Resistance to organophosphorus insecticides in the SKA strain of houseflies (Musca domestica)

The response of a susceptible strain and the diazinon-selected strain of houseflies, the SKA, to 20 organophosphorus insecticides was measured by topical application and injection.The SKA strain was very resistant to diazinon, ethyl chlorthion, chlorthion and parathion, moderately resistant to five other insecticides and only slightly resistant to the remainder. It was more resistant to the diethoxy than dimethoxy compounds and was only very slightly resistant to malathion. The compounds, especially the phosphates, were more toxic when injected than when applied topically. Additions or substitutions in the phenyl ring of the parathion molecule resulted in compounds less toxic than parathion, particularly to susceptible flies.

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Zusammenfassung Die Reaktion eines anfälligen und des auf Diazinonresistenz ausgelesenen Stammes SKA der Stubenfliege auf Organophosphor-Insektizide wurde nach topischer Applikation und Injektion gemessen.Der SKA-Stamm war sehr widerstandsfähig gegen Diazinon, Äthylchlorthion, Chlorthion und Parathion, mäßig resistent gegen fünf andere Insektizide und nur schwach resistent gegen die übrigen. Er war gegen die Diäthoxy-Verbindungen widerstandsfähiger als gegen Dimethoxy-Verbindungen und gegen Malathion nur sehr schwach resistent. Die Verbindungen, insbesondere die Phosphate, wirkten injiziert giftiger als bei äußerer Applikation. Additionen und Substitutionen im Phenyl-Ring des Parathion-Moloküls ergaben Verbindungen, die besonders gegen anfällige Fliegen weniger toxisch wirken.

     

Characterization of alanyl aminopeptidase from insecticide resistant and susceptible strains of Musca domestica L.

To investigate the high activity of intracellular proteases in insecticide resistant strains of Musca domestica L., purification by anion‐exchange chromatography and gel filtration of one of the enzymes, alanyl aminopeptidase (Ala AP), in three strains of Musca domestica was carried out. The fractions collected by gel filtration of soluble homogenates of the three strains (571ab, 17bb and Cooper) showed a single peak of Ala AP activity. Partially purified Ala AP of the three strains showed high activity at pH 7.5. The presence or absence of Ca²⁺ in the assay medium did not produce any difference in activity of Ala AP in the 571ab and Cooper strains, but there was a significant difference in the 17bb strain. The activity of Ala AP in all three strains was essentially unaltered in the presence of inhibitors of serine (PMSF), cysteine (E‐64) proteases and carboxypeptidases (pepstatin). Ala AP hydrolyzed alanine amino methylcoumarin (Ala‐AMC) maximally, followed by phenyl alanine amino methylcoumarin (Phe‐AMC), leucyl amino methylcoumarin (Leu‐AMC) and ornithine amino methylcoumarin (Orn‐AMC). Ala AP from the three strains showed differential activity towards various substrates. The comparison of alanyl aminopeptidase's activity from different sources is discussed.     

Insecticide-insensitive acetylcholinesterase from a laboratory selected and a field strain of housefly (Musca domestica) (L.)

1. Acetylcholinesterase from the heads of a strain of houseflies selected for resistance to the carbamate insecticide methomyl, and from a methomyl-resistant field strain was found to be less sensitive to inhibition by methomyl than that from a susceptible strain. 2. The enzyme from resistant insects was also more tolerant to malaoxon, dichlorvos and bomyl but not to azamethiphos. 3. The decrease in sensitivity to inhibition appeared to be due to an increase in affinity for substrate.     

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.     

Biochemistry and genetics of esterases in houseflies (Musca domestica) with special reference to the development of resistance to organophosphorus compounds

The paper reviews and reconsiders the experimental work on esterases in the housefly performed in the Laboratory for Research on Insecticides in Wageningen (formerly in Utrecht). It is attempted to integrate the different aspects of former studies, adding new data where necessary and possible. Two subjects that are intimately connected with esterases are briefly discussed, viz. the mode of action of organophosphorus insecticides and the mechanism of resistance to these compounds. A discussion on the possible functions of the esterases studied concludes the paper.

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Zusammenfassung Die Arbeit gibt eine Übersicht über die experimentellen Untersuchungen an Esterasen der Stubenfliege, die im Laboratorium für Insektizid-Forschung durchgeführt wurden. Es wurde versucht, die verschiedenen Aspekte früherer Arbeiten zu integrieren; nötigenfalls sind neue Daten beigefügt. Zwei Probleme, die sehr eng mit der Aktivität von Esterasen verknüpft sind, nämlich der Wirkungsmechanismus der Phosphorsäureester und die Resistenz gegen diese Substanzen werden ebenfalls besprochen. Eine Betrachtung über die Funktion der Esterasen beschließt die Arbeit.

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The material presented here was presented originally at the Symposium on the Function of Esterases in Animals, University of Recife, Recife, Pernambuco, Brazil, September 8–12. 1963.

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Deceased on October 3, 1964.     

House Fly (Musca domestica L.) Attraction to Insect Honeydew

House flies are of major concern as vectors of food-borne pathogens to food crops. House flies are common pests on cattle feedlots and dairies, where they develop in and feed on animal waste. By contacting animal waste, house flies can acquire human pathogenic bacteria such as Escherichia coli and Salmonella spp., in addition to other bacteria, viruses, or parasites that may infect humans and animals. The subsequent dispersal of house flies from animal facilities to nearby agricultural fields containing food crops may lead to pre-harvest food contamination with these pathogens. We hypothesized that odors from honeydew, the sugary excreta produced by sucking insects feeding on crops, or molds and fungi growing on honeydew, may attract house flies, thereby increasing the risk of food crop contamination. House fly attraction to honeydew-contaminated plant material was evaluated using a laboratory bioassay. House flies were attracted to the following plant-pest-honeydew combinations: citrus mealybug on squash fruit, pea aphid on faba bean plants, whitefly on navel orange and grapefruit leaves, and combined citrus mealybug and cottony cushion scale on mandarin orange leaves. House flies were not attracted to field-collected samples of lerp psyllids on eucalyptus plants or aphids on crepe myrtle leaves. Fungi associated with field-collected honeydews were isolated and identified for further study as possible emitters of volatiles attractive to house flies. Two fungal species, Aureobasidium pullulans and Cladosporium cladosporioides, were repeatedly isolated from field-collected honeydew samples. Both fungal species were grown in potato dextrose enrichment broth and house fly attraction to volatiles from these fungal cultures was evaluated. House flies were attracted to odors from A. pullulans cultures but not to those of C. cladosporioides. Identification of specific honeydew odors that are attractive to house flies could be valuable for the development of improved house fly baits for management of this pest species.     

The survival of ingested Serratia marcescens in houseflies (Musca domestica L.) after electrocution with electric fly killers

Electric fly killers (EFKs) are commonly used to control flying insects that enter food establishments. For establishment of the incidence of pathogen-bearing insects in food establishments, insect samples obtained from EFK trays could be used. The principal difficulty with this approach is that the survival time of microorganisms on or within insect corpses after electrocution is unknown. This study determined the survival of Serratia marcescens (as a representative of the enteric bacteria) within houseflies following their electrocution by a commercial EFK. S. marcescens was successfully ingested by houseflies and survived on and within the corpses after electrocution for up to 5 weeks. Maximal levels of bacteria were recovered 24 h postelectrocution. The study also demonstrates the ability of ingested S. marcescens to out-compete resident microbial flora within houseflies. The findings are intended to pave the way for further research to determine the incidence of pathogen-laden flying insects in food establishments.     

Molecular Polymorphism of Head Acetylcholinesterase from Adult Houseflies (Musca domestica L.)

Acetylcholinesterase (AChE) from housefly heads was purified by affinity chromatography. Three different native forms were separated by electrophoresis on polyacrylamide gradient gels. Two hydrophilic forms presented apparent molecular weights of 75,000 (AChE1) and 150,000 (AChE2). A third component (AChE3) had a migration that depended on the nature and concentration of detergents. In the presence of sodium deoxycholate in the gel, AChE3 showed an apparent molecular weight very close to that of AChE2. Among the three forms, AChE3 was the only one found in purified membranes. The relationships among the various forms were investigated using reduction with 2-mercaptoethanol or proteolytic treatments. Such digestion converted purified AChE3 into AChE2 and AChE1, and reduction of AChE3 and AChE2 by 2-mercaptoethanol gave AChE1, in both cases with a significant loss of activity. These data indicate that the three forms of purified AChE may be classified as an active hydrophilic monomeric unit (G1) plus hydrophilic and amphiphilic dimers. These two components were termed G2s and G2m, where "s" refers to soluble and "m" to membrane bound.     

Superoxide dismutase in the housefly,Musca domestica (L.)

Four superoxide dismutase (SOD) (E.C. 1.15.1.1) isozymes were present in whole tissue homogenates of Musca domestica when examined by polyacrylamide gel electrophoresis. One of the isozymes contained manganese, and the other three contained copper and zinc. All were observed in each of the body tagma (head, abdomen, and thorax) and at each developmental stage (egg to adult). The copper- and zinc-containing isozymes purified from newly emerged, adult M. domestica had a relative molecular weight of 34,800 as determined by gel filtration chromatography but consisted of two equal-size subunits of 16,000 as measured by sodium dodecylsulfate polyacrylamide gel electrophoresis. An isoelectric point between 4.8 and 5.1 was measured. Approximately 2 mol each of copper and zinc were present per dimer. The three copper, zinc isozymes were identified as charge variants. The amino acid composition of the enzyme was similar to that of copper, zinc-containing superoxide dismutases from other sources. Purified housefly copper, zinc superoxide dismutase was neither deactivated nor able to protect lactic dehydrogenase against deactivation in the presence of light and rose bengal, a known generator of singlet oxygen. The role of SOD in the phototoxic reaction involving rose bengal is discussed.     

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.