Overproduction of a P450 that metabolizes diazinon is linked to a loss-of-function in the chromosome 2 ali-esterase (MdalphaE7) gene in resistant house flies.

Up-regulation of detoxifying enzymes in insecticide-resistant strains of the house fly is a common mechanism for metabolic resistance. However, the molecular basis of this increased insecticide metabolism is not well understood. In the multiresistant Rutgers strain, several cytochromes P450 and glutathione S-transferases are constitutively overexpressed at the transcriptional level. Overexpression is the result of trans-regulation, and a regulatory gene has been located on chromosome 2. A Gly137 to Asp point mutation in alphaE7 esterase gene, leading to the loss of carboxylesterase activity, has been associated with organophosphate resistance in the house fly and the sheep blowfly. We show here that purified recombinant CYP6A1 is able to detoxify diazinon with a high efficiency. We also show that either the Gly137 to Asp point mutation in alphaE7 esterase gene or a deletion at this locus confer resistance and overproduction of the CYP6A1 protein. Based on these findings, we propose it is the absence of the wild-type Gly137 allele of the alphaE7 gene that releases the transcriptional repression of genes coding for detoxification enzymes such as CYP6A1, thereby leading to metabolic resistance to diazinon.

Effects of exposure to cypermethrin on saxitoxin binding in susceptible and pyrethroid-resistant houseflies.

Saxitoxin (STX) binding was measured in susceptible (SBO) and pyrethroid-resistant (KDR) female houseflies having only target site insensitivity as a resistance mechanism. In KDR flies, there was a quantitative decrease in STX binding capacity (Bmax) relative to SBO flies coupled with an increase in binding affinity (Kd). Treatment of SBO flies with sublethal doses of cypermethrin resulted in a large decrease in the number of STX binding sites and an increase in STX binding affinity. In KDR flies, identical treatments had the opposite effects. Treatment of both strains with higher doses of cypermethrin resulted in smaller decreases in Bmax values coupled with decreases in binding affinities. The results show that physiological changes in STX binding occur upon exposure to extremely low doses of cypermethrin. The data suggest that the kdr resistant gene may be expressed as changes in STX binding kinetics and that measurements of STX binding in pyrethroid-treated insects may be a useful approach for studying pyrethroid's mode of action and resistance.

Constitutive overexpression of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides.

Messenger RNA levels of the cytochrome P450 gene CYP6A1 were measured in the insecticide resistant Diazinon-R 'Rutgers' strain and in the susceptible strain sbo of the house fly with a cloned cDNA probe. The constitutive expression of the CYP6A1 gene was at least 10 times higher in the Rutgers strain than in the sbo strain. In both strains, CYP6A1 was inducible by phenobarbital treatment of the flies. Analysis of genomic DNA from the two strains indicated that there was no amplification of the CYP6A1 gene in the Rutgers strain. A developmental analysis revealed that CYP6A1 is expressed larvae and adults, and very low levels of CYP6A1 mRNA were detected in eggs and pupae. The constitutive overexpression of CYP6A1 in the Rutgers strain was observed in both larvae and adults. Crosses between the Rutgers strain and the multiply marked sbo strain revealed that the high constitutive expression of CYP6A1 in the Rutgers strain is controlled by one or more loci located on chromosome II.

Biochemistry and genetics of thiodicarb resistance in the house fly (Diptera: Muscidae).

The biochemistry and genetics of thiodicarb resistance were studied in the house fly, Musca domestica L. Bioassays with several strains revealed that a thiodicarb-resistant strain was also resistant to carbaryl and to several organophosphates. Bioassays with a thiodicarb-synergist combination showed that resistance was only partially blocked by use of a synergist, suggesting the presence of target site resistance. The inheritance of resistance to thiodicarb was studied through crosses of the resistant parent to a multimutant, susceptible strain. Bioassays of backcrosses and the F2 generation showed that the major gene(s) conferring resistance to thiodicarb were on chromosome II. Cholinesterase assays showed that the enzyme of the resistant strain was several times more resistant to inhibition by carbamates than the enzyme of a susceptible strain or a strain with only metabolic resistance to insecticides. The data indicate that resistance to thiodicarb in the house fly was mainly because of target site changes. In addition, metabolism may contribute to resistance.

Insect glutathione S-transferases. Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides.

Two classes of glutathione transferases have been identified and purified from Musca domestica. The first, designated as GST1, migrates as a single band of 28 kDa in SDS-gel electrophoresis, and the second, designated as GST2, migrates as a 32-kDa band. Antisera prepared against each class have no immunological cross-reactivity, and heterodimeric associations between the two classes have not been detected. Each class is composed of several isoforms: GST1 is composed of forms with isoelectric points from 4 to 9, whereas all the forms of GST2 have acidic pI values. Screening of cDNA libraries yielded clones coding for GST1, and the gene was sequenced and expressed in Escherichia coli. The high activity found in an insecticide-resistant strain (Cornell R) is correlated with high level of GST1 transcript.

Formamidines as synergists of cypermethrin in susceptible and pyrethroid resistant house flies (Diptera: Muscidae).

Mechanisms of formamidine synergism of pyrethroid insecticides were investigated in the house fly, Musca domestica L. A bioassay method was developed to show formamidine synergism of cypermethrin in adult house flies. Flies were exposed to a residue of cypermethrin with and without formamidine for 30 min and then transferred to a clean container. Mortality was recorded 24 h later. Synergism of cypermethrin occurred in flies exposed simultaneously to cypermethrin plus formamidines and in flies exposed to cypermethrin either before or after exposure to a formamidine. Synergism ranged up to 11.8-fold and was greater in susceptible than in resistant house flies. A monomeric derivative of amitraz (BTS 27271) was the most active synergist, chlordimeform was intermediate, and amitraz was least active. Synergism of cypermethrin also occurred in flies injected with octopamine and then exposed to cypermethrin, suggesting that formamidines may be acting as octopamine agonists. Measurements of the effects of formamidines on uptake of cypermethrin showed that BTS 27271 increased uptake less than chlordimeform and that amitraz had almost no effect. Both target site and behavioral effects are discussed as possible mechanisms of formamidine synergism of cypermethrin and other pyrethroids.

Cyromazine resistance in the house fly (Diptera: Muscidae): genetics and cross-resistance to diflubenzuron.

Larvae of a house fly, Musca domestica L., strain collected in a chicken house near Pittsburg, Tex, after a control failure with the poultry feedthrough insecticide cyromazine showed 6.5-fold resistance to cyromazine and 10-fold resistance to diflubenzuron. Adults of the strain showed high levels of resistance to carbaryl, DDT, and diazinon; moderate resistance to cypermethrin and permethrin; and low resistance to dieldrin. In contrast, no resistance to cyromazine was observed in eight laboratory house fly strains with resistance to four groups of conventional insecticides. When the genetics of cyromazine resistance was investigated in crosses to susceptible strains with visible mutant markers, results indicated cyromazine resistance was incompletely dominant over susceptibility and the resistance gene was on chromosome V. The same or a closely linked gene conferred resistance to diflubenzuron. A strain containing only chromosome V from the original resistant strain was resistant to cyromazine and diflubenzuron, but not to other insecticides except for low level resistance to DDT and carbaryl. Resistance to the latter insecticides appeared to be due to a linked, but distinct, gene. Therefore, resistance to cyromazine and probably diflubenzuron appears to be genetically distinct from other types of insecticide resistance.

Genetics of organophosphate resistance in field populations of the house fly (Diptera: Muscidae).

The genetics of resistance to the organophosphate insecticide diazinon were investigated in four populations of the house fly, Musca domestica L., collected in the southern United States. Crosses were made between individual females of lines derived from each population and males of a susceptible strain with three recessive mutants on chromosome II. Individual F1 females were crossed to mutant males, and the progenies were scored for resistance to diazinon and for the presence of mutant phenotypes. A major chromosome II gene for resistance to diazinon was present in all populations at an overall frequency of 83%. Map distances between the resistance gene and the mutant aristapedia and between the mutants aristapedia and stubby wing were highly variable in all populations. Recombination among the visible mutants was usually reduced in resistant progenies relative to susceptible progenies. The data suggest that a single major gene for resistance to diazinon was present on chromosome II in all test populations at variable map positions and is usually associated with a chromosome rearrangement, probably an inversion. The results are similar to those obtained earlier with house fly populations selected for resistance to insecticides in the laboratory; therefore, they seem to be characteristic of field and laboratory populations of the house fly. Overall, the data offer an explanation for previous results suggesting the existence of multiple, closely linked genes for metabolic resistance to insecticides on house fly chromosome II.

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 semi-dominant 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.

Induction of multiple cytochrome P-450 species in housefly microsomes--SDS-gel electrophoresis studies.

1. Microsomal fractions isolated from various housefly strains have been characterized with respect to multiple forms of cytochrome P-450 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 2. Susceptible NAIDM houseflies were pretreated with known inducers of cytochrome P-450, and their microsomal electrophoretic profiles were compared to control NAIDM microsomes, using as standards partially purified cytochrome P-450s from noninduced NAIDM houseflies. 3. Tentatively, at least five different species of cytochrome P-450 may exist in the NAIDM housefly strain. 4. A comparison of the microsomal electrophoretic profile of different housefly strains also indicates the presence of at least two additional cytochrome P-450 species. 5. Induction with alpha-pinene and phenobarbital was expressed by a shift of the maximum absorbance at 452 nm in the CO-difference spectrum to lower wavelengths in the NAIDM strain; whereas, beta-naphthoflavone, although increasing the amount of cytochrome P-450, did not change the wavelength of maximum absorbance. 6. Cytochromes of the P-452 type appear to predominate in the susceptible NAIDM strain, while cytochromes of the P-450 and P-448 types predominate in resistant strains.

Snakes as indicators of environmental contamination: relation of detoxifying enzymes and pesticide residues to species occurrence in three aquatic ecosystems.

Pesticide detoxifying enzymes and residues of p,p'-DDE and PCB were measured in snakes of two genera collected in three aquatic ecosystems in central Texas. Snake genera were Agkistrodon (water moccasins and copperheads) and Natrix (water snakes). One collecting site had heavy exposure to insecticides, one to PCBs and one to neither. Objectives were to correlate pesticide residues and detoxifying enzymes with species occurrence in different ecosystems, to evaluate the usefulness of snakes as indicators of environmental contamination, and to establish a biochemical basis for the differential distribution of snakes between ecosystems. Agkistrodon spp. had several times the NADPH-dependent oxidative detoxifying activity of Natrix spp. Within Natrix, oxidase activity was significantly higher in erythrogaster and fasciata than in rhombifera. Alkyltransferase activity was higher in A. piscivorus (water moccasins) than in any other species. There were no consistent differences between sexes in levels of detoxifying enzymes and enzyme activity was similar in snakes of each species from all collecting sites, indicating no induction had occurred as a result of heavy exposure to insecticides or PCBs. DDE residues were highest in snakes collected adjacent to a cotton agro-ecosystem. PCB residues occurred only in snakes collected near a heavily-traveled highway. Residues in A. piscivorus were only half those in Natrix spp. from the same site, indicating the former is more efficient as disposing of residues. The data indicate that snakes with high levels of detoxifying enzymes, particularly microsomal oxidases, are more likely to occur in a contaminated ecosystem than snakes with less active detoxifying enzymes and that residues are lower in the better detoxifiers. Snakes are apparently excellent indicators of environmental contaminated by insecticides and other persistent chemicals.