Mutations in acetylcholinesterase genes of Rhopalosiphum padi resistant to organophosphate and carbamate insecticides.

Apple grain aphid, Rhopalosiphum padi (Linnaeus), is an important wheat pest. In China, it has been reported that R. padi has developed high resistance to carbamate and organophosphate insecticides. Previous work cloned from this aphid 2 different genes encoding acetylcholinesterase (AChE), which is the target enzyme for carbamate and organophosphate insecticides, and its insensitive alteration has been proven to be an important mechanism for insecticide resistance in other insects. In this study, both resistant and susceptible strains of R, padi were developed, and their AChEs were compared to determine whether resistance resulted from this mechanism and whether these 2 genes both play a role in resistance. Bioassays showed that the resistant strain used was highly or moderately resistant to pirimicarb, omethoate, and monocrotophos (resistance ratio, 263.8, 53.8, and 17.5, respectively), and showed little resistance to deltamethrin or thiodicarb (resistance ratio, 5.2 and 3.4, respectively). Correspondingly, biochemistry analysis found that AChE from resistant aphids was very insensitive to the first 3 insecticides (I50 increased 43.0-, 15.2-, and 8.8-fold, respectively), but not to thiodicarb (I50 increased 1.1-fold). Enzyme kinetics tests showed that resistant and susceptible strains had different AChEs. Sequence analysis of the 2 AChE genes cloned from resistant and susceptible aphids revealed that 2 mutations in Ace2 and 1 in Ace1 were consistently associated with resistance. Mutation F368(290)L in Ace2 localized at the same position as a previously proven resistance mutation site in other insects. The other 2 mutations, S329(228)P in Ace1 and V435(356)A in Ace2, were also found to affect the enzyme structure. These findings indicate that resistance in this aphid is mainly the result of insensistive AChE alteration, that the 3 mutations found might contribute to resistance, and that the AChEs encoded by both genes could serve as targets of insecticides.     

Effects of some organophosphate and carbamate insecticides on nitrification and legume growth

Summary Nine insecticides (six organophosphates and three carbamates) were tested for their effects on soil nitrification, growth of legume seedlings, and growth of four species of rhizobia bacteria. No inhibition of nitrification was found at normal field rates (5 ppm) of application. Some instances of inhibition were observed at 50 ppm and at 500 ppm. Similarly, 5 ppm applications did not inhibit growth of alfalfa or sweetclover seedlings ... with one exception. Disc inhibition tests of the rhizobia bacteria showed thatRhizobium leguminosarum andRhizobium trifolii were most sensitive to the pesticides.Rhizobium meliloti, and particularlyRhizobium japonicum, were resistant to the insecticides. No consistent correlation was observed between tests on the nodulating bacteria and the tests on legume growth.Published with the approval of the Director of the North Dakota Agricultural Experiment Station as Journal Article No.309. Portion of a thesis presented by the senior author in partial fulfillment of the requirements for the M.S. degree in bacteriology at North Dakota State University.     

Mechanism of inhibition of cyclo-oxygenase in human blood platelets by carbamate insecticides.

Carbamates are a widely used class of insecticides and herbicides. They were tested for their ability to affect human blood platelet aggregation and arachidonic acid metabolism in platelets. (1) The herbicides of the carbamate type have no, or only little, influence up to a concentration of 100 microM; the carbamate insecticides, however, inhibit both aggregation and arachidonic acid metabolism in a dose- and time-dependent manner. (2) Carbaryl, the most effective compound, inhibits platelet aggregation and cyclo-oxygenase activity completely at 10 microM. The liberation of arachidonic acid from phospholipids and the lipoxygenase pathway are not affected, whereas the products of the cyclo-oxygenase pathway are drastically decreased. (3) By using [14C]carbaryl labelled in the carbamyl or in the ring moiety, it could be proved that the carbamyl residue binds covalently to platelet proteins. In contrast with acetylsalicylic acid, which acetylates only one protein, carbaryl carbamylates a multitude of platelet proteins. (4) One of the carbamylated proteins was found to be the platelet cyclo-oxygenase, indicating that carbaryl resembles in this respect acetylsalicylic acid, which is known to inhibit this enzyme specifically by acetylation.     

苏云金杆菌预处理小菜蛾对有机磷和氨基甲酸酯杀虫剂的增效作用

研究了小菜蛾Plutella xylostella幼虫经苏云金杆菌Bacillus thuringiensis预处理后,对有机磷和氨基甲酸酯杀虫剂敏感性的变化以及预处理对小菜蛾幼虫体内乙酰胆碱酯酶、羧酸酯酶、谷胱甘肽S-转移酶和谷胱甘肽的含量的影响。结果表明：苏云金杆菌预处理抗性小菜蛾幼虫后,其对甲胺磷、水胺硫磷和克百威的敏感性分别为未处理组的6.74、8.83和8.50倍;处理敏感小菜蛾幼虫后则分别为未处理组的2.96、1.69和3.88倍。苏云金杆菌预处理抗性小菜蛾,未处理组乙酰胆碱酯酶的K_m和V_max值分别为预处理组的1.86和1.56倍,所使用的6种杀虫剂对乙酰胆碱酯酶的K_I值,处理组为未处理组的1.80～2.66倍,苏云金杆菌预处理抗性小菜蛾对羧酸酯酶的K_m、K_I影响不大,但能显著地抑制羧酸酯酶和谷胱甘肽S-转移酶的活性并导致谷胱甘肽含量下降(对照分别为处理的2.02、1.76和1.66倍)。苏云金杆菌预处理敏感小菜蛾,对乙酰胆碱酯酶的K_m、V_max、K_I值和羧酸酯酶的K_m、K_I值以及谷胱甘肽含量影响不大,但能显著地抑制羧酸酯酶和谷胱甘肽S-转移酶的活性(对照分别为处理的1.54和1.64倍)。     

Hydrolysis of organophosphate insecticides by an immobilized-enzyme system.

An enzyme preparation that could detoxify parathion and eight other organophosphate pesticides was covalently bound to either porous glass or porous silica beads. This immobilized-enzyme system was examined for its use in detoxification of pesticides in production wastewaters. The kinetics of parathion hydrolysis were examined at flow rates up to 96 liter/hr and at influent substrate concentrations ranging from 10--250 mg/liter. The enzyme reactor was able to hydrolyze 95% or more of the parathion added to industrial wastewaters generated during its production, thus reducing the effluent parathion concentration to below 500 ppb. Laboratory continuous-flow experiments were conducted for 70 days with industrial wastewater and indicated no loss in immobilized-enzyme activity. The influence of pH, temperature, solvents, and detergents on enzyme stability and activity and enzyme reactor kinetics will be discussed.     

Determination of organophosphorous and carbamate insecticides by flow injection analysis.

A flow injection system, incorporating an acetylcholinesterase (AChE) single bead string reactor (SBSR), for the determination of some organophosphorous (azinphos-ethyl, azinphos-methyl, bromophos-methyl, dichlorovos, fenitrothion, malathion, paraoxon, parathion-ethyl and parathion-methyl) and carbamate insecticides (carbofuran and carbaryl) is presented. The detector is a simple pH electrode with a wall-jet entry. Variations in enzyme activity due to inhibition are measured from pH changes when the substrate (acetylcholine) is injected before and after the passage of the solution containing the insecticide. The percentage inhibition of enzyme activity is correlated to the insecticide concentration. Several parameters influencing the performance of the system are studied and discussed. The detection limits of the insecticides ranged from 0.5 to 275 ppb. The determination of these compounds was conducted in Hepes buffer and a synthetic sea water preparation. The enzyme reactor can be regenerated after inhibition with a dilute solution of 2-PAM and be reused for analysis. The immobilized enzyme did not lose any activity up to 12 weeks when stored at 4 degrees C.     

Identification of isoenzymes in cholinesterase preparations using kinetic data of organophosphate inhibition

Commercial preparations of acetylcholinesterase (EC 3.1.1.7) and of cholinesterase (EC 3.1.1.8) were characterized by organophosphate inhibition. Cholinesterase activities were inhibited by varying organophosphate concentration and time of inhibition. Bimolecular rate constants were determined by plotting log activity vs inhibitor concentration or inhibition time. Inhibition of acetylcholinesterase from bovine erythrocytes by diethyl p-nitrophenyl phosphate (Paraoxon), diisopropylphosphorofluoridate (DFP), and N,N′-diisopropylphosphorodiamidic fluoride (Mipafox) in semilogarithmic plots showed a linear decay of activity. Inhibition of acetylcholinesterase from electric eel (Electrophorus electricus) and of cholinesterases from horse serum and from human serum did not show linear characteristics, indicating the presence of more than one single enzyme in these preparations. The corresponding inhibition curves were resolved by subtraction of exponential functions. In each case two different activity components were identified and characterized in respect to partial activity, substrate specificity, and reactivity with organophosphorous compounds. The suitability of the method for application on crude homogenates is discussed.     

Determination of organophosphate and carbamate resistance allele frequency in diamondback moth populations by quantitative sequencing and inhibition tests

A quantitative sequencing (QS) protocol was established for predicting the frequencies of the A298S and G324A mutations in the diamondback moth (Plutella xylostella) type-1 acetylcholinesterase (AChE) locus, putatively involved in organophosphate (OP) and carbamate (CB) insecticide resistance. The nucleotide resistant signal ratio at each mutation site was generated from sequencing chromatograms and plotted against the corresponding resistance allele frequency. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were highly correlated with resistance allele frequencies (r² > 0.987). QS analysis of 15 representative regional field populations of DBM in Korea revealed that the allele frequencies of both A298S and G324A were over 70% in most field populations, implying the prevalent state of these resistance-associated mutations. In the AChE inhibition assay, all populations showed reduced sensitivity to paraoxon, DDVP, carbaryl, and carbofuran, supporting the notion that DBM resistance to OPs and CBs is widespread in Korea.     

Novel carbamate cholinesterase inhibitors that release biologically active amines following enzyme inhibition

Conjugation of the phenol derived from rivastigmine with amphetamines gave access to novel carbamate cholinesterase inhibitors. All compounds possessed increased affinity and selectivity for AChE compared to rivastigmine and were orally bioavailable. Compound 4a, incorporating d-amphetamine, caused significant inhibition of cholinesterase in vivo at doses that were well tolerated. Release of amphetamine from 4a was demonstrated following in vitro and in vivo inhibition of cholinesterase. Compound 4a was also effective in alleviating scopolamine induced amnesia in a rat passive avoidance model.     

The inhibition and reactivation of human maternal and fetal plasma cholinesterase following exposure to the organophosphate, dichlorvos.

Spontaneous and chemically-induced reactivation of organophosphate-inhibited cholinesterase were studied using as an enzyme source plasma obtained from non-pregnant females, pregnant females at term and their respective neonates, sampled immediately following delivery. Aliquots of plasma were incubated with dichlorvos (10^?6M) for 5 min at 37°C resulting in a 96 percent inhibition of cholinesterase activity in all three groups at which time either pralidoxime chloride (10^?3M) or an equivalent volume of saline was added to the reaction flask and the restoration of cholinesterase activity was monitored over the next 120 min. Pralidoxime-mediated cholinesterase reactivation in ‘non-pregnancy’ plasma was significantly greater than that observed in either ‘maternal’ or ‘fetal’ plasma, however, no significant difference was noted in reactivation rates for these latter two groups. Significant differences were also observed in the rates of spontaneous reactivation, however, after correcting for this, there were still significant differences in the rates of pralidoxime-mediated reactivation (non-pregnant > pregnant ≥ fetal).     

Detection of organophosphate and carbamate pesticides in vegetable samples by a photothermal biosensor

Previously developed photothermal biosensor was optimised by determining the most suitable enzyme substrate (acetylthiocholine iodide) and the optimal carrier buffer (0.05 M phosphate buffer, pH 8.0). Excitation laser operating at 488 nm and 120 mW power provided the highest biosensor sensitivity. The biosensor was tested for detection of toxic organophosphate and carbamate compounds present in samples of salad, iceberg lettuce, and onion. Sufficient sensitivities to different pesticides (carbofuran, propamocarb, oxydemeton-methyl and parathion-ethyl) were achieved without time-consuming sample preparation procedures. The results show good agreement with the concentrations of pesticides determined with standard GC-MS detection method. The developed photothermal biosensor offers new low cost means to detect low concentrations of pesticides in vegetable samples with high throughput and little or no sample pretreatment.     

有机磷类杀虫剂对美洲斑潜蝇的防治效果

在山东青州以西葫芦为试验害虫寄主 ,测定了毒死蜱、三唑磷、乙酰甲胺磷、辛硫磷、马拉硫磷对美洲斑潜蝇幼虫LiriomyzasativaeBlanchard的田间防效。毒死蜱 (2 4g (a .i.) 667m2 )、三唑磷 (1 0g (a.i.) 667m2 )、乙酰甲胺磷 (2 0g (a .i.) 667m2 )第 3～ 7d校正防效为 97 0 9%～ 97 1 4% ,86 80 %～87 0 4% ,85 99%～ 86 2 5 %。此 6个处理药效顺序为毒死蜱 >三唑磷 >乙酰甲胺磷 (2 0g(a.i.) 667m2 )>乙酰甲胺磷 (1 5g(a.i.) 667m2 ) >辛硫磷 >马拉硫磷。在河南洛阳以甘蓝为试验害虫寄主 ,乙酰甲胺磷 (1 5g(a.i.) 667m2 )、敌敌畏 (2 5g(a.i.) 667m2 )、敌百虫 (4 5g(a .i.) 667m2 )对美洲斑潜蝇幼虫第 7d校正防效分别为 76 98% ,82 42 % ,71 1 1 %。毒死蜱对在不同寄主上美洲斑潜蝇的防效均较好     

Cyclic acyl guanidines bearing carbamate moieties allow potent and dirigible cholinesterase inhibition of either acetyl- or butyrylcholinesterase

A series of cyclic acyl guanidine with carbamate moieties have been synthesized and evaluated in vitro for their AChE and BChE inhibitory activities. Structure−activity relationships identified compound 23 as a nanomolar and selective BChE inhibitor, while compound 32 exhibited nanomolar and selective AChE inhibition, selectivity depending on both the structure of the carbamate substituent as well as the position of guanidines-N substitution. The velocity of enzyme carbamoylation was analyzed and showed similar behavior to physostigmine. Phenolic compounds formed after carbamate transfer to the active site of cholinesterases showed additional neuroprotective properties on a hippocampal neuronal cell line (HT-22) after glutamate-induced intracellular reactive oxygen species generation.     

Serum cholinesterase inhibition by boronic acids

Horse serum cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) was reversibly inhibited by a variety of alkyl- and areneboronic acids with Ki values ranging from 6.2 mM (methaneboronic acid) to 3.1 microM (diphenylboric acid). Binding to the enzyme was apparently at the active center, because inhibition obeyed competitive kinetics and because boronic acids protected the enzyme from inactivation by phenylmethanesulfonyl fluoride. Boronic acids should prove useful in probing the active center of serum cholinesterase.     

Characterization of plasma cholinesterase activity in the Eurasian Griffon Vulture Gyps fulvus and its in vitro inhibition by carbamate pesticides

The legal and illegal use of organophosphorus and carbamate pesticides represents one of many threats to birds. The activity of the cholinesterase enzyme in plasma is used as a non‐destructive biomarker to diagnose the exposure of birds to these pesticides. Scavengers are one of the most important bird groups threatened by the use of baits poisoned with anticholinesterase pesticides. Knowledge of the characteristics of this enzyme in each bird species is crucial, as several studies indicate that more than one cholinesterase form may be present in the plasma of birds. In this study, cholinesterase activity was characterized in the plasma of the Eurasian Griffon Vulture Gyps fulvus by using several substrates and inhibitors of the enzyme, and its normal activity value was also determined. The in vitro sensitivity of Gyps fulvus plasma cholinesterase to carbamate insecticides (aldicarb, carbaryl and methomyl) was also investigated. The results indicated that propionylthiocholine iodide was the preferred substrate to determine plasma cholinesterase activity, followed by acetylcholine iodide and S‐butyrylcholine iodide, and acetylcholinesterase was the predominant enzymatic activity in Gyps fulvus plasma. Aldicarb was the most potent in vitro inhibitor of plasma cholinesterase activity in this species. However, cholinesterase enzymatic activity was significantly inhibited by all tested carbamates, providing further evidence that this biomarker is a suitable tool to monitor the exposure to these poisons in the field, highlighting its utility in conservation programmes.