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.

     

Cytogenetic activity of various organophosphorus insecticides

The action of certain insecticides belonging to the group of organophosphorous compounds (chlorophos, metaphos, etaphos, phosphamid, phosalone, carbophos, selecron) has been studied by the test of chromosome aberrations in Crepis capillaris cells. It is shown that these insecticides possess mutagenic activity in all phases of the cell cycle and are mutagens of the undelayed action.     

Microbial cleavage of various organophosphorus insecticides.

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as sole phosphorus sources were isolated from soil and sewage. Individual isolates used from 3 to 10 of these insecticides as sole phosphorus sources. The extent of growth of two Pseudomonas strains in media containing diazinon and malathion was in the range expected from the amount of insecticide supplied, and their proliferation resulted in disappearance of the chemical. Resting cells of the pseudomonads derived from cultures grown on diazinon or malathion but not orthophosphate caused extensive destruction of these two organophosphates in the presence or absence of chloramphenicol. Extracts of the two bacteria derived from organophosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate, Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysis suggested that the extracts formed dimethyl phosphate from azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from Trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethyl phosphate, dimethyl phosphorothioate , dimethyl phosphorodithioate, diethyl phosphate, and diethyl phosphorothioate were not used by the pseudomonads as sole phosphorus sources.     

Microbial cleavage of various organophosphorus insecticides.

Bacteria able to utilize Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, Trithion, dimethoate, Dylox, methyl parathion, and Vapona as sole phosphorus sources were isolated from soil and sewage. Individual isolates used from 3 to 10 of these insecticides as sole phosphorus sources. The extent of growth of two Pseudomonas strains in media containing diazinon and malathion was in the range expected from the amount of insecticide supplied, and their proliferation resulted in disappearance of the chemical. Resting cells of the pseudomonads derived from cultures grown on diazinon or malathion but not orthophosphate caused extensive destruction of these two organophosphates in the presence or absence of chloramphenicol. Extracts of the two bacteria derived from organophosphate-grown cultures catalyzed the disappearance of Aspon, Azodrin, Dasanit, diazinon, malathion, Orthene, parathion, and Trithion but not dimethoate, Dylox, methyl parathion, and Vapona. Results from gas chromatographic analysis suggested that the extracts formed dimethyl phosphate from azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from Trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Dimethyl phosphate, dimethyl phosphorothioate , dimethyl phosphorodithioate, diethyl phosphate, and diethyl phosphorothioate were not used by the pseudomonads as sole phosphorus sources.     

The interaction between organophosphorus insecticides and esterases in homogenates of organophosphate susceptible and resistant houseflies

Housefly homogenates contain different esterases, which compete for added organophosphates. Among these the cholinesterase and an ali-esterase rank first. If small amounts of organophosphate are added, the final result of the competition depends on the amounts of the esterases present and their reaction rates. The bimolecular rate constants (Table I) show that the ali-esterase is a powerful competitor of the cholinesterase for DDVP, paraoxon and diazoxon. Accordingly it was found that removal of the ali-esterase by denaturation (AD-treatment) strongly enhances the inhibition of the cholinesterase by these three compounds (Tables II, III and IV; Figs 1, 2 and 3), but not by tetram and QAT.In homogenates of organophosphate resistant flies, which as previously shown contain much less ali-esterase, cholinesterase inhibition was generally much stronger than in susceptible homogenates (Table IV: Figs 1, 2 and 3). It seemed difficult to reconcile these findings with previous observations showing that low ali-esterase activity and resistance are closely connected. Further studies, however, showed that resistant flies contain a modified ali-esterase which has the capacity of degrading the organophosphates (P=O compounds) to which resistance has developed. The change of the ali-esterase (in susceptible flies) into a phosphatase (the modified enzyme in resistant flies) is due to a single gene mutation, which in the two resistant strains investigated is also responsible for the resistance. In each of these two strains the phosphatase shows a very high affinity for its substrates-paraoxon and diazoxon.

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Zusammenfassung Wenn einem Stubenfliegen-Homogenisat Phosphorsäure-Ester zugesetzt werden, dann werden mehrere Esterasen in wechselseitiger Konkurrenz mit diesen Substanzen zu reagieren versuchen. Die wichtigsten sind die Cholinesterase und eine Aliesterase, die zur Hydrolyse des Methyl- und Phenylbutyrats und mehrerer anderen Ester befähigt ist. Nach Zusatz geringer Mengen von Phosphorsäure-Ester wird das Endergebnis dieses Wettbewerbes von den Quantitäten der Esterasen und ihren Reaktionsgeschwindigkeiten abhängig sein.Tabelle I zeigt die bimolekularen Geschwindigkeitskonstanten für die Reaktionen zwischen der Cholinesterase und der Aliesterase und den fünf untersuchten Estern: DDVP, Paraoxon, Diazoxon, Tetram und QAT (die quaternären Phosphonothiolat-Analoge von Tetram). Aus diesen Daten wird geschlossen, daß die Aliesterase ein wichtiger Konkurrent der Cholinesterase für DDVP, Paraoxon und Diazoxon darstellt. Dementsprechend wurde gefunden, daß ach Beseitigung der Aliesterase mittels einer Denaturierung (AD-Verfahren) die Hemmung der Cholinesterase durch diese drei Substanzen bedeutend höher ist (Tabelle II, III und IV; Fig. 1, 2 und 3), nicht aber jene durch Tetram und QAT.In Homogenisaten von Phosphorester-resistenten Fliegen, die — wie früher gezeigt wurde — viel weniger Aliesterase enthalten, war die Hemmung der Cholinesterase meistens viel stärker als in empfindlichen Homogenisaten (Tabelle IV; Fig. 1, 2 und 3). Es erschien schwer, diese Befunde mit dem früheren in Einklang zu bringen, daß niedrige Aliesterase-Aktivität und Resistenz eng verbunden seien. Weitere Versuche aber zeigten, daß resistente Fliegen eine modifizierte Aliesterase enthalten, die keine oder eine nur geringe Aktivität gegenüber Methyl- und Phanylbutyrat aufweist, aber die Fähigkeit besitzt, die Phosphorsäure-Ester, gegen welche Resistenz besteht, abzubauen. Die Substrat-Spezifizität und Aktivität wiesen eine deutliche Korrelation mit der Art und Höhe der Resistenz auf.Die Umwandlung der Aliesterase (in empfindlichen Fliegen) in eine Phosphatase (das modifizierte Enzym in resistenten Fliegen) wird durch eine einzige Gen-Mutation verursacht. Diese ist in den zwei untersuchten Stämmen auch für die Resistenz verantwortlich.Die Phosphatasen haben eine hohe Affinität für ihre Substrate, Paraoxon und Diazoxon. Die Abbau-Geschwindigkeit in vitro (0.01–0.04 g/St/Fliege) ist aber niedrig im Vergleich mit den gefundenen LD₅₀-Werten. Da ihre Bedeutung für den Mechanismus der Resistenz feststeht, muß entweder angenommen werden, daß der Abbau in vivo schneller verläuft oder daß strukturelle Faktoren in der lebenden Fliege einen wichtigen Einfluß ausüben.Weitere Betrachtungen ermöglichten die Berechnung der wahrscheinlichen Aliesterase- und Phosphatase-Konzentration und der Umsatzzahlen für Diazoxon in Homogenisaten.

     

Patterns of insecticide resistance to organo-phosphorus compounds in strains of houseflies from various sources

Four strains of housefly (Musca domestica L.), resistant to organo-phosphorus insecticides, have been maintained in laboratory culture, together with a normal colony. Resistance in one strain had been developed by laboratory selection and in the others by field usage of insecticides followed by laboratory selection. They originated in Denmark, Italy and U.S.A., mainly by the use of parathion, diazinon and malathion respectively. The resistance spectra of these strains were determined by measuring their relative resistance to 14 organo-phosphorus compounds, by a microdrop application test. The strains selected by parathion and diazinon gave rather similar resistant patterns, showing highest resistance to parathion, paraoxon and diazinon and low resistance to malathion. The American strain produced a different pattern with high resistance to malathion and low resistance to parathion, paraoxon and diazinon. It is believed that these two patterns reflect different resistance mechanisms.A detailed comparison of the two European strains revealed more divergence in compounds containing (EtO)₂P-groups than in those with (MeO)₂P—groups.Injection of parathion reduced its resistance level considerably and the resistance to paraoxon on injection was even more reduced.

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Zusammenfassung Vier gegen organische phosphorhaltige Insektizide resistente Stubenfliegen-Stämme wurden zusammen mit einer normalen Kolonie in Laboratoriumkultur gehalten. Die Resistenz des einen Stammes hatte sich durch Selektion im Laboratorium entwickelt, die der anderen durch den Feldgebrauch von Insektiziden und nachfolgender Selektion im Labor. Sie stammten aus Dänemark, Italien und USA, hauptsächlich nach Anwendung von Parathion, Diazinon bzw. Malathion. Die Resistenzspektren dieser Stämme wurden durch Messung ihrer relativen Resistenz gegenüber 14 organischen Phosphorverbindungen mit Hilfe eines Mikrotropfen-Applikator-Tests bestimmt. Die durch Parathion und Diazinon selektierten Stämme ergaben ziemlich ähnliche resistente Eigenschaften; sie zeigten höchste Resistenz gegen Parathion, Paraoxon und Diazinon und niedrige gegen Malathion. Der amerikanische Stamm ergab abweichendes Verhalten mit hoher Resistenz gegen Malathion und niedriger gegen Parathion, Paraoxon und Diazinon. Es wird vermutet, daß diese beiden Typen auf verschiedenen Resistenzmechanismen beruhen.Ein eingehender Vergleich der zwei europäischen Stämme erbrachte größere Unterschiede in Verbindungen mit (EtO)₂P-Gruppen als in solchen mit (MeO)₂P-Gruppen.Injektion von Parathion verminderte seinen Resistenzspiegel beträchtlich und die Resistenz gegen Paraoxon wurde bei Injektion noch mehr reduziert.

     

Studies of the acetylcholinesterase from houseflies (Musca domestica L.) resistant and susceptible to organophosphorus insecticides.

Acetylcholinesterase from the heads of insecticide-resistant and -susceptible houseflies (Musca domestica L.) was studied in vitro. The enzymes could not be distinguished electrophoretically, and their behaviour on polyacrylamide-disc-gel electrophoresis was influenced by the presence of Triton X-100 in both the homogenate and the gels. In the absence of detergent, the acetylcholinesterase was heterogeneous, but behaved as a single enzyme when it was present. By analogy with studies of acetylcholinesterase from other sources, these observations were attributed to aggregation of the enzyme when not bound by membranes. The enzyme from resistant flies was more slowly inhibited than the susceptible enzyme, bimolecular rate constants (ki) differing by approx. 4-20-fold for a range of organophosphorus compounds. The kinetics of inhibition of acetylcholinesterase were consistent with the results of electrophoresis, i.e. they corresponded to those of a single enzyme in the presence of Triton X-100, but a mixture of enzymes in its absence. The susceptibility of the more sensitive components in these mixtures was determined.     

Metabolism of organophosphorus insecticides

Summary The metabolism of ³²P-Malathion in Rhizobium leguminosarum and Rhizobium trifolii has been investigated. In addition to inorganic phosphates and/or thiophosphates, 5 hydrolytic metabolites could be identified. The carboxylic acid derivatives constituted the major portion (35–40% of the total metabolites output) suggesting the presence of powerful carboxyesterases in both Rhizobium spp. Malaoxon could not be detected in the media of both organisms.     

Resistance to two organophosphorus insecticides in New Zealand populations of the Australian sheep blowfly, Lucilia cuprina

Abstract. A treated-surface test was used in a bioassay to detect insecticide resistance to the organophosphorus compounds diazinon and propetamphos, in larvae of field strains of Lucilia cuprina in New Zealand during 1990/91 and 1991/92 (December-April). Mean LC₅₀s for both diazinon and propetamphos showed a significant increase between seasons although this is not necessarily indicative of a trend. Mean LC₅₀s for diazinon increased from 1.16 to 1.60 mg/1 (P< 0.001) and for propetamphos from 0.49 to 0.59 mg/1 (P< 0.005) in pooled populations. Diazinon and propetamphos resistance was significantly correlated in both seasons, suggesting side-resistance between the two insecticides.     

有机磷对抗拟除虫菊酯家蝇的毒力

测定敏感、抗溴氰菊酯(Del-R)、抗氯菊酯(2Cl-R)的家蝇品系对有机磷杀虫剂敌敌畏、辛硫磷及马拉硫磷的LD₅₀,α-乙酸萘酯（α-NA）酯酶动力学,酯酶的活性和酯酶的抑制作用。Del-R和2Cl-R的家蝇品系对三种有机磷杀虫剂的抗性倍数为0.966～7.190倍,均为低抗水平。三个家蝇品系的羧酸酯酶活性水平与抑制中浓度存在正相关性,说明羧酸酯酶在抗拟除虫菊酯家蝇对有机磷杀虫剂的抗性中起一定的作用。     

On the orientation of houseflies to various surfaces

The attraction of houseflies (Musca domestica L.) to surfaces was investigated by repeated counting of flies on samples of materials. The materials tested were mainly such as ordinarily occur in stables. The observations were made partly in a big cage in the laboratory and partly in a stable. The mechanism of attraction to some surfaces was analyzed by counting the number of flies arriving per time unit, and by measuring the duration of visits.Significant differences between materials were found. The brightness of the surfaces proved an important factor. The light materials, e.g. white tile and limewashed surfaces, were less visited than darker ones. Metallic surfaces were highly avoided. More flies were counted on very rough surfaces, straw, and jute sack, than on smoother ones, such as wood and concrete. The arrival of more flies per time unit to dark surfaces was demonstrated in some cases, and so was a tendency of staying longer on relatively rough surfaces, but the differences of attraction were not completely explained by these two reactions.

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Zusammenfassung Die Bevorzugung verschiedener Oberflächen durch die Stubenfliege (Musca domestica L.) wurde durch wiederholte Auszählung der Fliegen auf Proben verschiedener Substrate untersucht. Die Materialen waren hauptsächlich solche, die oft in Ställen vorkommen, welche bei uns nämlich den wichtigsten Aufenthaltsort der Stubenfliegen darstellen. Die Beobachtungen wurden teils im Laboratorium in einem großen Käfig, teils in einem Stall vorgenommen, wo der Besuch der dort freilebenden Fliegen registriert wurde. Vier Substrate wurden in jedem einzelnen Versuch verglichen, wobei jedes von ihnen in vier Stücken vertreten war. In allen Versuchen wurde Holz als Vergleichsbasis benutzt. Bei einigen Oberflächen wurde versucht, ihre Anziehungskraft durch Zählung der Fliegen näher zu analysieren, die pro Zeiteinheit zu diesen Materialproben kamen, sowie durch Messung der Aufenthaltsdauer auf diesen Substraten. Bei dieser Analyse wurde jeweils eine einzige Materialprobe in einem Käfig beobachtet und mit einem Standardstück aus Holz verglichen. Mehrere der Oberflächen zeigten in bezug auf Fliegenbesuche signifikante Unterschiede. Als ein wichtiger Faktor erwies sich die Helligkeit, indem helle Substrate, wie weiße Fliesen und getünchte Oberflächen, weniger als die dunkleren besucht wurden. In einem Versuch mit Kartonstücken verschiedener Tönung (weiß, hellgrau, dunkelgrau und schwarz) ergab sich, daß unter den betreffenden Versuchs-bedingungen allein die Farbe Unterschiede derselben Größenordnung verursachen konnte wie die Unterschiede zwischen den natürlichen Materialien. Metallische Oberflächen wurden weitgehend vermieden. Auf unebenen Oberflächen, Stroh und Jutesack, wurden mehr Fliegen als auf den glatteren, wie z.B. Holz und Zement, gezählt. Bei den Zählungen der pro Zeiteinheit ankommenden Fliegen ergab sich in mehreren Fällen, daß von dunkleren Oberflächen mehr Fliegen angelockt wurden. Bei Messung der Aufenthaltsdauer zeigte sich in einigen Fällen eine Tendenz zu längerem Verweilen auf unebenen Oberflächen. Jedoch liefert diese Analyse der Präferenz für Oberflächen nicht in allen Fällen eine Erklärung für die Unterschiede im Fliegenbesuch, die bei wiederholten Zählungen der Fliegen auf den Substratstücken festgestellt wurden.

     

Resistance to heavy metals by some Nigerian yeast strains

The heavy metal resistance of yeasts isolated from sugary substrates such as orange, palm wine and pineapple and identified asSaccharomyces carlsbergensis andS. cerevisiae was studied. The yeast isolates were tested against different concentrations of cadmium, copper, manganese, silver and zinc salts ranging from 1 to 20 mmol/L. Local yeasts showed resistance to 3–15 mmol/L cadmium, 18–20 copper, 16–20 manganese, 1–9 silver and 16–19 for zinc. The significance of the results is discussed in relation to the effects of heavy metals on growth of microorganisms and selection of yeasts for the brewing industry in Nigeria.     

Anticholinesterase effect of the organophosphorus and pyrethroid insecticides to some phytophages species,their predators and parasites

It has been determined catalytic activity of cholinesterases for several insect species (Apanteles glomeratus L., Coccinella septempunctata L, Rhopalosiphumpadi L. and Pieris brassicae L.) that varies in norm from 57 to 199 mmol/hour per 1 gr. It has been calculated the constants of bimolecular interaction (K_ii) for insecticides Aztek, Mavric and Bi‐58 new with cholinesterase of the insects. It occurred to be the most sensitivity to Bi‐58 new is peculiar to this ferment in Coccinella 7‐punctata L. K_ii (4.54 ± 0.23) 10⁴; whereas cholinesterase of Rhopalosiphum padi L. is the least sensitive to Aztek ‐ K_ii (2.9 ± 0.14) 10⁵. Determination of anticholinesterase action coefficient has revealed the value of this indice 118.9 for the preparation Aztek in the system "Rhopalosiphum padi ‐Coccinella 7‐punctata”;. Supposedly, anticholinesterase activity of Aztek is the base of its mechanism action on above‐mentioned species of insects.     

Resistance of various strains of mycobacteria to killing by activated macrophages in vivo

A variety of experimental infections with pathogenic mycobacteria are associated with the development of persistent disease, in which little or no changes in the numbers of the infectious organism can be detected. This report describes a simple experimental model designed to test the hypothesis that this persistence may reflect in part the ability of these organisms to resist the enhanced bacteriostatic and bactericidal properties acquired by host macrophages as a result of these mycobacterial infections. To examine this possibility mice were inoculated with test organisms at a time when these animals were expressing very high levels of nonspecific resistance, and hence macrophage activation, as a result of a prior intravenous infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG). The results show that the test organisms fall into three groups; (a) those, such as Mycobacterium tuberculosis, which were sensitive to the presence of activated macrophages, (b) those, such as Mycobacterium avium and Mycobacterium kansasii, which were insensitive, and (c) one organism, Mycobacterium intracellulare, in which progressive growth of the infection was significantly improved. These results are consistent with the hypothesis that some mycobacteria, particularly those associated with persistent disease, possess an intrinsic resistance to host bactericidal and bacteriostatic mechanisms in vivo.     

Acute toxicity of organophosphorus and pyrethroid insecticides to Bombyx mori

Acute toxicities of two organophosphorus insecticides (dichlorvos and phoxim), four pyrethroid insecticides (permethrin, tetramethrin, bifenthrin, and ethofenprox), and their combined uses to the third instar of the silkworm, Bombyx mori (L.), were determined by feeding the insect with the insecticide-treated mulberry, Morus albus (L.), leaves. Twenty-four and 48 h after treatment, toxicity levels of all insecticides to the silkworm were in the very high or high range, and the LC50 values of permethrin, tetramethrin, bifenthrin, ethofenprox, dichlorvos, and phoxim were 1.60 and 0.75, 3.86 and 2.83, 0.09 and 0.06, 2.87 and 0.80, 6.63 and 4.11, and 1.05 and 0.45 mg liter(-1), respectively. The toxicity levels of 50:50 binary mixtures of organophosphorus and pyrethroid insecticides to the silkworm were in the high or middle range. Twenty-four and 48 h after treatment, the LC50 values of 50:50 binary mixtures permethrin + phoxim, permethrin + dichlorvos, tetramethrin + phoxim, tetramethrin + dichlorvos, bifenthrin + phoxim, bifenthrin + dichlorvos, ethofenprox + phoxim, and ethofenprox + dichlorvos to the silkworm were 1.49 and 0.85, 1.24 and 0.79, 2.20 and 1.08, 14.62 and 13.16, 0.33 and 0.13, 0.12 and 0.10, 2.81 and 1.37, and 4.82 and 3.00 mg liter(-1), respectively. Based on the combinations coefficient values, the toxicities of binary mixtures of organophosphorus and pyrethroid insecticides had additive effect except for the binary mixture of etramethrin + dichlorvos, which showed antagonism effect.     

Resistance of some strains ofBacteroides to ampicillin,methicillin and cloxacillin

Twenty-three strains of thefragilis-convexus group ofBacteroides and two strains of thethetaiotaomicron group have been tested for sensitivity to ampicillin, methicillin and benzylpenicillin. Thirteen of these strains (twelve of thefragilis-convexus group and one of thethetaiotaomicron group) have been tested to cloxacillin, tetracycline, chloramphenicol and erythromycin.The strains proved to be resistant to benzylpenicillin, ampicillin, methicillin and cloxacillin.     

Toxicity of organophosphorus insecticides to diazinon-reversely-selected,pesticide-pressure-relaxed and field-resistant strains of bulb miteRhizoglyphus robini (Acari: Acaridae)

Some field strains ofRhizoglyphus robini in Taiwan, developed high multi-resistance to organophosphorus insecticides including diazinon, dimethoate, fenitrothion, malathion, methyl parathion, omethoate, parathion and prothiophos. The organophosphorus resistance of bulb mites remained stable when the pesticide pressure was relaxed. Changes of the susceptibility to several organophosphorus insecticides for a strain after relaxation of selection pressure for 3 and 27 generations were compared. Since susceptible bulb mites could not be found in the field, a reverse selection for susceptibility to diazinon was designed and conducted. Although heterogeneity was observed in the progeny of the mixed populations, the susceptibility of these reversely-selected mites increased slowly. A stable and susceptible strain was obtained after 26 generations of selection.Contribution No. 1412 from Taiwan Agricultural Research Institute.