Abamectin resistance in Drosophila is related to increased expression of P-glycoprotein via the dEGFR and dAkt pathways

Many insects have evolved resistance to abamectin but the mechanisms involved in this resistance have not been well characterized. P-glycoprotein (P-gp), an ATP-dependent drug-efflux pump transmembrane protein, may be involved in abamectin resistance. We investigated the role of P-gp in abamectin (ABM) resistance in Drosophila using an ABM-resistant strain developed in the laboratory. A toxicity assay, Western blotting analysis and a vanadate-sensitive ATPase activity assay all demonstrated the existence of a direct relationship between P-gp expression and ABM resistance in these flies. Our observations indicate that P-gp levels in flies' heads were higher than in their thorax and abdomen, and that both P-gp levels and LC₅₀ values were higher in resistant than in susceptible and P-gp-deficient strains. In addition, P-gp levels in the blood–brain barrier (BBB) of resistant flies were higher than in susceptible and P-gp-deficient flies, which is further evidence that a high level of P-gp in the BBB is related to ABM resistance. Furthermore, we found greater expression of Drosophila EGFR (dEGFR) in the resistant strain than in the susceptible strain, and that the level of Drosophila Akt (dAkt) was much higher in resistant than in susceptible flies, whereas that in P-gp-deficient flies was very low. Compared to susceptible flies, P-gp levels in the resistant strain were markedly suppressed by the dEGFR and dAkt inhibitors lapatinib and wortmannin. These results suggest that the increased P-gp in resistant flies was regulated by the dEGFR and dAkt pathways and that increased expression of P-gp is an important component of ABM resistance in insects.     

Dieldrin-resistant cabbage root fly in England

The existence of dieldrin resistance in Erioischia brassicae has been investigated in the main areas in England and Wales where brassicas are grown for human consumption. Dieldrin-susceptible and dieldrin-resistant flies were collected and bred as separate strains in the laboratory. In toxicity tests adult flies were treated with topically applied doses of insecticide in microdrops of cellosolve, and larvae were dipped in solutions of insecticide in 70% acetone and 30% water. Adults of the laboratory resistant strain showed a high level of resistance to dieldrin and aldrin and a lower level of resistance to γ-BHC. There was no cross-resistance with chlorfenvinphos or diazinon. Larvae of the resistant strain were shown to be resistant to dieldrin. Dieldrin resistance has been found in four areas in southern England, but not in a fifth major area, and resistant flies did not increase as rapidly in some areas as others. Sufficient migration of flies appeared to take place to produce similar percentages of resistant flies over areas of several square miles, but considerable differences were found in fly populations about 10 miles apart. It is suggested that the speed of development of insecticide resistance is affected by the proportion of brassica crops which are treated in an area, the amounts of insecticide applied to individual crops, and hence the insecticidal pressure applied to the E. brassicae population.     

Aspects of insecticide resistance in the melon and cotton aphid, Aphis gossypii (Hemiptera: Aphididae)

Strains of the aphid Aphis gossypii, taken from cucumber in UK glasshouses were tested for resistance to pirimicarb, diazinon and heptenophos. Compared to a susceptible strain, resistance factors to diazinon of up to 659 were found in populations collected between 1987 and 1989. Compared to the same susceptible strain, diazinon-resistant populations also showed a reduced response to heptenophos. All field populations tested against pirimicarb were resistant. Host plant preference tests showed that all strains (including stock cultures) tested could readily breed on cotton. However strains originally collected from chrysanthemum did not transfer to cucumber and likewise strains from cucumber would not colonise chrysanthemum. Polyacrylamide gel electrophoresis of four of the aphid strains showed differences in migration rates and intensity of staining of esterase bands. Intensity of staining was correlated with the degree of diazinon resistance.     

Acetylcholinesterase. Two types of modifications confer resistance to insecticide.

Quantitative and qualitative changes in acetylcholinesterase confer resistance to insecticides. We have constructed several Drosophila melanogaster strains producing various amounts of enzyme by P-mediated transformation. Toxicological analysis of these strains demonstrates that resistance to organophosphorus insecticides is correlated with the amount of acetylcholinesterase in the central nervous system. Resistance may also be qualitatively determined. Comparison of the Drosophila acetylcholinesterase gene between a resistant strain caught in the wild and a wild type susceptible strain only revealed one nucleotide transition resulting in the replacement of a phenylalanine by a tyrosine. Flies mutant for acetylcholinesterase and rescued with a minigene mutagenized for this same transition produced an altered enzyme which renders flies resistant to pesticides.     

Response of insecticide-resistant and susceptible houseflies (Musca domestica) to a commercial granular bait formulation containing methomyl

Female houseflies (Musca domestica L.) from a susceptible and a multi-insecticide-resistant strain were used to evaluate the relative toxicity of an insecticide bait formulation of the carbamate insecticide methomyl. Individual flies were allowed to feed on bait granules for an unrestricted period or for 5 s. Resistant flies took longer than susceptible flies to initiate a feeding response. When allowed to feed continuously, those from the resistant strain spent longer feeding than susceptible ones. The time taken to knock-down (KD), including feeding times, was significantly greater for resistant than susceptible flies (P less than 0.001), but once the proboscis was withdrawn from the granule there was no difference in KD times between the strains. All flies from both strains were knocked down, and only a very small number of resistant and susceptible flies recovered. The toxic effects of methomyl on flies which were restricted to a 5 s feed ranged from no observed effect to KD in less than 1 min. After feeding for 5 s, 81% of resistant and 98% of susceptible flies developed signs of methomyl poisoning. More resistant than susceptible flies recovered from KD, giving final mortalities of 46% and 88% respectively. With both feeding regimes, some flies of both strains which had apparently recovered from KD had lost their ability to fly. Observations have also shown that 8% of resistant flies may have been repelled by methomyl granules. The implication of these results on the survival of M. domestica in intensive animal units following exposure to methomyl bait is also discussed.     

Biochemical genetics of altered acetylcholinesterase resistance to insecticides in the house fly

Resistance to the organophosphate insecticide tetrachlorvinphos was examined in a house fly (Musca domestica L.) strain with an altered acetylcholinesterase (AChE) of decreased sensitivity to inhibition by the insecticide. Genetic tests showed that both resistance and the altered AChE were controlled by semidominant gene(s) on chromosome II. The gene for resistance was five crossover units from the mutant marker stubby wing (stw). A house fly strain was prepared in which resistance was introduced in to a susceptible stw strain by recombination. Biochemical assays revealed that the altered AChE was introduced along with resistance. Assays of the AChE of resistant and susceptible stw strains by two independent methods showed that the enzyme from resistant flies was 30 times more slowly inhibited by tetrachlorvinphos than the enzyme from susceptible flies.This work was supported in part by NIH Grant ES 00901.Technical Article 13340, Texas Agricultural Experiment Station.     

Organophosphorus resistance in Aphis gossypii (Hemiptera: Aphididae) on chrysanthemum in the UK

The FAO standard dip-test was used to test resistance of glasshouse populations of Aphis gossypii from chrysanthemums to the organophosphorus insecticides heptenophos and diazinon. Compared with a susceptible population collected in 1981, resistance factors to diazinon of up to 538 were found in populations collected after 1984. Resistance to heptenophos was less than that to diazinon but some populations collected after 1984 were 34 times as resistant as the susceptible strain. Possible reasons for the sudden increase in resistance in A. gossypii and current prospects for control of resistant populations are discussed.     

Polygenic and Single Gene Responses to Selection for Resistance to Diazinon in Lucilia Cuprina

Following mutagenesis with ethyl methanesulfonate, selection in a susceptible strain with a concentration of the insecticide diazinon (0.0004%, w/v) above that required to kill 100% of the susceptible strain, the LC100 of that strain, resulted in a single gene response. The resultant four mutant resistant strains have equivalent physiological, genetical and biochemical profiles to a diazinon-resistant strain derived from a natural population and homozygous for the Rop-1 allele. Modification of the microsomal esterase E3 is responsible for resistance in each case. The Rop-1 locus maps approximately 4.4 map units proximal to bu on chromosome IV. Selection within the susceptible distribution, at a concentration of diazinon [0.0001% (w/v)] less than the LC100, resulted in a similar phenotypic response irrespective of whether the base population had been mutagenized. The responses were polygenically based, unique to each selection line and independent of Rop-1. The relevance of the results to selection for insecticide resistance in laboratory and natural populations is discussed.     

Comparative analysis of proteinase activities of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae)

Proteinase activities were compared in soluble and membrane fractions of guts obtained from larvae of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis. Overall, serine proteinases from soluble fractions of the susceptible strain were more active than those of the resistant strain. The soluble trypsin-like proteinase activity of the resistant strain was approximately half that of the susceptible strain. The number and relative molecular masses of soluble and membrane serine proteinases were different. However, there were no significant differences in the activities of serine proteinases and aminopeptidases extracted from midgut membranes of the two strains. Cry1Ab protoxin hydrolysis by soluble proteinase extracts of the resistant strain was reduced approximately 20-30% relative to that of the susceptible strain. Reduced protoxin processing due to decreased activities of Bt protoxin activation proteinases may be associated with resistance to Bt toxin in this resistant strain of O. nubilalis.     

Control of insecticide resistance in a field population of houseflies,Musca domestica,by releasing susceptible flies

Susceptible houseflies, Musca domestica, were released at a waste disposal site to control insecticide resistance in a field housefly population. In the first experiment, a total of 163,000 pupae of the susceptible Takatsuki strain were released in October–November 1977. LD₅₀ values to fenitrothion and diazinon decreased to about one-sixth in April 1978, five months after the releases, of those before the releases. For the second experiment, a susceptible colony was derived by cross and backcross between a white-eyed substrain of the Takatsuki and a field colony. This susceptible colony consisted of whiteeyed flies with low activity and normal-eyed flies bearing no or one white eye gene. The results of large cage experiments suggested that the normal-eyed males of the susceptible colony had half the mating competitiveness of wild males. Approximately 31,000–46,000 susceptible pupae were used in each of five releases from October to November 1980. The population number of each sex, estimated by a mark-release-recapture method, increased from 12,000 in late September to 35,000–43,000 in middle November and then decreased to 5,000–8,000 in early December. The frequency of field-collected males bearing one white eye gene and those bearing one male determining factor, which were characteristics of the susceptible colony released, increased gradually during the period of releases. The susceptibility of the field population to fenitrothion and diazinon was examined five times in the period from September to December 1980. With time, the dosage-mortality regression gradually shifted towards that of the susceptible colony after starting the releases. LD₅₀ values to fenitrothion and diazinon decreased to about one-sixth and one-fifth, respectively, in June 1981, six months after the second series of susceptible fly releases, of those before the releases. Ratios of the wild flies to the released fiies were estimated to be between 4.7∶1 and 9.8∶1 in males and between 3.0∶1 and 3.9∶1 in females by taking the quality of the released colony and the population parameters of the field houseflies into consideration. Under several assumptions, the manner of resistant phenotype reduction was discussed, based on the dosage-mortality regressions and the ratios of released flies. These results showed that the releases of susceptible flies were successful in suppression of insecticide resistance in the field housefly population.     

Genetics of cytochrome P450 in two insecticide-resistant strains of the housefly,Musca domestica L.

A susceptible strain of Musca domestica containing visible mutant markers on chromosomes II, III, and V was crossed with multiresistant R-Fc and R-diazinon strains. F₁ flies were backcrossed to the mutant parent, resultant progenies were isolated according to phenotype, and substrains were established. The level of resistance to diazinon, aldrin epoxidase activity, and cytochrome P450 difference spectra of microsomes from each substrain were measured. Titers of cytochrome P450, measured as CO spectra, as well as type I, type II, and type III cytochrome P450 substrate difference spectra were compared in microsomal preparations obtained from phenotypes containing vatious resistant chromosome combinations. In both resistant strains, high levels of cytochrome P450 were controlled by a gene(s) on chromosome II. In R-diazinon, qualitative spectral changes were also controlled by chromosome II, whereas in R-Fc both chromosomes II and V contributed to qualitative changes in cytochrome P450. Both quantitative and qualitative characteristics were intermediate in heterozygous flies, suggesting incomplete dominance for their inheritance. Findings are discussed in relation to known genetics of microsomal resistance to insecticides.     

Monitoring for insecticide resistance in field-collected strains of the German cockroach (Dictyoptera: Blattellidae)

Forty-five field-collected strains of German cockroaches, Blattella germanica were tested for resistance to 12 different insecticides by the time-mortality response method in comparison with a known susceptible strain. Only low to moderate resistance to diazinon, chlorpyrifos, and acephate was detected. Resistance to malathion was widespread; about half of the strains tested showed high resistance. High resistance to the carbamates propoxur and bendiocarb also occurred. High resistance was uncommon with propoxur, but about 35 strains were highly resistant to bendiocarb. High resistance to pyrethrins was observed in about of the strains tested. Resistance to the pyrethroids allethrin, permethrin, phenothrin, fenvalerate, and cyfluthrin was detected in some of the strains examined. All of the strains tested were susceptible to one or more of the insecticides used. These results indicate that, although resistance is a serious problem in this species, satisfactory control should be possible by selection of an appropriate insecticide.     

家蝇中乙酰胆碱酯酶、羧酸酯酶和多功能氧化酶活性与抗药性的关系

本文对几种抗药性和敏感性家蝇品系的乙酰胆碱酯酶(AChE)、羧酸酯酶及多功能氧化酶(MFO)进行了测定.结果表明:①抗药性品系和敏感性品系的AChE活力差异不大, 有机磷抗性品系的AChE对对氧磷的不敏感性比敏感性家蝇明显增大.②某些抗药性家蝇的羧酸酯酶活力比敏感性家蝇大.③抗药性家蝇的MFO活力(O-脱甲基和环氧化)比敏感性家蝇均有不同程度的增高.④二氯苯醚菊酯抗性家蝇对有机磷有负交互抗性.     

Genetics of resistance to insecticides of the SKA strain of Musca domestica II. Isolation of the dominant factors of resistance to diazinon

The dominant factors for resistance to diazinon, i.e. R₃ on the III linkage group, and gene a for low ali-esterase on V linkage group were isolated in the homozygous condition from the single mutant progenies of a cross between diazinon-resistant SKA flies, and a susceptible strain marked with the recessive mutants, ocra (III linkage group) and ar (V linkage group). Each resistance factor segregated in opposition to the marker of the same linkage group; thus ar mutants carried only R₃ and ocra mutants only gene a. Each of the two incompletely dominant factors when homozygous conferred a similar but small resistance to diazinon (R.F. 13–34) and very little resistance to diazoxon (R.F. 3–5), and differed in their response to diazinon in the presence of sesamex. Sesamex synergised diazinon very much against flies with R₃ only, but increased resistance to diazinon by x 2 in flies with gene a only. n-propyl paraoxon was almost equally effective as a synergist with diazinon against susceptible flies, and strains with R₃ or gene a only (S.F. 1.4–3.6).A third factor segregates independently of the other two factors and confers very slight resistance to diazinon (c. x 3).

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Zusammenfassung Die dominanten Faktoren für Diazinon-Resistenz, d.h. R₃ in der IV.Koppelungsgruppe und Gen a für geringe Aliesterase in der V.Koppelungsgruppe, wurden unter homozygoten Bedingungen aus den Einzelmutanten-Nachkommenschaften einer Kreuzung zwischen Diazinonresistenten SKA-Fliegen und einem anfälligen Stamm isoliert, der mit den rezessiven Mutanten ocra (III. Koppelungsgruppe) und ar (V.Koppelungsgruppe) markiert war. Jeder Resistenzfaktor sonderte sich im Gegensatz zu dem Markierungsgen der gleichen Koppelungsgruppe ab; so trugen ar-Mutanten nur R₄ und ocra-Mutanten nur Gen a. Jeder der beiden unvollkommenen dominanten Faktoren verlieh im homozygoten Zustand eine ähnliche, aber geringe Diazinon-Resistenz (R.F. 13–34) und sehr wenig Resistenz gegen Diazoxon (R.F. 3–5). Sie unterschieden sich in ihrer Reaktion zu Diazinon in Gegenwart von Sesamex, Sesamex wirkte sehr stark synergistisch gegenüber Fliegen, die nur R₃ tragen, verstärkte aber die Diazinon-Resistenz um das Zweifache bei Fliegen, die nur Gen a besitzen. n-propylparaoxon war als Synergist für Diazinon fast gleich wirksam gegenüber anfälligen Fliegen wie Stämmen mit R₃ oder nur Gen a (1,4–3,6).Ein dritter Faktor spaltete sich unabhängig von den zwei anderen Faktoren ab und verlieh sehr schwache Diazinon-Resistenz (c. x 3).

     

Effect of synergists on the oral and topical toxicity of azamethiphos to organophosphate-resistant houseflies (Diptera: Muscidae).

Dermal and oral toxicities of azamethiphos were determined in two organophosphate-resistant and one susceptible strain of houseflies, Musca domestica L. The 594vb strain was 1,967-fold more resistant to azamethiphos when compared with the susceptible Chemical Specialties Manufacturers Association (CSMA) strain by dermal application. When the compound was administered orally to the 594vb strain, we observed only a 15-fold resistance. In contrast, the Yachiyo strain, which show 1,500-fold resistance to diazinon and which has known multiple mechanisms for organophosphate resistance, showed only 6-fold resistance to azamethiphos by topical application and 4-fold resistance by oral administration. Azamethiphos administered dermally and orally was equally toxic to the CSMA and Yachiyo strains. However, when azamethiphos was administered to the 594vb strain, the insecticide was 71 times more toxic orally than by the dermal route. This result indicated involvement of a cuticular penetration factor in the resistance mechanism. The effect on azamethiphos toxicity of piperonyl butoxide (PB), an inhibitor of the monooxygenases, and tributylphosphorotrithioate (DEF), an esterase inhibitor, was investigated in the three strains. Pretreatment of the flies with PB, DEF, or PB+DEF before topical application of azamethiphos resulted in a significant decrease in LD50s in all the strains. The degree of synergism, however, varied depending upon the strains and the synergists. Similar results were obtained when azamethiphos was administered orally following pretreatment of the flies with PB+DEF. We attribute the high level of azamethiphos resistance in the 594vb strain partly to increased degradation by oxidative and hydrolytic enzymes. The hydrolytic enzymes are more important, but other factors including reduced cuticular penetration and insensitive acetylcholinesterase may be involved.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of sequential exposure of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and imidacloprid against susceptible and resistant strains of <Emphasis Type="Italic">Musca domestica</Emphasis>

Insecticide resistance in the housefly Musca domestica is hampering pest management. However, entomopathogens, possibly in combination with insecticides, may have control potential against resistant houseflies. This study investigates the combination of the entomopathogenic fungus Beauveria bassiana and the neonicotinoid insecticide, imidacloprid against a susceptible and a resistant housefly strain, respectively under laboratory conditions. The fungus and insecticide were tested alone and in combinations at LC₃₀. Significant and synergistic interactions between B. bassiana and imidacloprid were observed with increased mortality rates of the combined treatment as compared to individual treatment in housefly strains 772a (susceptible) and 766b (resistant). Significant differences in the GST and P450 activities for both strains were found. Female 766b flies caused 15- to 237-fold increases in gene expression of xenobiotic response genes for B. bassiana and 23- to 120-fold changes for imidacloprid. The combination of B. bassiana and imidacloprid caused significant synergistic interaction when applied against two housefly strains irrespective of order of application. The effect was highest when the insecticide was applied first. The resistant housefly strain had elevated detoxification enzymes and higher expression of detoxification genes, but showed the same level of susceptibility to the combined fungus/insecticide treatment as the susceptible strain.     

Larval competition within and between insecticide resistant and susceptible individuals in the oriental fruit fly,Bactrocera dorsalis

Insecticide resistance is a serious issue in agriculture. Ecological interactions may be used to manage the spread of resistance, but little information is currently available. In particular, virtually none is known about interactions between resistant and susceptible individuals. This study investigated competition between resistant and susceptible oriental fruit fly larvae (Bactrocera dorsalis). Competition in the larval stage was examined with an explicit consideration of individual identity (resistant or susceptible). Guava fruits were inoculated with eggs of susceptible and/or resistant flies, and their development and survival were monitored. Egg density influenced the time that larvae stayed in fruits as well as their survival. In addition, susceptible flies survived better when interacting with resistant flies than with other susceptible flies, indicating that susceptible flies are competitively superior to resistant flies. The results suggest that artificially creating environments that induce competition between susceptible and resistant flies can be useful for the management of insecticide resistance.     

Carbaryl resistance in Mexican strains of the southern cattle tick (Acari: Ixodidae)

Susceptibility to carbaryl in six Mexican strains of the southern cattle tick, Boophilus microplus (Canestrini), was evaluated with the Food and Agricultural Organization larval packet test. Tick strains from the cattle fever tick quarantine zone in Texas were more susceptible to carbaryl than to coumaphos or diazinon. Compared with the susceptible reference (Gonzalez) strain, Mexican tick strains demonstrated 10.9-59.5-fold resistance to carbaryl. Significant cross-resistance was found between carbaryl and the organophosphate acaricides coumaphos and diazinon. Bioassay results with synergists suggested that metabolic detoxification mechanisms did not play a major role in carbaryl resistance. Resistance to carbaryl was likely conferred by insensitive acetylcholinesterase. The implications of carbaryl resistance in tick eradication and control also are discussed.