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.     

The genetic basis of insecticide resistance in the house fly: Evidence that a single locus plays a major role in metabolic resistance to insecticides

Genetic evidence indicates that insecticide resistance in insects is controlled by relatively few genes. In the house fly, Musca domestica L., major resistance genes include one for decreased uptake of insecticides, three for changes at the target sites of insecticide action, and a single gene for metabolic resistance to multiple types of insecticides. The latter gene, which is located on chromosome II, interacts with minor genes located on other chromosomes. The product of the major gene for metabolic resistance appears to be a receptor protein which recognizes and binds xenobiotics, including insecticides and plant defense substances, and then induces synthesis of appropriate detoxifying enzymes.     

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.     

An evaluation of the role of oxidative enzymes in Colorado potato beetle resistance to carbamate insecticides

Carbofuran and carbaryl LD₅₀ values were determined with and without piperonyl butoxide pretreatment for a resistant (New Jersey) and two susceptible (Utah and Netherland) populations of Colorado potato beetle larvae. Similar bioassays were conducted with carbofuran for resistant (Rutgers) and susceptible (NAIDM) adult house flies. The degree of resistance development by New Jersey Colorado potato beetles (RR = 848) was greater than that of the laboratory-selected colony of Rutgers house flies (RR = 583). Comparisons of synergist difference calculations including “percentage synergism” (%S), “log percentage synergism” (L%S), and “relative percentage synergism (R%S) for the resistant (R) and the susceptible (S) populations indicated the possibility that monooxygenases and other resistance mechanisms may be involved in Colorado potato beetle resistance to these carbamates. Monooxygenase involvement in resistance of Rutgers house flies was demonstrated in vitro by a 4-fold enhancement of p-nitroanisole O-demethylation over that of NAIDM house flies. O-demethylation of p-nitroanisole could not be demonstrated for potato beetle larvae. Colorado potato beetle resistance was associated with increases in microsomal levels of NADPH-cytochrome c reductase (ca. 2-fold) and NADPH oxidation (1.2-fold). The inability to measure O-demethylation in Colorado potato beetles may have been due to the solubilization of NADPH-cytochrome c reductase during microsomal preparation. Significant differences between resistant and susceptible Colorado potato beetle larvae were not observed in the penetration of [¹⁴C]carbaryl. Excretion of the radiocarbon may have been significantly greater in the resistant New Jersey population, but some of the insecticide may have also rubbed off the cuticle. This increased capacity for excretion, combined with increased levels of monooxygenase enzymes, could account for the high resistance level of this population.     

Studies on hydrolases in various house fly strains and their role in malathion resistance

Aliesterase, carboxylesterase, and phosphorotriester hydrolase activities in six house fly strains were studied in relation to malathion resistance. Selection of two susceptible strains with malathion for three generations resulted in an increase in both carboxylesterase activity and LD₅₀ of malathion, indicating that the increased detoxication by the enzyme was the major mechanism selected for malathion resistance. With the highly resistant strains, however, the carboxylesterase activity alone was not sufficient to explain the resistance level, and the involvement of additional mechanisms, including phosphorotriester hydrolase activity, was suggested. The E₁ strain, which had high phosphorotriester hydrolase activity but normal or low carboxylesterase activity, showed a moderate level, i.e., sevenfold resistance. Upon DEAE-cellulose chromatography, two or three esterase peaks were resolved from susceptible, moderately resistant, and highly resistant strains. The substrate specificity, the sensitivity to paraoxon inhibition, and the $\text{α}\text{β}$ ratio of malathion hydrolysis were studied for each esterase peak from the different strains. The results suggested the existence of multiple forms of esterases with overlapping substrate specificity in the house fly.     

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.     

Acetylcholinesterase isozymes of the house fly thorax: In vivo inhibition by organophosphorous insecticides

Polyacrylamide gel electrophoresis of the supernatant fraction of house fly thoracic homogenates demonstrated five electrophoretic bands having cholinesterase activity. The five esteratic bands were considered to be isozymes of acetylcholinesterase based on their staining properties with acetylthiocholine. There appeared to be no visual or densitometric difference in the intensity of staining of the isozymes when acetylthiocholine was compared with butyrylthiocholine as substrate. Selective inhibition of these isozymes in vivo by organophosphate insecticides may contribute only in small part to the mode of action of organophosphate insecticides.     

Mechanisms responsible for high levels of permethrin resistance in the house fly

The mechanisms responsible for > 6000-fold permethrin resistance in a pyrethroid-selected strain of house fly, Learn-PyR, were investigated. Through electrophysiological, in vitro metabolism, in vivo penetration and synergism studies it was demonstrated that the resistance mechanisms consisted of enhanced metabolic detoxification via the mixed-function oxidase (MFO) system, target-site insensitivity and decreased cuticular penetration. The major resistance mechanism was the MFO-mediated detoxification. The elevated MFO activity was correlated with higher levels of cytochrome P-450, cytochrome b₅ and NADPH-cytochrome c reductase activity. The kinetics of the latter showed similar K_m but greater V_max values in the Learn-PyR than in the susceptible strain, suggesting that the elevated activity was due to an altered amount, but not an altered form, of the enzyme. The Learn-PyR strain showed widely varying levels of resistance to the pyrethroids tested. Comparison of the pyrethroid structures with the resistance ratios revealed that resistance was highest in the presence of an unsubstituted phenoxybenzyl alcohol moiety. Substitution or certain modifications of the alcohol moiety reduced the level of resistance. Structure of the acid moiety or the presence or absence of an a-CN group did not affect the resistance level. These results are discussed with reference to the resistance mechanisms present.     

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.     

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.     

Muscarinic receptor in house fly brain and its interaction with chlorobenzilate

Membranes from house fly heads were tested for the presence of mucarinic acetylcholine receptors using as a probe [³H]quinuclidinyl benzilate ([³H]QNB). Based on the presence of saturable and reversible high-affinity binding of [³H]QNB, which is inhibited by muscarinic drugs, it is suggested that these sites may be muscarinic receptors. However, these putative muscarinic receptors differ in several characteristics from the ones in mammalian brain. They have lower affinities for muscarinic drugs and lower stereoselectivity, a relatively higher affinity for the nicotinic antagonist d-tubocurarine, a lower Hill coefficient for binding of muscarinic antagonists, and a lower concentration relative to α-bungarotoxin binding sites in the same membranes. Also, unlike mammalian muscarinic receptors, they are sensitive to treatments with N-ethylmaleimide and 5,5′-dithiobis(2-nitrobenzoic acid). The effect of reduction of disulfide bonds by dithiothreitol or mercaptoethanol suggests that only the insect receptor has one or more disulfide bonds which are important to binding. On the other hand, the putative muscarinic receptors of both insect and mammalian brains have important SH group(s), whose alkylation by p-chloromercuribenzoate inhibits binding. Also, chlorobenzilate is equally effective in inhibiting [³H]QNB binding to muscarinic putative receptors of house fly and bovine brains.     

The role of tyrosinase in the inactivation of house fly microsomal mixed-function oxidases

The low mixed-function oxidase activity of house fly microsomes has been associated with low cytochrome P-450 content and NADPH-cytochrome c reductase activity. The microsomal cytochrome P-450 content and NADPH-cytochrome c reductase activity could be decreased by the addition of catechol and increased by the addition of cyanide to the homogenates. Similar results were obtained with rat liver microsomes treated with tyrosinase and catechol. During the inactivation of rat liver microsomal enzymes by tyrosinase and catechol, crosslinking of microsomal proteins occurred. These results suggest that the instability of house fly microsomal mixed-function oxidase may be due in part to the action of contaminating tyrosinase on endogenous substrates.     

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.     

Resynthesis of multiple resistance to organophosphorus insecticides from strains with factors of resistance isolated from the SKA strain of house flies

Individual factors of resistance to insecticides attributable to chromosomes II, III and ^V of the SKA strain of houseflies (Musca domestica L) were combined in pairs to determine how their presence affects resistance. The re-synthesised strains with resistance factors on chromosomes II and V, and on chromosomes III and V, were tested with several organophosphorus insecticides and DDT. The penetration delaying mechanism Pen on chromosome III, which alone gives little or no resistance, slightly increased the resistance of flies with the microsomal detoxifying factor Ses on chromosome V to diazinon and malaoxon-ethyl (c. × 1.5), but was more effective in increasing resistance to DDT (× 6). There was no effect on the response to other insecticides tested. The combined effect of the mechanisms of resistance on chromosome II (glutathione S-ethyl transferase and phosphatase) and on chromosome V (microsomal detoxication) approximated to the product of the resistance conferred by each of these mechanisms singly, suggesting that the mechanisms of resistance on the two chromosomes act independently. Therefore, most of the strong resistance to organophosphorus insecticides in the SKA strain results from the interaction between delayed penetration (chromosome III) and the factors of resistance on chromosome II, and the independent action of the resistance factors on chromosomes II and V.     

Effects of insecticides and GABAergic agents on a house fly [35S]t-butylbicyclophosphorothionate binding site

Specific binding of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) to a house fly thorax-plus-abdomen membrane preparation at 20°C is characterized by apparent K_d and B_max values of 0.21 μM and 2.5 pmol/mg protein, respectively, an association half-time of 13 min at 2 nM, and a biphasic dissociation curve showing half-times of 15 and 35 min. Specific binding is reduced at 37°C apparently due to instability of the receptor-ligand complex and at 0°C as the result of very slow association. [³⁵S]TBPS binding is diminished by detergents, stimulated by GABA at low ligand concentration, and inhibited by picrotoxinin and certain barbiturates, benzodiazepines, bicyclophosphorus compounds, and polychlorocycloalkane insecticides. The potency of TBPS and three related phosphorothionates in displacing [³⁵S]TBPS parallels their toxicity on injection into house flies; the corresponding bicyclophosphates are less active in both assays. Cyclodienes of low toxicity are generally poor inhibitors of radioligand binding. α-Endosulfan and syn-12-hydroxyendrin are more potent than their β and anti isomers, respectively, both as inhibitors of TBPS binding and as toxicants. Analysis of Scatchard plots indicates that picrotoxinin and heptachlor epoxide are non-competitive inhibitors of [³⁵S]TBPS binding. The [³⁵S]TBPS binding site of the house fly membrane preparation differs from that extensively studied in mammalian brain with respect to their responses to many insecticides and GABAergic agents.     

DDT and pyrethroids: Receptor binding in relation to knockdown resistance (kdr) in the house fly

The nature of target site or knockdown resistance (kdr) to DDT and pyrethroids was studied by investigating specific binding of [14_C] DDT and [14_C] cis-permethrin to the previously established membrane receptors from the heads of susceptible (sbo) and resistant (kdr) strains of the house fly, Musca domestica L. In vivo studies showed the heads from sbo flies bound two to three times more DDT than those from kdr flies at all doses tested. Reduced binding was also observed in kdr flies in in vitro [14_C] DDT binding assays. Scatchard analysis indicated that kdr flies have the same affinity but fewer receptors per milligram protein in the CNS than sbo flies. Assays with [14_C] cis-permethrin also showed binding was much reduced in kdr flies in comparison with sbo flies. Based on these results, the nature of the target site insensitivity of kdr flies may relate to their having a reduced number of receptors for the insecticides.     

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.