Toxicokinetics of carbofuran in house fly, Musca domestica L

The kinetics of accumulation and elimination of lethal doses of [¹⁴C]carbofuran in the hemolymph of the house fly suggest a one-compartment open model. Carbofuran in the hemolymph appeared to be in equilibrium with that in the tissues very soon after treatment.Following topical application of carbofuran, the rate of onset of symptoms of poisoning was correlated with the amount of carbofuran in the hemolymph, and the onset of convulsions only occurred after the concentration of carbofuran in the hemolymph reached μM levels. This value correlated well with neurobioassays of known concentrations of carbofuran perfused in saline onto the isolated thoracic ganglion.Following topical doses, carbofuran concentration in the hemolymph reached a peak within an hour and then gradually declined. At an LD₆₀ dose, the initial decline in carbofuran concentration in the hemolymph over time was significantly slower than the decline after an LD₁₀ dose, suggesting saturation kinetics.Hemolymph was collected from house flies for up to 3 hr following topical application of toxic amounts of carbofuran. Thereafter, hemolymph volume decreased and blood samples could not be collected. Curiously, hemolymph samples could be collected for 5 hr from house flies that were injected with toxic doses of carbofuran.     

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.     

Different forms of insensitive acetylcholinesterase in insecticide-resistant house flies (Musca domestica)

A new form of acetylcholinesterase insensitive to organophosphorus inhibitors was identified in house flies collected from animal farms in the U.K. Its degree of insensitivity to a number of inhibitors ranged from 4-fold (omethoate) to >40-fold (tetrachlorvinphos and dichlorvos) compared with our standard susceptible enzyme, and its spectrum of response to inhibitors differed widely from that of a previously studied insensitive form. In addition, the form of acetylcholinesterase reported here is unusual in having a 3.4-fold greater affinity (lower K_m) for acetylthiocholine than the normal enzyme. The possible toxicological significance of this is discussed.     

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.     

Characterization of multiple glutathione transferases from the house fly, Musca domestica (L)

Glutathione transferases have been purified to a high degree of homogeneity from three strains of house fly by a procedure involving affinity chromatography on glutathione-sulfobromophthalein conjugate immobilized on Sepharose 4B, followed by preparative isoelectrofocusing. The affinity chromatography yielded purifications of between about 10- and 100-fold, depending on the strain and the substrate with which activity was measured. Each strain was shown to possess several proteins with glutathione S-transferase activity which fell into two clearly defined groups. The first group, of relatively low isoelectric point, showed activity with CDNB but little with DCNB, p-nitrobenzylchloride, or 1,2-epoxy-3-(p-nitrophenoxy)propane, whereas the second group, of higher isoelectric points, showed substantial activity with all substrates tested. Studies on the subunit structure of these enzymes demonstrated the existence of three different sized subunits of M_r 20,000, 22,000, and 23,500. From the experimental evidence recorded here, the existence of at least three functionally different glutathione transferases is inferred.     

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.     

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.     

Neural and behavioral correlates of pyrethroid and DDT-type poisoning in the house fly, Musca domestica L

Intact house flies were observed during poisoning caused by several pyrethroid and DDT-type insecticides. The two insecticide classes could be generally distinguished from each other based on differences in symptoms and several physiological correlates. Both insecticide types caused motor unit repetitive backfiring, but the temporal development and stability of repetitiveness were distinctly different between the two classes. Repetitive backfiring always disappeared at low temperatures, but DDT-type backfiring disappeared at lower temperatures than the pyrethroids. trans-Tetramethrin caused a threshold increase in flight motor nerve endings which did not occur in DDT or trans-Barthrin poisoning. Pyrethroids caused “uncoupling” of the flight motor pattern, while DDT-types did not. trans-Barthrin, a methylenedioxyphenyl pyrethroid, was unique in causing both symptoms and physiological aberrations which more closely resembled those of the DDT-types than the pyrethroids.     

细辛醚对家蝇和淡色库蚊的生物活性

为了进一步确定细辛醚的作用谱和作用方式,测定了其对家蝇和淡色库蚊的触杀、熏蒸和忌避活性。结果表明:细辛醚对家蝇4 h的触杀LD50值为3.45 μg/头;对家蝇、淡色库蚊4 h的熏蒸LC50值分别为5.77和4.24 μL/L;对两者在LC80剂量下熏蒸处理的击倒中时(KT50)值分别为11.64和4.80 min;在1 mg/mL的浓度下,对家蝇的忌避率为81.82%。中毒家蝇依次表现出兴奋、痉挛、麻痹和死亡等症状。     

Frequencies and evolution of organophosphate insensitive acetylcholinesterase alleles in laboratory and field populations of the house fly, Musca domestica L.

Resistance to organophosphate (OP) and/or carbamate insecticides can be due to mutations in the acetylcholinesterase gene (Ace). Genotypes of house fly, Musca domestica L., Ace were determined in twelve laboratory maintained strains (originally from North America, Europe and Asia) and two field collected populations from New York and Florida. There were 15 Ace alleles found and 11 of the alleles coded for a susceptible form of the enzyme (i.e., V260, A316, G342 and F407). Three of the four resistance alleles were previously described, while one is new. Phylogenetic analysis of the alleles suggests multiple origins of the F407Y mutation and multiple origins of the G342A mutation that confer OP resistance. Genotyping of field collected house flies from New York and Florida populations revealed the presence of only one resistance allele, Acev10 (containing the non-synonymous mutations for A342 and Y407). All other alleles detected from the field-collected flies coded for a susceptible AChE. Thus, we were able to categorize individual flies as having homozygous susceptible (Ace^S/Ace^S), homozygous insensitive (Ace^I/Ace^I or Acev10/Acev10) or heterozygous AChE. The frequencies of Ace^S and Ace^I were not different between the NY2002 and FL2002 populations. Both populations were out of Castle-Hardy-Weinberg equilibrium, having an excess of Ace^S/Ace^I individuals and very few Ace^S/Ace^S individuals. Comparison of Ace, Vssc and CYP6D1 genotypes indicates individual house flies commonly have resistance alleles at multiple loci. Comparison of genotype data with bioassays, as well as the use of genotype data in resistance studies is discussed.     

Comparative metabolism of six ethoxychlor analogs and DDT in DDT-resistant and susceptible strains of the house fly Musca domestica L

The metabolic fate of six ³H-ring-substituted ethoxychlor analogs with altered aliphatic moieties and [¹⁴C]p,p′-DDT was investigated in susceptible and DDT-resistant strains of the house fly Musca domestica Linnaeus. The chloroalkane analogs, dichloroethane, chloropropane, and dichloropropane were primarily metabolized to the corresponding dehydrochlorinated products. This pathway was relatively more prominent in the resistant strain than in the susceptible strain. Biotransformation and detoxication of the isobutane, nitropropane, and neopentane derivatives was through microsomal oxidation (O-deethylation) of aryl ethoxy degradophores, and oxidation of the aliphatic moieties to produce the corresponding benzophenones, with no substantial differences between the resistant and susceptible strain. There was a strong correlation between the Taft ($\text{σ}{}^1$) values for the altered aliphatic moieties of chloroalkane analogs and their rate of dehydrochlorination in both the strains. These results suggest the importance of altered aliphatic moieties in developing resistance-proof DDT derivatives.     

六种植物精油对家蝇的熏蒸及触杀毒力测定

测定了6种植物精油对家蝇的熏蒸及触杀作用。结果表明,6种精油均对家蝇有较强的熏蒸活性,且表现出一定的触杀活性。熏蒸毒力次序为:黄樟油(LC50＝1.7695 μL/L)＞八角叶油(1.8930 μL/L)＞肉桂油(5.8074 μL/L)＞留兰香油(6.2123 μL/L)＞香茅油(6.5801 μL/L)＞薄荷素油(9.4500 μL/L);触杀活性较好的为黄樟油,其次为 留兰香油、香茅油和薄荷素油,而八角叶油和肉桂油对家蝇的触杀作用较差。     

Structure-activity relationships of 1,2,3-benzothiadiazoles as synergists for carbaryl against the house fly (Musca domestica)

Fifty six 1,2,3-benzothiadiazoles and related compounds were evaluated as carbaryl synergists against the house fly (Musca domestica). Many of these were excellent synergists, the most active being those containing various combinations of halogen, alkyl, or alkoxy substituents in the 5- and/or 6-positions of the ring.Regression analysis on the data from 14 compounds for which substituents constants were available established that synergistic activity can be satisfactorily described by equations in terms of the hydrophobic bonding constant (π) and the homolytic free radical constant (σ ·).The results with compounds related to the 1,2,3-benzothiadiazoles suggest that synergistic activity is associated primarily with the diazosulfide moiety.     

Choline acetyltransferase in organophosphorus-resistant and -susceptible strains of the cattle tick, Boophilus microplus

Choline acetyltransferase was demonstrated in homogenate extracts of larvae and adult brains of the cattle tick. The enzyme activity was approximately 15 and 100 μmol of acetylcholine synthesized/g of tissue/hr for larvae and adult brains, respectively. Comparisons of five strains showed that, despite organophosphate selection for acetylcholinesterases with wide differences in activity and inhibition kinetics, the choline acetyltransferase activity was statistically uniform between strains. it is concluded that the two enzymic components of the cholinergic system are controlled independently.     

Comparative inhibition of the juvenile hormone esterases from Trichoplusia ni,Tenebrio molitor, and Musca domestica

Twenty-seven compounds were screened as potential inhibitors of juvenile hormone esterases. Of these compounds O-ethyl-S-phenyl phosphoramidothiolate provided the best inhibition for the cabbage looper, Trichoplusia ni (Hubner), and the yellow mealworm, Tenebrio molitor L., while the juvenile hormone esterases of the house fly, Musca domestica L., were best inhibited by a juvenoid carbamate (1-(m-phenoxy-N-ethyl carbamate)-3,7-dimethyl-7-methoxy-2E-octene). The inhibition patterns of T. ni and T. molitor are similar, while those of M. domestica are relatively different. Further studies on the juvenile hormone and α-napthyl acetate esterases of T. ni showed that they could be differentially inhibited. Diisopropyl phosphorofluoridate and an alkyl trifluoromethyl ketone selectively inhibit the hydrolysis of α-naphthyl acetate and juvenile hormone, respectively, while O-ethyl-S-phenyl phosporamidothiolate inhibits both enzymes. The juvenile hormone esterases of T. ni also appear to be unique enzymes that are selective for juvenile-hormone-like molecules. The in vivo inhibition of T. ni juvenile hormone esterases by O-ethyl-S-phenyl phosphoramidothiolate slows the in vivo hydrolysis of juvenile hormone and results in delayed pupation and malformed larvae that resemble larval-pupal intermediates. Thus, the esterases involved in juvenile hormone metabolism appear to be important in juvenile hormone regulation.     

Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China

Insecticides have been extensively used for house fly control in China, with dichlorvos and deltamethrin being widely used. Knowledge about the current status of insecticide resistance and the underlying genetic changes is crucial for developing effective fly control strategies. The susceptibility to dichlorvos and deltamethrin, and the frequencies of genetic mutations involved in insecticide resistance were studied in five field populations of the house fly collected across China. Bioassay results show that flies exhibit 14- to 28-fold resistance to dichlorvos and 41- to 94-fold resistance to deltamethrin, indicating that dichlorvos and deltamethrin resistance are common in house fly populations in China. Molecular analysis reveals that flies from the five various locations carry resistance alleles at multiple loci and have diverse allelic types, different relative frequencies and combinations of each allele. Four non-synonymous single nucleotide polymorphisms (SNPs) (i.e. V260L, G342A/V, F407Y) in acetylcholinesterase (Ace) and two mutations (W251L/S) in a carboxylesterase (MdαE7) were commonly present in the field house flies. The L1014H rather than L1014F mutation in the voltage sensitive sodium channel gene (Vssc) was widely distributed in Chinese house flies. CYP6D1v1, which confers pyrethroid resistance, was found in all the five tested populations in China, although its frequency in house fly from Shandong province was very low. Our results suggest that resistance monitoring and management of house flies should be customized for a given location.     

Beta-cypermethrin resistance associated with high carboxylesterase activities in a strain of house fly, Musca domestica (Diptera: Muscidae)

A housefly strain, originally collected in 1998 from a dump in Beijing, was selected with beta-cypermethrin to generate a resistant strain (CRR) in order to characterize the resistance and identify the possible mechanisms involved in the pyrethroid resistance. The resistance was increased from 2.56- to 4419.07-fold in the CRR strain after 25 consecutive generations of selection compared to a laboratory susceptible strain (CSS). The CRR strain also developed different levels of cross-resistance to various insecticides within and outside the pyrethroid group such as abamectin. Synergists, piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF), increased beta-cypermethrin toxicity 21.88- and 364.29-fold in the CRR strain as compared to 15.33- and 2.35-fold in the CSS strain, respectively. Results of biochemical assays revealed that carboxylesterase activities and maximal velocities to five naphthyl-substituted substrates in the CRR strain were significantly higher than that in the CSS strain, however, there was no significant difference in glutathione S-transferase activity and the level of total cytochrome P450 between the CRR and CSS strains. Therefore, our studies suggested that carboxylesterase play an important role in beta-cypermethrin resistance in the CRR strain.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Characterisation of insecticide-insensitive acetylcholinesterase: Microcomputer-based analysis of enzyme inhibition in homogenates of individual house fly (Musca domestica) heads

A spectrophotometer interfaced with a microcomputer to collect and analyse data automatically was used to study the inhibition kinetics of house fly acetylcholinesterase by malaoxon and dichlorvos in the presence of substrate. This system simply and unequivocally identified insecticide-insensitive enzymes from single fly heads, even when these enzymes were hitherto uncharacterised. As well as identifying individual insects homozygous for a particular form of acetylcholinesterase, the method also recognised heterozygotes because the mixture of enzymes then present gave a heterogeneous response to inhibitors. In the latter case, each component enzyme present could be identified. The technique is valuable for characterising heterogeneous insect populations collected from the field, for establishing homozygous strains, and for more detailed biochemical study.     

Effect of dietary benzoxadiazole on larval development, cuticle enzyme and antioxidant defense system in housefly (Musca domestica L.)

The toxicity and influence on chronic development regulation of dietary benzoxadiazole as well as the subsequent action on cuticle enzyme and antioxidant defense system in feed-thru housefly larvae are investigated. Dietary benzoxadiazole shows limited larvicidal activity and weak interference on larval pupation, but strong blockage against the succedent eclosion process. It does not change the content ratio of protein/chitin in the larval cuticle, but strongly regulates the constituents of cuticle proteins. Moreover, chitinase activities in the integument of third-instar larvae, in vivo, are enhanced and gradually decreased whereas phenoloxidase activities are inhibited and the inhibitory rates are gradually increased. Glutathione S-transferase activities are strongly improved whilst peroxidase activities decrease from about 42.25% to 17.36%, catalase activities decrease from about 80.31% to 27.98% and superoxide dismutase activities are almost unchanged during the different treatment procedures. Peroxidase SDS-PAGE analysis shows that band photodensities of 200.0 and 10.3 kDa proteins in feed-thru larvae are significantly weaker than the corresponding control band. Results suggest that dietary benzoxadiazole might exert strong regulation on larvae cuticle metabolism, interfere with cuticle enzymatic browning and protein sclerotization and weaken the self-protection of larvae against endogenetic oxidative damages.