Expression and characterization of a codon-optimized butyrylcholinesterase for analysis of organophosphate insecticide residues

Organophosphate insecticide residues on vegetable, fruit, tea and even grains are primary cause of food poisoning. Organophosphate compounds can cause irreversible inhibition of the activity of acetylcholinesterase and butyrylcholinesterase（BChE, EC 3.1.1.8）, which are both candidates for rapid detection of organophosphate pesticides. To develop an easy-tohandle method for detecting organophosphate pesticides using BChE, BChE from human was optimized according to the codon usage bias of Pichia pastoris and successfully expressed in P. pastoris GS115. The codon-optimized cDNA shared 37.3% of the codon identity with the native one. However, the amino acid sequence was identical to that of the native human butyrylcholinesterase gene（h BCh E） as published. The ratio of guanine and cytosine in four kinds of bases（（G＋C） ratio） was simultaneously increased from 40 to 47%. The recombinant hBChE expression reached a total protein concentration of 292 mg m L^–1 with an activity of 14.7 U m L^–1, which was purified 3.2×10^3-fold via nickel affinity chromatography with a yield of 68% and a specific activity of 8.1 U mg^–1. Recombinant hBChE was optimally active at pH 7.4 and 50°C and exhibited high activity at a wide pH range（〉60% activity at pH 4.0 to 8.0）. Moreover, it had a good adaptability to high temperature（〉60% activity at both 50 and 60°C up to 60 min） and good stability at 70°C. The enzyme can be activated by Li^＋, Co^＋, Zn^2＋ and ethylene diamine tetraacetic acid（EDTA）, but inhibited by Mg^2＋, Mn^2＋, Fe^2＋, Ag^＋ and Ca^2＋. Na^＋ had little effect on its activity. The values of h BChE of the Michaelis constant（Km） and maximum reaction velocity（Vm） were 89.4 mmol L^–1 and 1 721 mmol min^–1 mg^–1, respectively. The bim olecular rate constants（K_i） of the hBChE to four pesticides were similar with that of electric eel AChE（EeAChE） and higher than that of horse BChE（HoBChE）. All vlues of the half maximal inhibitory concentration of a substance（IC50） for hBChE were lower than those for HoBChE, but most IC50 for hBChE were lower than those for EeAChE except dichlorvos. The applicability of the hBChE was further verified by successful detection of organophosphate insecticide residues in six kinds of vegetable samples. Thus, hBChE heterologously over-expressed by P. pastoris would provide a sufficient material for development of a rapid detection method of organophosphate on spot and produce the organophosphate detection kit.

Indoor Toxicity of Emamectin Benzoate and Chlorantraniliprole Against Spodoptera litura and Their Residues on Lotus Plants

Spodoptera litura is the most threatening pest in lotus production,seriously affecting the lotus yield and quality.Emamectin benzoate and chlorantraniliprole,the main insecticides for controlling S.litura on vegetables,are widely used by farmers to control S.litura on lotus plants.To determine the application concentrations,control effects,and safety of the two insecticides in lotus fields,indoor experiments were conducted to determine the control effects of 200 g/L chlorantraniliprole(SC)and 5%emamectin benzoate(WDG),and the residues of the two insecticides in the water,lotus leaves,and lotus seeds after field application were determined by HPLC-MS/MS.The indoor experiment results showed that chlorantraniliprole and emamectin benzoate both had good control effects on S.litura,with the median lethal concentrations(LC50)of 17.700 and 1.694 mg/L,respectively.After unmanned aerial vehicle spraying of emamectin benzoate at 20 g/667m^(2),there was no residue of emamectin benzoate in the water or lotus leaves after 5 d.After spraying of chlorantraniliprole at 20 mL/667m^(2),the residual amounts in the water and lotus leaves after 9 d were 0.005 and 0.007 mg/L,respectively.No residue of the two insecticides was detected in lotus seeds(dry and fresh)2 h after spraying.Therefore,it was recommended that chlorantraniliprole and emamectin benzoate can be used to control S.litura in lotus fields during the growth period,while attention should be paid to the application interval for safety.Considering the safe harvesting of lotus seeds and leaves,it was recommended that the preharvest intervals of chlorantraniliprole and 5%emamectin benzoate should be 9 d.

The integrated use of chemical insecticides and the entomopathogenic nematode, Steinemema carpocapsae （Nematoda： Steinernematidae）, for the control of sweetpotato whitefly, Bemisia tabaci （Hemiptera： Aleyrodidae）

The integration of chemical insecticides and infective juveniles of the entomopathogenic nematode Steinernema carpocapsae （Wesier） （Nematoda： Steinernematidae）, to control second instars of the sweetpotato whitefly, Bemisia tabaci Gennadius （Hemiptera： Aleyrodidae） was investigated. Using a sand bioassay, the effects of direct exposure of S. carpocapsae for 24 h to field rate dilutions of four insecticides （spiromesifen, thiacloprid, imidacloprid and pymetrozine） on infectivity to Galleria mellonella larvae were tested. Although all chemicals tested, except spiromesifen, produced acceptable nematode infectivity rates, they were all significantly less than the water control. The effect of insecticide treatment （dry residues of spiromesifen, thiacloprid and pymetrozine and soil drench of imidacloprid） on the efficacy of the nematode against B. tabaci was also investigated. Nematodes in combination with thiacloprid and spiromesifen gave higher B. tabaci mortality （86.5% and 94.3% respectively） compared to using nematodes alone （75.2%） on tomato plants. There was no significant difference in B. tabaci mortality when using the chemicals imidacloprid, pymetrozine and spiromesifen in conjunction with nematodes compared to using the chemicals alone. However, using thiacloprid in combination with the nematodes produced significantly higher B. tabaci mortality than using the chemical alone. The integration of S. carpocapsae and these chemical agents into current integrated pest management programmes for the control of B. tabaci is discussed.

Diatomaceous earths as alternatives to chemical insecticides in stored grain

Diatomaceous earth （DE） is a natural inert dust used to control insect pests in stored grain as an alternative to synthetic residual insecticides. Various DE formulations are now registered as a grain protectant or for structural treatment in many different countries throughout the world. The mode of action of DE is through the absorption of cuticular waxes in the insect cuticle, and insect death occurs from desiccation. The main advantages of using DE are its low mammalian toxicity and its stability. The main limitations to widespread commercial use of DE are reduction of the bulk density and flowability of grain, irritant hazards during application and reduction in efficacy at high moisture contents. This paper is an updated review of published results of researches related to the use of DEs and discusses their potential use in large-scale, commercial storage and in small scale applications.