The biochemical nature of malathion resistance in Anopheles stephensi from Pakistan

Malathion resistance in Anopheles stephensi from Pakistan was synergized by triphenyl phosphate, primarily a carboxylesterase inhibitor. There was a slight degree of antagonism with piperonyl butoxide. The major metabolite of malathion in larvae of both the resistant and susceptible strains was malathion monocarboxylic acid. Resistant larvae produced about twice as much of this product as the susceptible larvae. This suggests that a qualitative or a quantitative change in a carboxylesterase enzyme may be the basis of malathion resistance in this strain. Analysis of general esterase levels to α- and β-naphthyl acetate showed that there was no quantitative change in the amount of carboxylesterase enzyme present in the resistant strain as compared to the susceptible.     

Mosquito larvicidal activity of alkaloids from Zanthoxylum lemairei against the malaria vector Anopheles gambiae

Four alkaloids, 10-O-demethyl-17-O-methylisoarnottianamide 1, 6-acetonyl-N-methyl-dihydrodecarine 2, nitidine 3, and chelerythrine 4 were isolated from the plant Zanthoxylum lemairei (Rutaceae) and evaluated for mosquito larvicidal activity against the malaria vector Anopheles gambiae. The mortalities of the larvae were determined after 24 h. The results of the larvicidal tests demonstrated that compounds 1 and 2 were the most potent with mortality rates of 96.7% and 98.3% at a concentration of 250 mg/L, respectively. Compound 3 was less potent with a mortality of 28.3% at the same concentration. The percent mortality of 100% was observed at a concentration of 500 mg/L. The least potent of the four alkaloids was compound 4, which achieved 100% mortality at 1000 mg/L. These findings could be useful in the research for newer more selective, biodegradable and natural larvicidal compounds or can be used as lead compounds for the development of larvicides.     

Cytochrome P-based resistance mechanism and pyrethroid resistance in the field Anopheles albimanus resistance management trial

The relative rates of cytochrome P⁴⁵⁰ selection in southern Mexican Anopheles albimanus populations were investigated during a 3 years indoor residual house spraying intervention with a pyrethroid (PYR) or DDT, a mosaic of organophosphate (OP)-PYR, and the annual rotation of OP-PYR-carbamate (CARB). This insecticide resistance mechanism, initially evenly spread in the mosquito population, correlated with PYR resistance during the second treated year, when cytochrome P⁴⁵⁰ contents increased in most villages of the PYR, rotation and mosaic schemes. However, by the third year, mean cytochrome P⁴⁵⁰ contents declined to susceptible levels in mosquitoes of the rotation and one mosaic group but not in the PYR-treated villages. In DDT-treated villages, a continuous decrement of cytochrome P⁴⁵⁰ levels occurred since the first treatment year, and susceptible levels were observed at the end of the intervention. Most correlations of cytochrome P⁴⁵⁰ levels and PYR resistance were lost during the third year, indicating that another mechanism evolved in resistant mosquito populations.     

Development of high-throughput real-time PCR assays for the identification of insensitive acetylcholinesterase (ace-1) in Anopheles gambiae

Resistance to the organophosphate and carbamate insecticides through insensitivity of the target site enzyme, acetylcholinesterase has recently been reported in Anopheles gambiae populations in West Africa. To date, screening for the mutation (G119S of the ace-1 gene) conferring this insensitivity has employed a simple PCR-RFLP diagnostic. However, this has the disadvantage of requiring digestion of the amplified fragment and subsequent gel electrophoresis of the products. To overcome this, and thus increase throughput and reduce costs, we have developed two assays based on real-time PCR (TaqMan and melt-curve) that represent true ‘closed-tube’ approaches. The two new platforms were compared to PCR-RFLP to genotype over 280 samples. The two new methods compared favourably with PCR-RFLP with the TaqMan assay delivering the greatest specificity and sensitivity of the three approaches. This assay is also cheaper to run than PCR-RFLP and results are obtained in a single step.     

The biochemistry of insecticide resistance in Anopheles sacharovi: Comparative studies with a range of insecticide susceptible and resistant Anopheles and Culex species

Fourth instar larvae, the progeny from wild-caught Anopheles sacharovi females, were subjected to a number of biochemical tests and the results were compared to those from similar tests on laboratory insecticide resistant and susceptible strains of anopheline and culicine mosquitoes. DDT resistance in An. sacharovi is associated with the ability to rapidly metabolise DDT to DDE. The organophosphorus and carbamate resistance was not associated with quantitative changes in esterases, multifunction oxidases, or glutathione S-transferase. The acetylcholinesterase was less sensitive to malaoxon and propoxur than laboratory susceptible An. albimanus, and plots of inhibition suggest that the population was polymorphic for more than one form of acetylcholinesterase. Metabolism studies on malathion and pirimiphos methyl did not indicate resistance due to increased metabolism. There was no evidence of penetration barriers contributing to resistance to either DDT or malathion, and there was no indication of any resistance to pirimiphos methyl in our tests.     

Inhibition of Penicillium duponti carboxylesterase by the fungicides captan and folpet

Captan, folpet, and perchloromethylmercaptan were effective inhibitors of Penicillium duponti p-nitrophenylpropionate esterase activity (I₅₀ = 0.5 – 2 μM) whereas α-naphthyl acetate esterase activity was not affected by the presence of these compounds. Captan and folpet are both equally effective at pH 7.3 and 8.3. The ionic composition of the medium had strong effects on the degree of inhibition produced by all inhibitors but did not alter esterase activity. Neither succinamide nor phthalimide caused inhibition of the p-nitrophenylpropionate esterase activity: The trichloromethylmercaptan portion of these fungicides appears to be responsible for the observed inhibition. The rapidity of captan and folpet inhibition of esterase activity (complete in < 1 min) compared to the rates of spontaneous decomposition ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{> 1}\text{min}$) and the insensitivity of captan and folpet inhibition to hydrogen ion concentration suggest that generation of spontaneous decomposition products is not required for inhibition. The results are consistent with a mechanism in which the entire fungicide molecule binds to the protein followed by enzyme-promoted reactions of captan and folpet which result in loss of esterase activity.     

华北大黑鳃金龟Holotrichia oblita中肠羧酸酯酶基因 HoCL1的克隆与序列分析

利用粉纹夜蛾(Trichoplusia ni)围食膜蛋白多克隆抗体,从已构建的华北大黑鳃金龟 Holotrichia oblita 中肠cDNA表达文库中筛选得到1个编码羧酸酯酶的cDNA克隆 HoCL1 ,其开放阅读框(ORF)长1 599 bp,编码532个氨基酸,推导的蛋白质分子质量为59.5 kDa,等电点(p I)为4.5。 HoCL1蛋白具有羧酸酯酶的保守结构域:1个二硫键形成的位点和1个丝氨酸活性中心,三联体催化活性中心位于Ser207、Asp333和His422上,不含有氮联糖基化位点和氧联糖基化位点,只含有3 个半胱氨酸残基。依据氨基酸序列同源性分析和保守结构域分析,HoCL1属于B类酯酶,与赤拟谷盗 Tribolium castaneum 羧酸酯酶相似性最高,为35.2%。通过与其他昆虫羧酸酯酶序列比对及构建系统发育树,发现HoCL1羧基端的氨基酸序列保守性低,但靠近N端的活性中心处的氨基酸序列则高度保守,可与赤拟谷盗、异色瓢虫 Harmonia axyridis 羧酸酯酶聚类在一起。羧酸酯酶 HoCL1 基因的克隆鉴定为进一步研究该基因在华北大黑鳃金龟体内的表达及功能奠定了基础。     

Selection of Anopheles stephensi with DDT and dieldrin and cross-resistance spectrum to pyrethroids and fipronil

The field strain of Anopheles stephensi, the main malaria vector in south of Iran, was colonized in laboratory and selected with DDT and dieldrin in two separate lines for 3 generations to a level of 19.5- and 14-fold for DDT and dieldrin resistance, respectively. Synergist tests with chlorofenethol (DMC) and piperonyl butoxide (PBO) on the selected strains indicated that dehydrochlorination and oxidative detoxification might be the underlying mechanisms involved in the resistance to dieldrin and DDT in selected strains. DDT selection decreased susceptibility to DDT and pyrethroids including lambdacyhalothrin, permethrin deltamethrin and cyfluthrin. The result also showed that selection with dieldrin caused negative and positive cross-resistance to pyrethroid and fipronil, respectively. Based on these results, it can be concluded that besides metabolic resistance mechanisms, other factors such as mutation in γ aminobutyric acid (GABA) and voltage-gated sodium channels (Kdr) might be involved.     

Effect of celangulin V on detoxification enzymes in Mythimna separata and Agrotis ypsilon

Celangulin V (CA-V), a β-dihydroagrofuran sesquiterpene polyol ester, is extracted from the root bark of Chinese bittersweet, Celastrus augulatus Maxim. It exhibited selective toxicity against different insects. By CO-difference spectral and biochemical method, the effects of CA-V on two kinds of detoxification enzymes, cytochrome P450 (P450 and NADPH-cytochrome P450 reductase) and glutathione S-transferase, were investigated in oriental armyworm, Mythimna separata and black cutworm, Agrotis ypsilon. CA-V showed higher induction against P450 of M. separata than that of A. ypsilon. Treated by CA-V, the maximum absorption of M. separata increased 1.2 and 0.8 nm than the control, respectively. Meanwhile, compared with the control, the P450 content and NADPH-P450 reductase activity in treated M. separata larvae increased 1.46-, 2.26- and 1.26-, 2.56-fold, respectively. But in treated A. ypsilon larvae, they all increased a little more than those of control. So far as M. separata and A. ypsilon, whether there is exposure of CA-V or not, the P450 content and GST activity in A. ypsilon were obviously higher than those in M. separata. It suggested that the content or activity difference of these two kinds of detoxification enzymes may have important roles in the selective toxicity of CA-V in M. separata and A. ypsilon.     

Frequency detection of pyrethroid resistance allele in Anopheles sinensis populations by real-time PCR amplification of specific allele (rtPASA)

To investigate the level of pyrethroid resistance in Anopheles sinensis Wiedemann 1828 (Diptera: Culicidae), a major malaria vector in Korea, we cloned and sequenced the IIS4-6 transmembrane segments of the sodium channel gene that encompass the most widely known kdr mutation sites. Sequence analysis revealed the presence of the major Leu-Phe mutation and a minor Leu-Cys mutation at the same position in permethrin-resistant field populations of An. sinensis. To establish a routine method for monitoring resistance, we developed a simple and accurate real-time PCR amplification of specific allele (rtPASA) protocol for the estimation of resistance allele frequencies on a population basis. The kdr allele frequency of a field population predicted by the rtPASA method (60.8%) agreed well with that determined by individual genotyping (61.7%), demonstrating the reliability and accuracy of rtPASA in predicting resistance allele frequency. Using the rtPASA method, the kdr allele frequencies in several field populations of An. sinensis were determined to range from 25.0 to 96.6%, suggestive of widespread pyrethroid resistance in Korea.     

绿色木霉对小麦防御酶系的影响

木霉Trichoderma spp.对病原菌拮抗作用的研究较多,而对植物影响的研究则较少.用哈茨木霉处理番茄、莴苣、烟草,能诱导植株对灰霉病的系统抗性,延缓和抑制病斑的形成和扩展[1].木霉能产生某些化合物,激发植物的防卫反应[2].作者研究了木霉菌对植物抗病性的诱导作用.     

不同抗性板栗品种的防御酶系对栗疫菌Cp-毒素的响应

对照.此外,抗病品种酶活性高于中抗品种和感病品种;防御酶的活性与品种抗病性之间呈正相关,毒素作用后防御酶活性的变化可作为衡量板栗抗病性强弱的指标.     

Toxicological effects of oxyfluorfen on oxidative stress enzymes in tilapia Oreochromis niloticus

Several environmental pollutants enhance the intracellular formation of reactive oxygen species, and can lead to the damage of macromolecules and a decrease in oxidant defences levels in fish. The effects of the herbicide oxyfluorfen on the activities of antioxidant enzymes such as catalase, superoxide dismutase, glutathione reductase, and glutathione S-transferase were evaluated in freshwater fish Oreochromis niloticus. These were determined in tilapia liver exposed to sublethal concentrations (0.3 and 0.6 mg/L at 7, 14, and 21 days of exposure. This study also analyzed the effects of oxyfluorfen on the total fatty acid profile. The results showed that CAT activity was higher in tilapia exposed to oxyfluorfen at the sampling days, except at the highest concentration after 21 days. Similarly, the enhancing effect of the herbicide was observed on the GR activity. However, its effect was moderate at the highest dose. On the contrary, fish treated with oxyfluorfen at both doses displayed a decrease in the SOD activity. After 7 days of treatment at both concentrations tilapia showed a significant increase in GST levels, although the enzymatic activity decreased at 14 and 21 days of exposition when compared with the control. The major saturated fatty acids measured in tilapia liver were the palmitic acid (C16:0; 17.9%) and stearic acid (C18:0; 8.7%). The exposure to oxyfluorfen caused a significant increase of the oleic acid (C18:1), whereas the amount of nervonic acid (C24:1) increased at all sampling data. The results of the present study should be taken in account when using tilapia as an environmental indicator species in studies of xenobiotic biotransformation and biomarker response, as well as in monitoring programmes.     

Induction of microsomal enzymes in the southern armyworm (Prodenia eridania)

Levels of microsomal epoxidation, N-demethylation, and cytochrome P-450 in the gut tissues of sixth instar southern armyworm larvae were considerably enhanced following oral in vivo treatment with a series of methylbenzenes. Induction increased with increasing methyl substitution and was maximal with pentamethylbenzene. The increase in microsomal activity occurred rapidly after initiation of treatment and the final levels of induction achieved were dependent on the concentration of the inducer in the diet and the time of exposure. Microsomal enzyme activity returned to control levels following termination of exposure and induction was blocked by puromycin and cycloheximide but not by actinomycin D. The in vivo tolerance of induced worms to orally administered carbaryl was increased in a manner reflecting the enhanced microsomal enzyme activity.     

A carboxylesterase with broad substrate specificity causes organophosphorus,carbamate and pyrethroid resistance in peach-potato aphids (Myzus persicae)

Carboxylesterase E4, the enzyme previously shown to cause resistance to organophosphorus insecticides in peach-potato aphids, Myzus persicae, was purified and characterized by electrophoretic and enzyme kinetic techniques. Its insecticidal substrate specificity, determined by following the rate of recovery of esterase activity after inhibition by a range of acylating inhibitors, included a variety of carbamates and organophosphates, although the catalytic center activity for these substrates was low. Radiometric measurement of hydrolysis of the pyrethroid, permethrin, showed that E4, whether purified or in crude aphid homogenates, hydrolyzed the (1S)trans enantiomer rapidly but hydrolysis of the other three isomers could not be detected. Such absolute specificity for one enantiomer of a pyrethroid is rare. The rates of hydrolysis of the various insecticidal classes correlated well with the relative degrees of resistance to them, and no other resistance mechanisms have been detected. Although the enzyme is relatively inefficient in degrading insecticidal esters, it is produced in very large quantity, accounting for approximately 3% of the total protein in very resistant aphids. Its effect is thus mediated not only by hydrolysis but also by sequestering a substantial proportion of a toxic dose of insecticide. It is effective in this respect because the molar amount present is similar to that of a lethal dose of insecticide. These results support earlier indirect evidence for “overproduction” of E4, probably because of structural gene duplication or amplification and have direct implications for strategies to delay the buildup of resistance or for developing synergists to overcome resistance.     

Induction of branchial enzymes in snake head (Channa striatus) by oxydemeton-methyl

The effects of acute and chronic exposures to a sublethal concentration (1.7 mg/liter) of oxydemeton-methyl (Metasystox) on the activities of branchial enzymes Na, K-ATPase, Mg-ATPase, alkaline phosphatase, acid phosphatase, aspartate aminotransferase, and alanine aminotransferase were determined. While chronic and acute treatments inhibited Na, K-ATPase and Mg-ATPase concentrations, an insignificant rise in the activities of alkaline and acid phosphatases was noticed in the acute treatment. However, both chronic exposures decreased the activities of these phosphatases. A progressive accumulation of AAT and AlAT, with a maximum occurring after 30 days, was recorded. The significance of the results are discussed.     

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.     

Differential influence of herbicide treatments on activity and kinetic parameters of C4 photosynthetic enzymes from Zea mays

The recommended field dose of rimsulfuron, imazethapyr, alachlor, atrazine or fluometuron differentially reduced shoot fresh and dry weight of 10-day-old maize seedlings as well as leaf protein content during the following 12 days. These reductions seemed consistent during the whole period with fluometuron, atrazine and alachlor but appeared to be nullified by the 5th day of treatment with rimsulfuron and imazethapyr. On the other hand, all herbicides mostly provoked significant inhibitions in specific activities of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), malate dehydrogenase (MDH, EC 1.1.1.82), pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) in leaves during the first 2 days. Thereafter, the inhibition was recovered in samples treated with rimsulfuron and imazethapyr, leveled off with alachlor but consistently augmented with atrazine and fluometuron. The kinetic characterization showed that rimsulfuron or imazethapyr unchanged V_max of all enzymes in vitro, however, V_max of PEPC, PPDK and Rubisco were decreased in vivo. Nevertheless, atrazine or fluometuron substantially reduced V_max of all enzymes while alachlor showed a reduction in this value of PEPC, MDH and Rubisco. Thus atrazine, fluometuron and, to a lower extent, alachlor reduced concentrations of all enzymes as well as rimsulfuron and imazethapyr for only Rubisco. On the contrary, K_m values of all enzymes were progressively increased by all herbicides indicating that the different herbicides altered the structural integrity of all enzymes. These findings conclude that rimsulfuron or imazethapyr competitively inhibited MDH but revealed mixed inhibition to PEPC, PPDK and Rubisco. Atrazine or fluometuron revealed mixed inhibitions to all enzymes whereas alachlor seemed to be either a competitive inhibitor to PPDK or a mixed inhibitor to PEPC, MDH and Rubisco.     

Evidence that the elevated carboxylesterase (esterase 2) in organophosphorus-resistant Myzus persicae (Sulz.) is identical with the organophosphate-hydrolyzing enzyme

An enzyme hydrolyzing methylparaoxon in vitro in an organophosphorus-resistant strain of the peach potato aphid (Myzus persicae Sulz.) is present in the same electrophoretic fraction as a carboxylesterase (esterase 2) which has previously been shown to have characteristically increased activity in organophosphorus resistant strains of this aphid.No in vitro organophosphate hydrolysis was found in a susceptible strain with low carboxyl-esterase-2 activity. Carboxylesterase-2 and the methylparaoxon-hydrolyzing enzyme are both inhibited by n-propylparaoxon but not by methyl- and ethylparaoxon. This indicates that the two enzymes are identical.