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Mutations of acetylcholinesterase which confer insecticide resistance in Drosophila melanogaster populations.

BACKGROUND: Organophosphate and carbamate insecticides irreversibly inhibit acetylcholinesterase causing death of insects. Resistance-modified acetylcholinesterases(AChEs) have been described in many insect species and sequencing of their genes allowed several point mutations to be described. However, their relative frequency and their cartography had not yet been addressed. RESULTS: To analyze the most frequent mutations providing insecticide resistance in Drosophila melanogaster acetylcholinesterase, the Ace gene was cloned and sequenced in several strains harvested from different parts of the world. Sequence comparison revealed four widespread mutations, I161V, G265A, F330Y and G368A. We confirm here that mutations are found either isolated or in combination in the same protein and we show that most natural populations are heterogeneous, composed of a mixture of different alleles. In vitro expression of mutated proteins showed that combining mutations in the same protein has two consequences: it increases resistance level and provides a wide spectrum of resistance. CONCLUSION: The presence of several alleles in natural populations, offering various resistance to carbamate and organophosphate compounds will complicate the establishment of resistance management programs.

Acetylcholinesterase↗

High paraoxon-hydrolyzing activity in organophosphorus insecticide-resistant mosquitoes.

We found a strong paraoxon-hydrolyzing activity (23.4 +/- 8.50 nmol/h/individual and 137 +/- 86.2 nmol/h/mg protein) in the crude extract from larvae of Culex tritaeniorhynchus Toyama 89, which is markedly resistant to organophosphorous insecticides. The activity was higher than those from Cx. tritaeniorhynchus re-e-ae (0.175 +/- 0.0336 and 1.83 +/- 0.651), Anopheles omorii (0.112 +/- 0.0301 and 1.86 +/- 0.746) and An. stephensi (0.0651 +/- 0.0713 and 0.789 +/- 0.910), which are susceptible to organophosphorous insecticides. These facts suggest that the high paraoxon-hydrolyzing activity plays a role in the development of organophosphorous resistance in Cx. tritaeniorhynchus. The enzyme preparation obtained from Toyama 89 showed higher activity in the alkaline pH range and its Km values to paraoxon were 0.67 mM in larvae and 0.50 mM in adults. A calcium ion was strictly required for the hydrolysis of paraoxon. Fenitroxon was also hydrolyzed, in addition to paraoxon. However, it did not degradate parathion and fenitrothion at all. Dichlorvos and phenyl acetate competitively inhibited the enzyme. The phenyl acetate-hydrolyzing activity in the preparation of Toyama 89 was significantly (p less than 0.01) lower than those in susceptible strains, and was irreversibly inhibited by paraoxon. Therefore, the paraoxon-hydrolyzing activity belongs to the class of organophosphate compound hydrolases; it must be thus distinguished from bacterial phosphotriesterase.

Animals↗

Identifying insecticide-resistant greenbugs (Homoptera: Aphididae) with diagnostic assay tests.

Laboratory bioassays were used to develop a diagnostic assay test for identifying greenburg, Schizaphis graminum (Rondani), populations that are insecticide-resistant. Petri dish assays with chlorpyrifos showed greenbug mortality should be monitored after 2 h of exposure. One-hour exposure did not kill a high percentage of susceptible greenbugs, and a 3-h exposure killed too many resistant greenbugs. Ethanol and methanol were both good solvents for mixing with chlorpyrifos in the petri dish assay. From the laboratory bioassays, four diagnostic concentrations of chlorpyrifos (3, 10, 30, and 100 ppm) were evaluated in the field by Texas A&M University agricultural research and extension entomologists across the Texas High Plains. Results from the diagnostic assay tests were compared with gel-electrophoresis resistance tests to validate resistance detection. The diagnostic assay tests gave the same greenbug resistance identification as the gel-electrophoresis analysis in 21 of 22 field bioassays in 1994 and 35 of 39 field bioassays in 1995. Diagnostic concentrations of 30 and 100 ppm chlorpyrifos killed > or = 85 and > or = 90%, respectively, of greenbugs identified by gel-electrophoresis as susceptible and < 40% and < 55%, respectively, of resistant greenbugs. The diagnostic assay technique is a quick, reliable, and inexpensive method for detecting insecticide resistance in greenbug populations.

Animals↗

Negative correlations between resistance to three organophosphate insecticides and productivity within a natural population of Drosophila melanogaster (Diptera: Drosophilidae).

To investigate the relationship between resistance to organophosphate insecticides and fitness components, we first measured resistance to three organophosphates, malathion, prothiophos, and fenitrothion, and productivity, a measure of fitness components, for each of the isofemale lines from the same natural population of Drosophila melanogaster (Meigen). Pearson correlation coefficients indicated that positive correlations among resistance to the organophosphates and negative correlations between resistance to each of the organophosphates and the productivity existed within the natural population. We further investigated the genetic basis of the correlations among resistance to the organophosphates and the productivity, by using chromosome-substituted lines between a resistant and a susceptible inbred line established from the same natural population. Chromosomal analyses indicated that the third chromosome from the resistant line exhibited not only significant, positive effects on resistance to all of the organophosphates tested but also a significant negative effect on the productivity, suggesting positive genetic correlations between resistance to each organophosphate and negative genetic correlations between resistance to each organophosphate and the productivity. In addition, a significant negative effect on the productivity was also detected from the second chromosome, which did not exhibit significant major effects on resistance to the organophosphates. This suggests that fitness components of resistant lines could be also affected by factors independent of insecticide resistance. The dynamics of genetic variation in resistance to the organophosphates within the natural population of D. melanogaster are discussed from the standpoint of negative genetic correlations between resistance to the organophosphates and the productivity.

Animals↗

Managing head lice in an era of increasing resistance to insecticides.

Head lice are present in all age groups, however, the peak age for infestation is 7-8 years and the incidence varies throughout the year with higher incidence during the winter. Different insecticides have been used over the past 60 years to manage this condition. There is now strong evidence of insecticide resistance established in many countries to such an extent that some of these chemicals have become obsolete. Resistance to some pediculicides can vary from country to country and region to region within a country. The lack of a local monitoring system of resistance patterns means that parents and pupils are hampered in making an informed decision regarding how to treat head lice. One should no longer assume that treatment failure is due to poor treatment compliance or re-infestation. Clear treatment guidelines drawn up by healthcare professionals with an interest in head lice and taking into account regional/national resistance patterns should be implemented. These guidelines should combine chemical and non-chemical approaches to treatment and be coordinated and regularly reviewed by local public health departments. Drug companies should be made to provide up-to-date efficacy of their products.

Animals↗

Insecticide resistance in Culex pipiens from New York.

Insecticides are the primary means to control Culex pipiens, an enzootic vector of West Nile virus, in the USA. To better understand how the evolution of resistance might impact control of this insect, we investigated the levels of resistance in populations collected from 2 metropolitan areas (Albany and Syracuse, NY) to 4 larvicides (methoprene, phenothrin, Bacillus sphaericus [Bs], and Bacillus thuringiensis israelensis [Bti]) and 1 adulticide (phenothrin) registered for mosquito control in New York State. High levels of resistance were found only to Bti, and only at 1 site (Syracuse). Resistance levels to the other insecticides were less than 10-fold. Given the large difference in Bti resistance between Syracuse and Albany, it appears these populations of Cx. pipiens do not rapidly mix, leading to localization of resistant populations.

Animals↗

[Laboratory evaluation of alpha-cypermethrin insecticide efficacy on Anopheles gambiae populations of Côte d'Ivoire resistant to permethrin and deltamethrin].

Susceptibility tests were carried out in laboratory conditions to evaluate the efficacy of alpha-cypermethrin (a synthetic pyrethroid never used in Côte d'Ivoire) for malaria vector control. Five wild populations of Anopheles gambiae originating from M'bé, Yaokoffikro, Korhogo, Kafiné and Daola and two laboratory reared strains (Kisumu susceptible and Kou permethrin resistant selected strain) were tested. The diagnostic dosage of alpha-cypermethrin for the sensitive strain Kisumu was 2.5 x 10(-3)%. A comparative study of the susceptibility of samples of wild populations of An. gambiae was carried out according to the WHO standard susceptibility test. Impregnated papers with 4% DDT, 0.25% permethrin, 0.025% deltamethrin and 0.0025% alpha-cypermethrin were used. The results showed that except for mosquitoes from M'bé, all the other populations were resistant to these insecticides. Bioassays were carried out with alpha-cypermethrin at the operational dosage of 20 mg a.i./m2 on the same population and laboratory reared strains. The results showed the efficacy of this insecticide on both the Kisumu strain and the population from M'bé, a maintained efficacy for the Daloa, Kafiné and Korhogo mosquito populations, but the wild anopheline population from Yaokoffikro clearly appeared fully resistant.

Animals↗

[The effect of insecticide resistance on the demographic parameters of 3 strains of Culex quinquefasciatus (Diptera: Culicidae) under laboratory conditions].

The life tables of 3 strains of Culex quinquefasciatus were studied under laboratory conditions: Slab, susceptible to reference; Old Havana, treated with cypermethrin until its fourth generation; and Cotorro, from the field. It was found that the different levels of resistance to organophosphate insecticides and pyrethroids present in the Cotorro and Old Havana strains did not influence either on the duration of the period of development of the immature stages or in the sexual proportion of the emerged adults, but they had a favorable and directly proportional effect on the survival and longevity of the adults. However, they exerted a negative and inversely proportional influence on the reproduction and population growth of these strains. Useful data for the use and management of insecticides within the strategies of control of Culex quinquefasciatus, an important vector of lymphatic filariasis, are provided.

Animals↗

Susceptibility of Musca domestica L. (Diptera: Muscidae) from various breeding sites to commonly used insecticides.

Field strains of house flies, Musca domestica L., from three different breeding sites-garbage dump (IMR), poultry farm (Kundang, Selangor), and agricultural farm (Kampung Batu, Kuala Lumpur), were evaluated against five insecticides. Resistance status of adult female flies was determined using the modified WHO bioassay methods. The WHO susceptibility strain was used as a reference strain for comparison. Flies from the garbage dump and poultry farm were more resistant to the insecticides than the strain from the agricultural farm. Results obtained from bioassay tests were confirmed by in-vitro microenzyme assays of non-specific esterases and glutathione-S-transferases. Significant differences between the esterase levels of WHO and field strains were observed. Levels of glutathion-S-transferases were approximately the same, which may indicate that other enzymes are involved in house fly resistance.

Animals↗

Evaluation of a polymerase chain reaction assay for detection of pyrethroid insecticide resistance in the malaria vector species of the Anopheles gambiae complex.

A published polymerase chain reaction assay proved reliable for detecting nerve-insensitivity (kdr-type) resistant and susceptible alleles to the pyrethroid insecticide permethrin in the malaria vectors Anopheles gambiae s.s. and An. arabiensis and the nonvector species An. quadriannulatus. The assay detects pyrethroid susceptible and resistance alleles mediated by a mutation in region II of the para-type sodium channel gene, showing that resistance is conferred by pyrethroid target site insensitivity. Analysis of results of the assay suggests that more than 1 mechanism conferring pyrethroid resistance is operating in an An. gambiae s.s. strain from Côte d'Ivoire. The value of the assay as a tool for monitoring this mode of insecticide resistance in wild populations is discussed.

Animals↗

Insecticide cross-resistance spectra and underlying resistance mechanisms of Sri Lankan anopheline vectors of malaria.

Present status of insecticide resistance was investigated in two major vectors of malaria; An. culicifacies and An. subpictus, collected from a high malaria transmission area in Sri Lanka during 1996/1998. Adult and larval bioassays were carried out to obtain log-probit mortality lines for malathion, propoxur, permethrin and chlorpyrifos. Respective LD50 values were 4.45%, 0.002%, 0.16% and 0.001% for An. culicifacies and 0.66%, 0.004%, 0.004% and 0.04% for An. subpictus. Adults were also tested for WHO standard discriminating dosages of malathion, propoxur, permethrin, DDT, cypermethrin, deltamethlin and lambda cyhalothrin. Both populations were highly resistant to DDT. An. culicifacies was more resistant to malathion and An. subpictus was more resistant to chlorpyrifos. About 25% of both populations were resistant to permethrin. An. culicifacies was susceptible to propoxur, deltamethrin and lambda cyhalothrin and An. subpictus to cypermethrin and lambda cyhalothrin. Adult mosquitos were individually tested for their insecticide detoxifying enzyme activities and altered target-site, acetylcholinesterase. High general esterase activity indicated the presence of amplified esterase genes in both populations. Native gel electrophoresis resolved one elevated esterase isoenzyme, with high affinity to organophosphates, from each species. Malathion carboxylesterase mechanism was present in both populations. Higher glutathione-S-transferase activity was marked in An. subpictus. Synergistic studies showed the possible involvement of monooxygenases in resistance in both species. Acetylcholinesterase activity of approximately 80% of both populations was not inhibited by a standard dosage of propoxur. Low resistance to carbamates shows that the impact of agricultural pesticides is not significant in the development of resistance especially in An. culicifaies. Pyrethroids, other than permethrin, can be successfully used in vector control programs. Carbamates will be an alternative.

Acetylcholinesterase↗

Insecticide susceptibility tests of Anopheles minimus s.l., Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus in northern Thailand.

The susceptibility of Anopheles minimus s.l., Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus to insecticide in northern Thailand was monitored by using the WHO standard susceptibility test. One- to two-day old female mosquitos, which were reared from wild caught females or immature stages, were exposed to discriminating dosages of insecticides for recommended exposure periods, and the 24-hour mortality recorded. The results revealed that, in general, An. minimus s.l. was still susceptible to DDT and permethrin, except in some areas where a slight increase in tolerance to DDT was observed. Ae. aegypti and Ae. albopictus were both highly resistant to DDT, but in some areas the former was also resistant to permethrin and deltamethrin. Cx. quinquefasciatus was resistant to DDT and etofenprox, with a slight increase in tolerance to permethrin, deltamethrin, malathion and fenitrothion. No resistance to lambda-cyhalothrin was detected in any of the species studied.

Animals↗

A pilot project of residual-insecticide spraying to control malaria transmitted by the Anopheles punctulatus group in Netherlands New Guinea.

The authors report on preliminary observations carried out in three experimental houses in Netherlands New Guinea, the object of which was to determine whether residual spraying with insecticides might be expected to give good results in the control of malaria transmitted by mosquitos of the Anopheles punctulatus group and by A. karwari. One house was sprayed with DDT, another with dieldrin, and the third was left unsprayed. From the kills recorded of mosquitos entering these houses at night it is apparent that good results can be anticipated with both insecticides, although dieldrin is the more effective.Observations made at the same time on the behaviour of these mosquitos indicate that they have greater contact with the inside walls of houses than was previously thought.

Animals↗

Control of insecticide exposure in employment; a guide to physicians for dealing with organic phosphates.

Increasingly larger amounts of the highly efficient and highly toxic organic phosphate insecticides, particularly parathion, tetraethyl pyrophosphate, and Systox (demeton), are being applied on California's farms. These insecticides have become an economic necessity to agriculture. They can be used safely when rigid precautions are utilized and when medical supervision is provided for employees regularly working with these chemicals. The demand for the services of physicians prepared to supply this kind of industrial medical supervision is expected to increase in California. The basic procedures through which effective medical supervision can be provided are outlined.

Agriculture↗

Laboratory tests on mosquito tolerance to insecticides and the development of resistance by Aedes aegypti.

Three strains of Aëdes aegypti and a strain of Culex molestus were used in tests to determine the relative susceptibility levels of the larvae to various insecticides and to investigate the development of resistance in the laboratory through selection by larval treatment only. The procedures and test methods used are described and the results discussed and set out in tabular and graphic form.It was found, inter alia, that selection of fourth-stage larvae of a laboratory strain of Aëdes and Culex for 15 generations with DDT at the LC(50) level did not induce any appreciable decrease in susceptibility; that with another Aëdes strain, naturally three times as resistant to DDT as the laboratory strain, selection for eight generations further increased this resistance to seven times its natural level; that selection of the laboratory strain of Aëdes with gamma-BHC for 15 generations induced a twofold increase in resistance to this compound, which also extended to DDT and dieldrin; and that selection of the same strain with dieldrin, over 14 generations at a low mortality level to avoid after-effects of this insecticide, did not induce any appreciable decrease in susceptibility.

Aedes↗

Methods for measuring insecticide susceptibility levels in bed-bugs, cone-nosed bugs, fleas and lice.

A standard kit is prepared and distributed by WHO for testing insecticide resistance in adult mosquitos, and it would seem advantageous to be able to use the filter papers impregnated with DDT and dieldrin contained in this kit for testing resistance in other insects. Experiments have been successfully conducted with a view to developing methods based on the use of these papers for testing susceptibility levels in bed-bugs, cone-nosed bugs, fleas and lice. The designs of the various test methods and the results obtained are described in this paper. The tests for bed-bugs and fleas have been adopted as provisional methods by the WHO Expert Committee on Insecticides; those for cone-nosed bugs and lice have been designated tentative methods, requiring further investigation prior to the drawing up of specifications.

Animals↗

Insecticidal vapours for aircraft disinsection.

A general discussion of the problem of aircraft disinsection is presented. The weaknesses of the present aerosol method, the limitations imposed by airline operators, government officials, passengers, and others, and the potential value of insecticidal vapours as a means of aircraft disinsection are discussed. An apparatus is described for screening insecticides for their vapour toxicity to houseflies and mosquitos. This apparatus led to the discovery of DDVP (O,O-dimethyl-2,2-dichlorovinyl phosphate) and its remarkable vapour toxicity to insects, and to the subsequent studies of its potential use in the vapour state for aircraft disinsection.

Aerosols↗

A mechanical system for dispensing known amounts of insecticidal vapours.

The requirements for a self-contained semi-automatic insecticidal vapour dispenser for use in the disinsection of aircraft are presented. A prototype device meeting these requirements is described and data on its performance, using DDVP (O,O-dimethyl-2,2-dichlorovinyl phosphate) as the insecticide, are given. In this system a miniature air compressor forces air through a membrane impregnated with DDVP, and the vapour-laden air exits into the cabin through a tubular distribution system equipped with orifices. The vapour output is governed by the volume and the temperature of the air passing through the membrane, and the system is adaptable to all types of aircraft at present in use or projected for the near future. The system can also be adapted for use in the disinsection of other closed or semi-closed spaces.

Aerosols↗