. The strains represented a wide range of adult insecticide resistance and R-mechanisms. The larvicide tests were done by treating larval medium with serial or discriminating dosages of the larvicide, seeding it with eggs and calculating the mortality during development to adults. The WHO susceptible strain (S) was used as a reference. Dose-response tests with DFB gave resistance ratios (R/S) from 1·1 to 4·1 at LC₅₀ and 0·3 to 3·4 at LC₉₅ and, with CYR, R/S from 0·6 to 1·8 at LC₅₀ and 0·6 to 2·9 at LC₉₅. It was concluded that the relatively small variation in susceptibility between strains was not generally correlated with resistance in adult flies to organophosphorus, pyrethroid or other conventional insecticides (neurotoxins). Tests with discriminating dosages of DFB (59 farms) and CYR (63 farms) showed no indication of resistance to either product. The results of investigations by other workers on the relation between resistance to DFB or CYR and resistance to conventional insecticides are discussed.     

The pattern of cross-resistance to insecticides and juvenile hormone analogues in a diflubenzuron-resistant strain of the cotton leaf worm Spodoptera littoralis boisd

The rate of development of resistance to diflubenzuron in a laboratory susceptible strain of the cotton leafworm Spodoptera littoralis Boisd., the pattern of cross-resistance exhibited by the resistant strain to several insecticides and juvenile hormone analogues, as well as the synergistic action of piperonyl butoxide (PB) and S, S, S-tributyl phosphorotrithioate (TBP) with insecticides or diflubenzuron on both strains, were investigated. Resistance to diflubenzuron increased slightly in the first eight selected generations and was enhanced by further selection until in generation 30, the selected strain attained the high level of resistance of 290.7-fold, compared with the parent strain. The resistant strain when challenged with either insecticides or juvenile hormone analogues at selected generations (5, 10, 15, 20, 25 and 30) exhibited different levels of resistance to several insecticides representing organochlorine and organo-phosphorus compounds, carbamates and pyrethroids, but a clear case of negative correlation was indicated between resistance to diflubenzuron and juvenile hormone analogues. With regard to the synergistic action of PB and TBP on the toxicity of either diflubenzuron or insecticides against the fourth-instar larvae of the susceptible strain, methomyl showed slight levels of synergism when it was combined with them. With the exception of cypermethrin, which was not affected by the two synergists, lower levels of synergism were observed with the compounds endrin, diflubenzuron and fenvalerate when they were combined with the same synergists. These two synergists however, antagonised the toxic action of the organophosphorus compounds phosfolan and chlorpyrifos. Against the resistant strain, endrin was moderately synergised by TBP but only slightly by PB. Slight levels of synergism were observed when methomyl, phosfolan and diflubenzuron were combined with either synergist, but both antagonised chlorpyrifos and fenvalerate. Resistance to diflubenzuron and to the other tested chemicals in the resistant strain was scarcely affected by the two synergists.     

Effect of insecticide—synergist combinations on the survival of Spodoptera exigua

The chitin synthesis inhibitors diflubenzuron and teflubenzuron have recently become ineffective for the control of Spodoptera exigua in floricultural crops. An extended laboratory test with second-instar larvae of S. exigua on Vicia faba plants was carried out to determine the influence of synergists on the biological activity of three benzoylphenyl ureas (BPUs). The co-application of piperonyl butoxide, an oxidase inhibitor, did not increase the activity of diflubenzuron, teflubenzuron or hexaflumuron. The best results were obtained with diethyl maleate, for suppressing glutathione S-transferase activity, and with dimethoate, as a hydrolase inhibitor. A joint application of diflubenzuron (at a concentration which resulted in 43% survival) with diethyl maleate or dimethoate gave only 6.2 and 8.9% surviving larvae, respectively. In addition, development to fourth-instar larvae was inhibited. The more stable teflubenzuron was synergized by both compounds to a much lesser extent than diflubenzuron. None of the synergists had a significant effect on the activity of hexaflumuron, which was the most potent insecticide of the three BPUs tested against S. exigua.     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

Inhibition of chitin synthesis by benzoyl-phenylurea insecticides,III. Similarity in action in Pieris brassicae (L.) with Polyoxin D

The increase in cuticle thickness with age of fifth instar larvae of Pieris brassicae (L.) was measured microscopically. The injection of a lethal dose of either Polyoxin D or diflubenzuron revealed total inhibition of cuticular growth and caused comparable abnormalities in the cuticles.In a further experiment [¹⁴C]glucose was injected along with Polyoxin D into Pieris brassicae and the incorporation of radioactivity into various tissue fractions was measured. This revealed that the impairment of cuticular growth was due to inhibition of chitin synthesis.With the methods used the effects of Polyoxin D and two benzoylphenylurea insecticides appeared to be the same.     

Delayed cuticular penetration and enhanced metabolism of deltamethrin in pyrethroid-resistant strains of Helicoverpa armigera from China and Pakistan

The penetration and metabolism of [14C]deltamethrin was studied in susceptible and resistant Chinese and Pakistani strains of Helicoverpa armigera (Hübner), which were resistant to deltamethrin by 330- and 670-fold, respectively. The penetration of deltamethrin into resistant individuals was significantly slower than into susceptible individuals over a 24-h period. The time taken for 50% penetration of the applied deltamethrin was 1 h for the susceptible strain and 6 h for both of the resistant strains. The internal radioactivity was reduced by the larvae of resistant strains much faster than by the susceptible larvae. After 48 h, 40% of the penetrated deltamethrin was still inside the larvae of the susceptible strain, in comparison with 1.5-5% in the Pakistani strain and 8-14% in the Chinese strain. Both of the resistant strains produced methanol-soluble and water-soluble metabolites, but the susceptible strain produced methanol-soluble metabolites only. By 12, 24 and 48 h after dosing, the amount of methanol-soluble metabolites excreted by the resistant strains was almost double that of the susceptible strain. Both of the resistant strains also excreted 5-7% of the penetrated dose as a water-soluble metabolite after 48 h. In comparison with the Chinese strain, the Pakistani strain exhibited slower penetration, lower internal content and faster excretion of deltamethrin, which correlated with the higher resistance of the Pakistani strain. These findings show that the resistant Pakistani and Chinese strains of H. armigera possess mechanisms of reduced cuticular penetration and enhanced metabolism of deltamethrin and perhaps other pyrethroids.     

Ph 70–23: A new acaricide and insecticide interfering with chitin deposition

PH 70–23 is a new benzoylphenylurea interfering with chitin deposition. In larvae of Spodoptera littoralis, incorporation of N-acetyl-glucosamine into chitin was equally inhibited by PH 70–23 and diflubenzuron, the first commercially available benzoylphenylurea. In contrast to diflubenzuron, which is active only against insects and eriophyid mites, PH 70–23 has the advantage of also being highly active on spider mites. The latter activity might be partly due to leaf penetration of PH 70–23. Laboratory experiments indicate PH 70–23 to have a better ovo-larvicidal activity than the commercial acaricides tested (cyhexatin, dicofol, fenbutatin oxide and tetradifon) against Tetranychus cinnabarinus, T. turkestani, T. urticae and Panonychus ulmi. A method in which eggs were deposited on residues of cyhexatin or fenbutatin oxide resulted in much lower activities than application of the spray liquid after egg deposition. Activities of PH 70–23, dicofol and tetradifon were hardly influenced by this difference in the test method. The direct contact activity of PH 70–23 on eggs of mites is influenced by the relative humidity and the age of the eggs. The total ovolarvicidal activity of PH 70–23 is only slightly positively influenced by the relative humidity, however. Ovicidal activity of PH 70–23 is also achieved by transovarial transmission. The compound does not influence the fertility of the mites. Comparison of a strain of T. urticae, resistant to dicofol, parathion and tetradifon, with a susceptible strain indicated absence of cross-resistance to PH 70–23. Compared to diflubenzuron, PH 70–23 shows an interesting shift in the spectrum of insecticidal activity.