CSMA Strain Research Articles

Decapitation Impacting Effect of Topically Applied Chlorpyrifos on Acetylcholinesterase and General Esterases in Susceptible and Resistant German Cockroaches (Dictyoptera: Blattellidae)

The effect of topically applied chlorpyrifos on acetylcholinesterase and other esterases in heads and decapitated bodies of CSMA and Crawford German cockroaches was examined with spectrophotometric enzyme assay and native polyacrylamide gel electrophoresis. The toxicity of chlorpyrifos was greatly reduced in decapitated CSMA male cockroaches with LD50 value 17.1-fold higher than that of normal CSMA cockroaches. Acetylcholinesterase activity from heads was significantly higher in the Crawford compared with the CSMA strain and did not change until 24 h after chlorpyrifos in vivo treatment in both strains. The p-nitrophenyl butyrate (NPB) esterase activities from both heads and decapitated bodies of the resistant Crawford strain were significantly greater than the susceptible CSMA strain. The p-NPB esterase activity was significantly inhibited by chlorpyrifos in vivo treatment, and total p-NPB esterase activity was significantly reduced in decapitated bodies compared with heads of both strains. Native polyacrylamide gel electrophoresis (PAGE) analysis of extracts solubilized with Triton X-100 from heads and decapitated bodies revealed five major esterase bands and an acetylcholinesterase (AChE) band with a high capability of hydrolyzing alpha-naphthyl butyrate and acetylthiocholine, respectively. In the heads of susceptible CSMA male cockroaches, the activity of mobile isozymes d1 and d2 was completely inhibited at 24 h after chlorpyrifos application, and isozyme e was partially inhibited. In contrast, isozymes c1 and c2 from the decapitated bodies of CSMA cockroaches were mostly affected at 24 h after the topical application of chlorpyrifos. The activities of acetylcholinesterase and esterase isozymes a and b from the decapitated body remained uninhibited in both strains. Inhibition of isozymes d1 and d2 seems to be more important in chlorpyrifos intoxication than acetylcholinesterase.

In vivo study on combined toxicity of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) strain ESC-1 with sublethal doses of chlorpyrifos, propetamphos, and cyfluthrin against German cockroach (Dictyoptera: Blattellidae).

The effect of Metarhizium anisopliae (Metschnikoff) Sorokin strain ESC-1 alone and in combination with sublethal doses of commercial formulations of chlorpyrifos, propetamphos and cyfluthrin on mortality of CSMA strain of German cockroach, Blattella germanica (L.), was determined by conducting in vivo studies that included 3 bioassays. Spores of M. anisopliae cultured on SDAY media had germination of >90%. Based on bioassay 1, doses ranging from 0.5 to 300 ppm of chlorpyrifos and propetamphos, and 0.05 to 40 ppm of cyfluthrin were selected for bioassays 2 and 3. Cockroach mortality ranged from 5 to 20% for insecticides alone and 48 to 70% for insecticides + M. anisopliae in bioassay 2. In bioassay 3, mortality ranged from 15 to 60% for insecticides and 57.5 to 92.5% for insecticides + M. anisopliae. Percentage of cockroach mortality resulting from insecticide + M. anisopliae combinations was significantly higher than insecticide alone. Mortality was also significantly higher in certain insecticide + M. anisopliae combinations than M. anisopliae alone. There was no significant interaction between M. anisopliae and insecticides with their concentrations in bioassay 2, indicating an additive effect. But in bioassay 3, a significant interaction was observed when M. anisopliae was combined with multiple insecticide concentrations. The interaction indicated an additive effect for chlorpyrifos and cyfluthrin, and a synergistic effect for propetamphos. There were significant differences in LT50 among various treatment combinations. M. anisopliae alone or insecticide + M. anisopliae combinations did not affect body weight in treated German cockroaches.

Distribution and Inhibition of Esterases in Various Body Tissues of Susceptible and Resistant German Cockroaches (Dictyoptera: Blattellidae)

The distribution and inhibition of esterase activity among body of the resistant Crawford and susceptible CSMA strains of male German cockroach, Blattella germanica (L.), were evaluated with α- and β-naphthyl esters using a spectrophotometric assay and native polyacrylamide gel electrophoresis. The esterase activities in the Crawford strain were significantly higher than in the CSMA strain in most tissues except midgut and male genitalia, where the activities were similar. In both strains, isozyme a was dominant in the brain and fat body and isozymes c and d were more abundant in the midgut; but in addition to isozyme a, isozyme b was observed only in the Crawford strain. The α-naphthyl butyrate (α-NB) and β-naphthyl acetate (β-NA) esterase activities in the brain of both strains were completely inhibited by most inhibitors (DEF, 3,4-dichloroisocoumarine, paraoxon, pepstatin A, and propoxur). However, in the midgut tissue and contents, the activities and inhibition patterns of the α-NB and β-NA esterase were apparently different from the brain and between strains. The complete inhibition by paraoxon was observed regardless of the strain, substrate type, and body part for enzyme source. In esterase inhibition on native PAGE gels, paraoxon completely inhibited the activity of most esterases similar to the results from spectrophotometric assay. In addition, the esterase b was observed from the whole body homogenates of several German cockroach resistant strains on native PAGE analysis.

Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica)

Using reverse transcription polymerase chain reactions (RT-PCR), the DNA sequence for the main membrane-spanning region (IS3 through IVS6) of the gene encoding the alpha-subunit of the para sodium channel of the German cockroach, Blattella germanica, has been determined. The overall structure of the open reading frame region of this B. germanica gene is very similar to that of the para gene of Drosophila melanogaster, and that of the partially sequenced para gene of Musca domestica. On the other hand, it is distinctly different from that of the DSC gene (Drosophila sodium channel). As a result of a side-by-side comparison of the para gene sequences of the susceptible CSMA strain and the kdr resistant VT strain of B. germanica, one mutation (TTG to TTC) at the approximate center of the IIS6 membrane-spanning segment was found to result in an amino acid change from L to F. While the functional meaning of this mutation for the operation of the para sodium channel remains to be studied, this region is very highly conserved among all sodium channels identified so far, and is one of the most hydrophobic areas of the entire alpha-subunit. For comparison, we have studied the same region of the para sodium channel of both kdr and susceptible SBO strain of the housefly, Musca domestica. We found the homologous type of mutation, CTT to TTT, resulting in the same amino acid alteration (L to F) at this site. However, in the case of houseflies both kdr and susceptible strains contained both L and F versions of the protein. The ratio of TTT to CTT was significantly higher in the kdr strain of M. domestica than in the three susceptible strains examined.

Effects of Azadirachtin on Different Strains of German Cockroach (Dictyoptera: Blattellidae)

Technical grade azadirachtin (11.46% [AI]) was tested for its toxicity to insecticide-resistant and -susceptible strains of German cockroach, Blattella germanica (L.), by injection and diet incorporation. The effect of azadirachtin was dose dependent. The LC50 and LC90 for 3 strains ranged between 2.49 and 2.56 μg per insect and 6.49 and 6.92 μg per insect, respectively. Incorporation of Azadirachtin in food pellets resulted in LT50s of 22, 23, and 22 d for Dursban-R, CSMA, and Baygon-R strains, respectively. No significant differences were observed in toxicity of Azadirachtin to either insecticide-resistant or susceptible strains. The body weight of German cockroaches varied with time depending upon the dose administered. The roaches injected with 2 and 3 μg of Azadirachtin showed a continuous weight reduction. The toxicity of Azadirachtin to German cockroaches may be associated with an altered feeding behavior and a disruption of endocrinal events.

Separation of multiple forms of acidic glutathione S‐transferase isozymes in a susceptible and a resistant strain of house fly, Musca domestica (L.)

The acidic glutathione S-transferases from a CSMA (susceptible) strain and a Cornell-R (resistant) strain of houseflies were purified and separated utilizing affinity chromatography followed by chromatofocusing. Nine fractions were isolated from each house fly strain. Fraction 1 had the highest 1-chloro-2,4-dinitrobenzene vs. 1,2-dichloro-4-nitrobenzene ratio (CDNB/DCNB ratio) in both strains and the ratio of all the other fractions tended to decrease as the isoelectrical points decreased except for fractions 4 and 9. Most fractions from the CSMA strain had higher CDNB conjugation activities than the fractions from the Cornell-R strain, but all the fractions from the CSMA strain had lower DCNB conjugation activities than fractions from the Cornell-R strain. Steady-state kinetics of all the fractions were examined. The Km values obtained from both strains ranged from 0.36 to 1.12 mM, while the Vmax value ranged from 3.0 to 32.6 mumol/min/mg. In the 100,000 g supernatant, the CDNB specific activities in the CSMA strain was about 1/3 of the activity in the Cornell-R strain but it was about 1.5-fold following affinity chromatography. The specific activity for DCNB measured in the CSMA strain was only 1/5 of the activities of the Cornell-R strain in the 100,000 g supernatant, but was about the same after affinity chromatography. The difference was due to the selectivity of the affinity column used in the current study.

Immunological comparison of cytosolic glutathione S-transferases between rat and two strains of houseflies

Five different antisera, which include three antisera raised against rat liver glutathione S-transferases (GST), one antiserum raised against human pi GST, and one antiserum raised against housefly GST1, were used to examine their cross-reactivity with different classes of GST subunits isolated from rat liver and the housefly. Two classes of rat liver GSTs, alpha and mu, were isolated from rat liver and two classes of housefly GSTs, GST1 and GST2, were isolated from both CSMA and Cornell-R strains. Antiserum against GST 3-3 was the most reactive antiserum and reacted not only with the mu class of GSTs but also with the GST1 class from both CSMA and Cornell-R strains. Antiserum against human pi GST and antiserum against housefly GST1 had weak immunological reactivity toward the GST1 class from both strains of housefly. Antiserum against GST 4-4 and antiserum against GST 1-1 had immunological reactivity toward any class of GSTs from housefly. None of the five antisera had any immunological cross-reactivity toward subunit 2 of the alpha class of rat GST and the GST2 class of housefly GSTs from both strains.

Biochemistry and genetics of chlorpyrifos resistance in the German cockroach, Blattella germanica (L)

The mechanisms and genetics of chlorpyrifos resistance were studied in a strain (Dursban-R) of the German cockroach displaying 20-fold levels of resistance by topical application compared to a susceptible laboratory strain (CSMA). Dursban-R was equally resistant to both chlorpyrifos and chlorpyrifos oxon. Penetration rates of [ 14C]chlorpyrifos were similar for both strains. In vivo metabolism studies employing [ 14C]chlorpyrifos indicate that chlorpyrifos oxon was present at reduced levels in the resistant strain relative to the susceptible strain, and that elevated levels of the hydrolysis product, 3,5,6-trichloropyridinol, were also associated with resistance. Resistance to chlorpyrifos was partially suppressed by both piperonyl butoxide and DEF suggesting the involvement of both cytochrome P450-dependent monooxygenases (MFOs) and hydrolytic enzymes in the resistance mechanism. In vitro, the Dursban-R strain displayed higher rates of NADPH-dependent microsomal metabolism of [ 14C]chlorpyrifos associated with the formation of chlorpyrifos oxon, trichloropyridinol, and unknown metabolites, further implicating the monooxygenase system in the resistance. In vitro metabolism of [ 14C]-chlorpyrifos with cytosolic fractions was not detected. However, higher levels of hydrolysis were detected in the Dursban-R strain when [ 14C]chlorpyrifos oxon was used as substrate. These results indicate that increased levels of monooxygenase activity as well as increased hydrolysis of chlorpyrifos oxon are mainly responsible for resistance to chlorpyrifos in this strain. Reciprocal crosses of the resistant and susceptible strains and backcrosses of the F 1 progeny to the parental CSMA strain indicate that resistance is incompletely dominant, autosomal, and associated with more than one genetic factor.

Characterization of multiple forms of cytochrome P-450 from an insecticide resistant strain of house fly ( Musca domestica)

Components of the microsomal monooxygenase system derived from abdominal preparations of an insecticide resistant house fly ( Musca domestica, Rutgers strain) were characterized. Cytochrome P-450 isozymes with molecular weights of 49,000, 53,000, 54,000, and 58,000 were partially purified using a procedure involving chromatography on columns of phenyl Sepharose, DEAE-cellulose (DE-52), carboxymethyl-Sepharose, hydroxylapatite, and an affinity column prepared from rat liver cytochrome b 5. Activity toward a variety of pesticides, as well as other exogenous and endogenous monooxygenase substrates, was examined using intact microsomes or purified isozymes in reconstituted systems. Oxidation of benzo[ a]pyrene in microsomal preparations was high although neither O-dealkylation of 7-ethoxyresorufin nor N-dealkylation of p-chloromethylaniline could be detected. Other substrates metabolized by abdominal microsomes from Rutgers flies included testosterone, lauric acid (primarily at in-chain positions), phorate, p-nitroanisole, and aldrin. Activity of abdominal microsomes from the susceptible CSMA strain toward benzo[ a]pyrene and lauric acid was, surprisingly, higher than that from the resistant Rutgers strain. Synergism between NADPH and NADH was noted with microsomes from either strain. Reconstitution experiments were relatively unsuccessful but activity toward lauric acid was obtained using a system that included purified cytochrome P-450 fractions, purified house fly or rat liver NADPH-cytochrome P-450 reductase, an NADPH generating system, and a phospholipid. Unlike similar systems utilizing mammalian cytochrome P-450, phosphatidylethanolamine was more active than phosphatidylcholine.

Studies on the metabolism of vamidothion and its thioanalog in insecticide-resistant and susceptible house fly strains

The in vivo and in vitro metabolism of vamidothion [ O,O-dimethyl S-[2-(1-methylcarbamoyl)-ethylthio] ethylphosphorothiolate] as well as the in vitro metabolism of thiovamidothion [ O,O-dimethyl S-[2-(1-methylcarbamoyl)ethylthio] ethylphosphorodithioate] was investigated in insecticide-resistant and susceptible house fly strains. Vamidothion was converted in vivo to the sulfoxide, the principle metabolite, and subsequently to the sulfone at a slower rate. Vamidothion and vamidothion sulfoxide were hydrolyzed at the PS and SC bond. The resulting primary alcohol metabolite was further oxidized to a carboxylic acid followed by decarboxylation. No metabolism of vamidothion or thiovamidothion occurred in vitro without the addition of NADPH. The addition of NADPH resulted in rapid conversion of vamidothion to the sulfoxide, and thiovamidothion was oxidatively metabolized to six metabolic products. No qualitative differences were found between resistant and susceptible strains, but there were signficant quantitative differences. The metabolism was highest in the Rutgers strain followed by Cornell-R, Hirokawa, and then CSMA strain. The route of vamidothion and thiovamidothion metabolism was via the cytochrome P-450-dependent monooxygenase system, and none of the resistant strains showed glutathione S-transferase activity toward vamidothion or thiovamidothion. No further oxidation of vamidothion sulfoxide to the sulfone was observed and also no hydrolysis products were formed, in vitro.

The purification and characterization of esterases from insecticide-resistant and susceptible house flies

Four major esterases in one susceptible (CSMA) and two resistant (Hirokawa, E 1) house fly strains were separated by chromatofocusing. Of the four esterases, those with p I's of 5.1 and 5.3 accounted for 90% of the p-nitrophenyl butyrate hydrolyzing activity in the three house fly strains. They also accounted for 70% (Hirokawa, E 1) and 40% (CSMA) of the paraoxon-hydrolyzing activity as well as 87% (Hirokawa), 39% (E 1) and 66% (CSMA) of the malathion-hydrolyzing activity in microsomes as measured by esterase-antibody interaction. In the Hirokawa strain, the p I 5.1 esterase was the predominant esterase and was more active than that of the the CSMA strain. Different substrate specificities and a different K m toward acetylthiocholine, as well as different rates of malathion and paraoxon hydrolysis between the Hirokawa and CSMA strains, suggest a qualitative difference in the p I 5.1 esterase. For the p I 5.1 esterase from the E 1 strain, a different substrate specificity, a different K m for p-nitrophenyl butyrate, a different sensitivity to inhibitors, and a different rate of paraoxon hydrolysis suggest that it is a modified esterase. This esterase is not a phosphorotriester hydrolase, nor does it lack nonspecific esterase activity. It is a modified esterase which has a different substrate specificity when compared to the esterases from the other strains. The molecular weight of the esterases studied was approximately 220,000, with pH optima of about 7.0. The ratio of malathion α-monoacid to β-monoacid formation was about 9.0 for the p I 5.1 and 5.3 esterases and 1.5 for the p I 4.8 and 5.6 esterases. The existence of a higher α β ratio for the p I 5.1 and 5.3 esterases and their significant rate of malathion hydrolysis in the Hirokawa strain indicate that an increase in the α β ratio in house flies reported was due to the increase in the p I 5.1 esterase in the resistant strain.

Age-dependent variation in enzymatic and electrophoretic properties of house fly (M. domestica) carboxylesterases

1. The general and carboxylesterase activities of an insecticide resistant (Rutgers) and a susceptible (CSMA) strain of house flies were compared in 1–8 day adults and in last instar larvae. 2. The major difference between the two strains was the markedly higher, approximately 4-fold, carboxylesterase activity of the CSMA strain compared to the Rutgers strain, despite the much higher resistance, about 20-fold, of the latter strain to the organophosphate and carboxylic ester, malathion. 3. Both strains exhibited multiple forms of the esterase, as many as 17 isozymes being detected in the hemolymph during the 8-day period of examination of the adults. However, there was a daily variation in the number of isozymes detected, ranging from 8 to 14 in the CSMA strain and from 12 to 15 in the Rutgers strain. 4. The esterase data are compared with similar data from two other Diptera, the flesh fly and the blow fly.

The characterization by affinity chromatography of glutathione S-transferases from different strains of house fly

Glutathione S-transferases have been partially purified from five strains of house fly of defined resistance to insecticides. Apparent purification factors of 1.8–25 were obtained, the degree of purification depending on the assay substrate employed. Yields of up to 100% were obtained when 3,4-dichloronitrobenzene was used as substrate but with 1-chloro-2,4-dinitrobenzene as substrate the yields were of the order of 20–30%. Qualitative analysis of the proteins in these preparations indicated a high degree of similarity between the Cornell and Hirokawa strains and also a resemblance between the Fc and Rutgers strain. In the Cornell and Hirokawa strains, the bulk of the enzyme activity is attributed to two proteins (constituting a major fraction of the protein in the extracts) which appear to be of different isoelectric point from the proteins associated with enzyme activity in the Rutgers, Fc, and CSMA strains. A minor component in the Hirokawa and Cornell preparations appeared to correspond to the major catalytic protein in the Rutgers extract. On the basis of the above data, it is proposed that the various strains may possess a multiplicity of GSH S-transferases, common to all, individual proteins being present to varying extents in the different strains.

Characterization of Glutathione <i>S</i>-Transferases in Relation to Azinphosmethyl Resistance

A field population of the predaceous mite, Amblyseius fallacis (Garman), was 300, 000 fold resistant to azinphosmethyl. Demethylation of the insecticide by glutathione S-transferase appeared to be the major cause for resistance. Rutgers strain of the housefly was over 1, 600 fold resistant to azinphosmethyl comparing to CSMA strain. The major mechanism of the resistance was again attributed to an increased degradation of the compound by glutathione S-transferase. The enzyme catalysed only GS-methyl conjugation. Soluble fraction from Rutgers strain demonstrated higher activities toward all the substrates tested for glutathione-dependent reactions, i. e., azinphosmethyl, diazinon, methyl iodide, and 1, 2-dichloro-4-nitrobenzene (DCNB), than that from CSMA strain. Various chromatographies, interstrain comparisons, and genetic studies all failed to distinguish enzymes responsible for alkyl and aryl conjugations from each other. Glutathione S-transferase was partially purified from both housefly strains using DCNB as the substrate. A protein band extracted from unstained electrophoretic gels was active for methyl iodide and DCNB indicating both conjugations were catalysed by the same enzyme. The partially purified enzyme was active for several organophosphorus (OP) insecticides as well as for γ-BHC, though it was inactive for dehydrochlorination of p, p′-DDT. The ratio of O-alkyl and aryl conjugations with OP substrates varied depending upon the chemical structure. The enzyme was further purified from another housefly strain to study a possible existence of multiple forms. Results suggested multiple forms of the enzyme obtained upon electrophoresis were not evidence for different enzyme species.

Multiplicity of cytochrome P-450 in microsomal membranes from the housefly, Musca domestica

The heterogeneity of cytochrome P-450 in abdominal microsomes from the CSMA, SBO, Fc, Rutgers and Baygon strains of the housefly was examined by three different methods. Examination of ‘apparent absolute absorption spectra’ indicated at least two types of cytochrome in all strains, one with an absorption maximum at about 394 nm, being present in greater quantity in the insecticide-resistant strains, while the other, with an absorption maximum at about 412 nm, predominates in the insecticide-susceptible strains. Controlled tryptic digestion of microsomes followed by spectral examination at various time intervals indicated a heterogeneous population of cytochromes P-450 in CSMA, Fc and Rutgers strains. Subfractionation of microsomes from houseflies of the CSMA and Fc strains by a two-step discontinuous sucrose gradient centrifugation method provided evidence for cytochromes P-450 of different spectral characteristics. The concentration of cytochrome P-450, as well as its spectral characteristics varied between fractions and strains.

Absorption of Insecticides in Resistant and Susceptible House Flies12

Absorption of DDT, dieldrin, diazinon, parathion, and carbaryl in resistant (Rutgers and Fc) and susceptible (CSMA) strains of Musca domestica L. was studied in adult females, larvae, and puparia. Absorption of all insecticides (except DDT in the Fc strain) in adult flies of the resistant strains was slower than the susceptible strain. In larval and puparial stages no consistent trend of absorption related to resistance was found. Absorption in the CSMA strain was found to be highest in the adult and lowest in the puparial stages.

Reduced glutathione in the housefly: Concentration during development and variation in strains

The amount of glutathione present at various stages of development, body regions, sex and strains of the housefly was determined. An increase in glutathione occurred through the larval, pupal and adult stages. The female had approximately 1.5 times the amount of glutathione per individual as did the male due to the larger size of the female. High glutathione levels were found in the Rutgers and the Cornell-R strains, and lower but approximately equal amounts in DDT-Orlando, 49r 2, Fc and the CSMA strains. These differences were small when compared to the greater differences in the levels of glutathione transferase activity. The glutathione content in various strains of adult housefly was found to be higher than that previously reported in houseflies, other insects, ticks and certain mammalian tissues.

CSMA 系及び RP 系イエバエの殺虫剤抵抗性について(第 11 回大会講演要旨)

CSMA 系及び RP 系イエバエの殺虫剤抵抗性について(第 11 回大会講演要旨)