Effect of organophosphates in vivo upon acetylcholinesterase isozymes from housefly head and thorax

Four soluble acetylcholinesterase isozymes of head and three from the thorax of housefly (Musca domestica L.) were separated by polyacrylamide gel electrophoresis. Their inhibition in vivo was studied after poisoning with an LD₅₀ of four organophosphates: malaoxon, paraoxon, diazinon and dichlorvos. The isozymes differed greatly in their degree of inhibition. Thoracic isozymes were found to be more sensitive to inhibition than head isozymes. In surviving flies, the recovery rates of head isozymes were much faster than thoracic isozymes. In vitro studies showed that thoracic isozymes differed 4.1- and 2.9-fold in their K_m and V_max.     

Esterase isoenzymes and insecticide resistance in Frankliniella occidentalis populations from the south-east region of Spain

BACKGROUND: Frankliniella occidentalis (Pergande) is among the most important crop pests in the south‐east region of Spain; its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. To this end, F. occidentalis populations, collected from the field at different locations in south‐east Spain, were studied in terms of total esterase activity and esterase isoenzyme pattern. RESULTS: Individual thrips extracts were analysed by native polyacrylamide gel electrophoresis (PAGE) and stained for esterase activity with the model substrate α‐naphthyl acetate. Significant correlations were found between resistance to the insecticides acrinathrin and methiocarb and the presence of a group of three intensely stained bands, named Triplet A. For each individual thrips extract, total esterase activity towards the substrates α‐naphthyl acetate and α‐naphthyl butyrate was also measured in a microplate reader. Insects possessing Triplet A showed a significantly higher α‐naphthyl acetate specific activity and α‐naphthyl acetate/α‐naphthyl butyrate activity ratio. This observation allowed a reliable classification of susceptible or resistant insects either by PAGE analysis or by total esterase activity determination. CONCLUSION: The PAGE and microplate assays described can be used as a monitoring technique for detecting acrinathrin‐ and methiocarb‐resistant individuals among F. occidentalis field populations. Copyright © 2008 Society of Chemical Industry     

鱼类肝脏酯酶的同工酶构成及其对磷酸三苯酯和马拉氧磷的敏感性

用聚丙烯酰胺凝胶电泳法分离了麦穗鱼 (Pseudorasbora p arva)、金鱼 (Carassius auratus)、尼罗罗非鱼 (Tilapja nilotica)、食蚊鱼 (Gambusia affinis)、虹鳟 (Salmo gairdneri)等五种鱼的肝脏酯酶的同工酶 ,以乙酸α-萘酯为底物测定了它们的活性。在麦穗鱼、金鱼、尼罗罗非鱼体内发现两条酶带 ,在食蚊鱼和虹鳟体内发现一条酶带。离体抑制率实验表明 ,五种鱼肝脏内均含有对磷酸三苯酶敏感的酶带 ,而对马拉氧磷敏感的酶带只存在于麦穗鱼、金鱼、尼罗罗非鱼体内。酯酶同工酶分布型的意义及与马拉硫磷选择毒性的关系在本文中进行了讨论。     

Reactivity In vitro toward substrate and inhibitors of acetylcholinesterase isozymes from electric eel electroplax and housefly brain

Five acetylcholinesterase isozymes from electric eel eletroplax (Electrophorus electricus) and four from housefly brain (Musca domestica L.), were separated by polyacrylamide gel electrophoresis combined with specific substrate staining and gel scanning procedures. The K_m values for acetylthiocholine varied over a 2.4-fold range for eel and a 4.2-fold range for housefly. The sensitivities to inhibition also varied: for eel, the range was 2.2-fold for malaoxon, 2.6-fold for Tetram and 2.1-fold for eserine. The corresponding values for housefly were 2.3, 1.5, and 1.9.     

羊草发育过程中同工酶的研究

采用聚丙烯酰胺凝胶电泳法 ,对羊草不同物候期不同器官的酯酶和过氧化物酶同工酶的多态性研究结果表明 :羊草酯酶同工酶活性在地上与地下营养器官间存在明显的互补性 ;过氧化物酶活性与有性生殖器官的发育有一定的一致性。总的来看 ,过氧化物酶同工酶与羊草的生长发育的关系较酯酶密切 ,可以作为生长发育特性评价较好生理指标 ;酯酶的稳定性较强 ,标志性酶带较恒定 ,可以作为遗传鉴定的指标。     

Partial purification and characterization of a methyl-parathion resistance-associated general esterase in Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

Increased hydrolytic metabolism of organophosphate insecticides has been associated with resistance among Nebraska western corn rootworm populations. In this study, resistance-associated esterases were partially purified by differential centrifugation, ion exchange, and hydroxyapatite column chromatography, with a final purification factor of 100-fold and recovery of approximately 10%. Kinetic analysis of the partially purified enzyme indicated that the K_m of the group II esterases was identical for the two populations, although V_max was consistently threefold higher in the resistant population. A putative esterase, DvvII, was further purified to homogeneity by preparative polyacrylamide gel electrophoresis. DvvII is a monomer with a molecular weight of approximately 66 kDa, although three distinct isoforms with similar pIs were evident based on isoelectric focusing gel electrophoresis. Immunoassays with the Myzus persicae E4 antiserum indicated that group II esterases from D. v. virgifera were cross-reactive and expressed at much higher titers in the resistant population relative to the susceptible counterpart. These results suggest that the resistance is likely associated with overproduction of an esterase isozyme in resistant D. v. virgifera populations.     

Communication to the Editor Insecticide Resistance in a Strain of Aphis gossypii from Southern France

A strain (R) of Aphis gossypii from Southern France was found to be resistant to several insecticides, particularly to pirimicarb, as compared to a susceptible strain (S). Resistance levels were determined by biological tests, and the highest resistance factor (1350) was for pirimicarb. Resistance was mainly restricted to anticholinesterase inhibitors. Use of synergists, DEF and PB, suggested that resistance mechanisms based on detoxification were involved to a minor extent, since a good correlation was observed between I₅₀ values and k_i values of AChE and in-vivo bioassay data. The two strains differed in esterase activity, with a 27·7-fold increase in the R strain. Resolution of esterases by polyacrylamide gel electrophoresis showed different patterns in the S and R strains, and two isozymes were less sensitive to pirimicarb in the S strain; however, no in-vitro degradation of [¹⁴C]pirimicarb was observed. These data suggest that the main mechanism of resistance was through a decrease in the sensitivity of the target, AChE, to the insecticides. © 1997 SCI.     

Biochemical Characterization of Carboxylesterase in the Small Brown Planthopper Laodelphax striatellus (Fallén)

Several forms of carboxylesterase were observed in a malathion-resistant small brown planthopper, Laodelphax striatellus Fallén, by isoelectrofocusing. To study the mechanisms of increased esterase activity, esterases were purified and their biochemical characteristics were investigated in five active esterase isozymes of two resistant strains. These esterases have polymorphic characteristics and their molecular weights ranged from 66 to 70 kDa, due to variations in glycosylation. The pI values of these esterases ranged from 5.3 to 4.7. These esterases were immunologically related and NH₂-terminal amino acids were identical in all isozymes regardless of pI or molecular weight. No differences have been found in kinetic parameters (K_m and V_max) to α-naphthylacetate and specific activity toward α-naphthylacetate and malathion as a substrate in all isozymes. Resistant individuals showed high ali- and malathion carboxylesterase activities and these enhancements were caused by quantitative differences of carboxylesterases with several different pI.     

Characterizations of general esterases in relation to malathion susceptibility in two field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen)

General esterases were characterized and compared from two populations of the oriental migratory locust, Locusta migratoria manilensis, collected from Huanghua and Pingshan Counties, Hebei Province, China. General esterases were most concentrated in the thorax and abdomen, which contained 46.1 and 36.1% of total esterase activity in females, and 42.7 and 36.0% in males, respectively, when α-naphthyl acetate was used as a substrate. There was no distinct difference in esterase banding patterns in different body regions for the substrates α-naphthyl acetate and β-naphthyl acetate on non-denaturing polyacrylamide gel electrophoresis (PAGE). However, the general esterase activities in the Huanghua population were 1.8-fold higher than those in the Pingshan population in both females and males. Increased esterase activity in the Huanghua population appeared to be mainly due to several additional esterase bands detected on non-denaturing PAGE. Inhibition studies of general esterases using four inhibitors, including paraoxon, malaoxon, eserine, and carbaryl, indicated that most general esterases in the two populations were B-type. The increased esterase activity in the Huanghua population appeared to be associated with a 1.8-fold decreased susceptibility to malathion. Such differences may attribute to the difference in control practices for the locust between Huanghua and Pingshan Counties.     

Resistance to bifenthrin and resistance mechanisms of different strains of the two-spotted spider mite (Tetranychus urticae) from Turkey

Nine different strains of the two-spotted spider miteTetranychus urticae Koch (Acari: Tetranychidae) were collected on cotton from Adana, Antalya, Izmir, Manisa and Urfa in Turkey. Their responses to bifenthrin were investigated using conventional bioassay and biochemical assays. LC₅₀ and LC₉₀ values of bifenthrin were determined for all strains by using a residual bioassay with a petri dish-spray tower. Resistance ratios were determined by comparing the samples with a standard susceptible strain, GSS. The resistance ratios of the strains ranged from <1 to 669-fold (at LC₅₀). Of the investigated field strains, only three (two from Adana and one from Urfa) were resistant to bifenthrin. There was a correlation between esterase enzyme activity and bifenthrin resistance according to polyacrylamide gel electrophoresis and microtiter plate assays in the three resistant strains. http://www.phytoparasitica.org posting May 17, 2005.     

Increased activity associated with reduced sensitivity of acetylcholinesterase in organophosphate-resistant greenbug,schizaphis graminum (homoptera: aphididae)

The activity and sensitivity of acetylcholinesterase (AChE, EC 1.1.1.7) from an organophosphate-susceptible (OSS) and three resistant (OR-0, OR-1 and OR-2) strains of the greenbug (Schizaphis graminum) were biochemically compared. All three resistant strains displayed higher frequencies of individuals with respect to both increased activity of AChE toward the model substrate acetylthiocholine (ATC) and reduced sensitivity of AChE to inhibition by paraoxon as compared with the OSS strain. Kinetic study indicated that AChE from the OR-0, OR-1 and OR-2 strains had 3.3-, 2.7- and 2.3-fold, respectively, lower affinity, but 1.5-, 2.2- and 2.0-fold, respectively, higher catalytic activity toward ATC than AChE from the OSS strain. Significantly increased activity of AChE in the resistant strains was also confirmed by non-denaturing polyacrylamide gel electrophoresis, and appeared to be associated with the increase of general esterase activity. Inhibition kinetics revealed that AChE from the OR-0, OR-1 and OR-2 strains was 2.1-, 2.2- and 2.7-fold less sensitive to inhibition by paraoxon than that from the OSS strain. The study suggested that both qualitative and quantitative modifications of AChE had evolved in the resistant strains and were likely to significantly enhance the overall resistance level in greenbugs. © 1999 Society of Chemical Industry     

赖草发育过程中同工酶的研究

利用聚丙烯酰胺凝胶电泳技术 ,对赖草酯酶和过氧化物酶同工酶研究结果表明 :赖草不同发育期的酯酶和过氧化物酶同工酶酶谱及酶活性存在明显的器官间差异和多态性 ;其中过氧化物同工酶与赖草生长发育的关系比酯酶相对密切 ,同时稳定性也较强 ,可以作为赖草生长发育特性的生理指标和遗传鉴定指标     

Isolation and partial characterization of polypeptides associated with phytotoxin in cultures of the cool-temperature biotype of Stemphylium botryosum pathogenic on alfalfa

Stemphylium botryosum pathogenic on alfalfa (Medicago sativa) produced a phytotoxin in a defined liquid medium which caused symptoms similar in sequence and appearance to stemphylium leafspot lesion development on attached alfalfa leaflets. The molecular weight of the phytotoxin was estimated to be 19500 by gel filtration chromatography. Treatment of partially purified phytotoxin with proteinase K or subtilisin resulted in loss of phytotoxic activity. Action of the proteases in inducing loss of phytotoxicity was inhibited by phenylmethylsulphonyl fluoride. Concentrated culture filtrates were fractionated by chromatofocusing, HPLC ion-exchange and gel filtration. Gel filtration also was followed by non-denaturing electrophoresis or isoelectric focusing (IEF). Analysis of sequential fractions from each procedure by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that phytotoxic activity was consistently coincident with the presence of two major polypeptides (M_r = 26900 ± 2%; 19500 ± 4%) and a triplet of polypeptides (M_r = 15500). The constant association between the three polypeptides suggested an intermolecular interaction during fractionation. The phytotoxic activity eluted from the chromatofocusing column between pH 5·79 and 5·43 whereas IEF estimated the isoelectric point to be between 5·40 and 5·02.Immobilized concanavalin A and wheat germ lectin failed to bind phytotoxicity. Proteolytic activity in some phytotoxic gel filtration fractions was reduced by addition of phenyl-methylsulphonyl fluoride and separated from phytotoxicity by non-denaturing electrophoresis, indicating that phytotoxicity was not due to serine protease activity.Pathogenicity tests of the cool-temperature biotype on 20 plant species representing eight plant families resulted in a compatible interaction only on M. sativa and M. polymorpha. In contrast, sensitivity to the phytotoxin was observed on both pathogen-resistant and susceptible alfalfa clones along with all other plants tested with the exception of Triticum aestivum, Zea mays, Apium graveolens and Melilotus officinalis, indicating non-host-specificity of the phytotoxic polypeptides produced by the cool-temperature biotype.     

Resistance to chlorpyrifos and lambda-cyhalothrin along with detoxifying enzyme activities in field-collected female populations of European red mite

The levels of susceptibility of populations of the European red mite Panonychus ulmi (Koch) (Acarina: Tetranychidae) collected from apple orchards in the Bursa region of Turkey to the insecticides chlorpyrifos and lambda-cyhalothrin, were determined by a petri leaf disk—Potter spray tower method. When compared with the susceptible population, resistance ratios at the LC₅₀ level ranged from 6.0- to 35.6-fold, and from 0.7- to 5.7-fold for chlorpyrifos and lambda-cyhalothrin, respectively. Kinetic parameters of general esterase activity with α-naphthyl acetate as substrate indicated that an increased activity was present in the resistant populations compared with the susceptible populations. In these strains, 1.5- and 2.2-fold higher Glutathione S-transferase (GST) activity was also detected with the substrate 1-chloro-2,4-dinitrobenzene. General esterase activity gel profiles of these populations were studied by native polyacrylamide gel electrophoresis, but no relationship between resistance ratios and band patterns was detected. The results of this study document a decreased efficacy of chlorpyrifos and lambda-cyhalothrin in field populations of P. ulmi in Turkey, possibly linked to altered activities of esterases and GST.     

栗疫病菌表型特征的遗传分析

 本文对栗疫病菌的菌落形态、α-酯酶、β-酯酶及营养体亲和性(VC)特征进行了遗传分析。对有性后代的38个单子囊孢子菌株的研究结果显示:亲本菌株366与68的菌落形态、β-酯酶中(采用聚丙烯酰胺凝胶电泳法)相对迁移率为2.17和2.33的2条酶带的遗传分别由1个位点上的2个等位基因控制;α-酯酶中相对迁移为2.54与2.772条酶带的遗传由相互连锁的2个基因位点控制;α-酯酶与β-酯酶间存在完全连锁关系;菌株366与68之间至少存在4个VC基因差异。     

Characterization of a Trans-permethrin hydrolyzing enzyme from the midgut of Pseudoplusia includens (Walker)

The enzymatic hydrolysis of trans-permethrin by midgut homogenates of the soybean looper, Pseudoplusia includens (Walker), was characterized through gel electrophoresis, isoelectric focusing, gel filtration chromatography, and selective inhibition. All three separation techniques indicated that one enzyme (or closely related enzyme forms) was responsible for the observed hydrolytic activity. The molecular weight was approximately 80,000, the isoelectric point ranged from pH 4.6 to 4.8, and the apparent K_m was 59.5 ± 3.2 μM. Enzyme activity was inhibited by organophosphates, carbamates, and sulfhydryl group inhibitors, as well as some chelators. The hydrolysis of trans-permethrin was distinct from the majority of “general esterase” activity when enzyme activity was separated by electrophoretic techniques.     

Association of three RNA molecules with potato mop-top virus

Summary Potato mop-top virus particles, purified from systemically infectedNicotiana benthamiana plants and then disrupted by heating with sodium dodecyl sulphate and 2-mercaptoethanol, contained only a single polypeptide of M_r 19 100 detectable by polyacrylamide gel electrophoresis. Single-stranded RNA preparations from virus particles, when subjected to electrophoresis in an agarose gel containing methylmercuric hydroxide as a denaturant, were shown to contain approximately equal proportions of three RNAs of 6.5, 3.2 and 2.5 kb. Double-stranded RNA preparations extracted from systemically infectedN. benthamiana leaves or from locally infectedN. debneyi leaves, was shown by polyacrylamide gel electrophoresis to contain a major species of 3.2 kbp and two minor species of 6.5 and 2.4 kbp.     

星豹蛛猎物甜菜夜蛾电泳检测的初步研究

应用聚丙烯酰胺凝胶电泳技术,对星豹蛛(Pardosa astrigera)捕食猎物甜菜夜蛾(Laphygma exigua)前后酯酶同工酶的变化进行了电泳分析。结果表明:星豹蛛捕食猎物前后其酯酶同工酶在数目、迁移率及活性方面均有明显区别;应用电泳方法研究捕食者和猎物之间的关系,具有简便、直接且灵敏度高等优点。     

Biochemical studies on sheep body louse Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae): comparison of pyrethroid and organophosphate resistant and susceptible strains

Strains of sheep louse Bovicola ovis (Schrank) with various levels of resistance to pyrethroid and one strain with high degree of resistance to organophosphate (OP) insecticides were used to investigate the biochemical mechanisms of insecticide resistance, i.e., enhanced levels of general esterases, specific acetylcholinesterases (AChE), glutathione S-transferase (GST), and mixed function oxidases. Native gel electrophoresis combined with quantitative enzyme assays showed analogous expression profiles of several esterase isozymes in all the strains tested. The determination of the sensitivity of each esterase isozyme to five inhibitors (acetylthiocholine iodide, butyrylthiocholine iodide, paraoxon eserine sulfate, and pCMB) led to the identification of nine esterases in the B. ovis strain. Gel electrophoresis results are supported by enzyme assay studies where, except for the OP resistant strain, no differences in esterase activities were detected in all the pyrethroid resistant and susceptible strains assayed. Statistical analyses demonstrated that some strains have elevated GST activities compared to the susceptible reference strain.     

Development of microsatellite markers and their effectiveness in Lolium temulentum

Screening a genomic library of Lolium temulentum (darnel) with synthetic (GT)₁₅ and (GA)₁₅ oligonucleotides, we identified microsatellite‐containing clones. Based on the sequence data of the clones, specific primer pairs were synthesized and their effectiveness examined using 10 L. temulentum and two L. persicum accessions from Asia, Africa and Europe. Fifteen primer pairs amplified single fragments, and five of them showed polymorphism in polyacrylamide gel electrophoresis in the L. temulentum and L. persicum accessions. Fifteen primer pairs reported in L. perenne (perennial ryegrass) were also applied in this study. Nine of them amplified the microsatellite alleles, and two of the nine showed polymorphic alleles in L. temulentum and L. persicum. For the polymorphic markers, polymorphic information content values ranged from 0.60 to 0.86 with an average of 0.76 among 10 L. temulentum accessions. The microsatellite markers developed for L. temulentum and L. perenne should be informative and powerful in analysing the intraspecific genetic diversity and the phylogenetic relationships of L. temulentum and L. persicum.