| To search natural products for new insecticidal compositions is one of primary and effective ways to develop novel pesticides and overcome or postpone resistance. Tripterygium wilfordii Hook is an important resource to product botanical pesticides. The alkaloid from T. wilfordii had been proved to be the main insecticidal compositions by Research and Development Center of Biorational Pesticide, Northwest A&F University. To further veritify the insecticidal activities, mode of action and bioactive spectra of the total alkaloid from T. wilfordii, the toxicity to several standard insects were investigated under the laboratory conditions. Toxic symptom, pathological changes of tested insects and effect on some important physiological and biochemical criterions were carried out. Optimum extracting technology of the total alkaloid in the root bark of T. wilfordii with ethanol was studied. The total alkaloid had been formulated to emulsifier concentration (EC) by the way of combining researches in physical and chemical characters with laboratory bioassay and field trials. The main results and conclusions were given as follows:1. The total alkaloid from T. wilfordii had more modes of action and high toxicity to many pests. It would be prospective in the IPM. The total alkaloid had strong antifeedant effect on Plutella xylostella and Mythimna separate larvae. The antifeedant rate of third instar larvae of P. xylostella treated with 750 mg·L-1 were above 90%. The AFC50 values (24 h and 48 h) against third, fourth and fifth instars larvae of M. separate were 37.92, 50.23, 119.53 mg·L-1(24 h) and 42.39, 60.47, 122.91 mg·L-1(48 h), respectively. The LC50 values were 228.28, 307.61, 306.39 mg·L-1 and 653.00 mg·kg-1(48h) and 136.67, 238.18, 216.12 mg·L-1 and 463.08 mg·kg-1(72 h) against third instar larvae of P. xylostella, fifth instar larvae of Pieris rapae and third instar larvae of M. separate with oral application, respectively. The LC50 values were 435.90, 622.17 mg·L-1(48 h) and 309.67, 485.36 mg·L-1(72 h) against wingless adults of Myzus persicae, female adults of Tetranychus viennensis, respectively. It was also toxic to Musca domestica adults. The ND50 value against fifth instar larvae of M. separata was l35.36 ug·g-1. It imposed great influence on the growth of M. separata larvae. The treated third instar larvae showed big reduction on body weight, weight gain and relative growth rate comparing with the control. In the second day of treatment with the dose of 60 mg·L-1, the body weight,weight gain and relative growth rate reduced 26.13%, 42.74% and 22.26%, respectively. It could prolong the instar stage, reduce the survival rate and pupation rate of M. separate larvae. The emergence rate, fecundity and longevity of adults were lower than that of the control. It also imposed great influence on the growth of third instar larvae of H. armigera. It had a strong population inhibition effect on S. zeamais. The total alkaloid had no obvious touch toxicity to P. xylostella, Pieris rapae and M. separate larvae.2. Different kinds of insects showed similar toxic symptom to the total alkaloid. When fifth instar larvae of M. separata were fed with the total alkaloid, the vivo symptom was characterized by a definite sequence of events, starting with moving slowly, stopping feeding, flaccid paralysis and recovering (or died). Anabiotic insects started feeding, were paralyzed, recovered (or died) again and up to death. It was presumed the total alkaloid from T. wilfordii influenced the nerve system of insects.3. Neural enzymes and neurotransmitters in poisoned larvae of M. separate were assayed. The results showed AChE was not strongly inhibited or activated by the total alkaloid form T. wilfordii and ACh content wasn't significantly different with that of the control. Theγ-aminobutiric acid (GABA) content raised by 89.86%, 49.28% and 20.29% and the glutamate (Glu) content raised by 24.55%, 23.33% and 8.13% in weak, deep paralysis stages and recovery stage, respectively. The activities of glutamate-alanime transminase were remarkably inhibited by the total alkaloid and the activities of glutamic decarboxylase did not change. The activities of Na+,K+-ATPase and Ca2+,Mg2+-ATPase in the brain of tested larvae were inhibited, and those in the midgut did not change. Combining the changes of neural enzymes and neurotransmitters with the symptom, it was presumed that nerve-muscle junction was the target of the total alkaloid from T. wilfordii.4. Effect of the total alkaloid from T. wilfordii on the skeletal muscle in sixth instar larvae of M. separata were studied by means of microphotographics and biochemical analysis. Abnormal changes of skeletal muscles in poisoned larvae were found with optical microscope. It was observed with electric microscope that ongoing pathological changes occurred in the skeletal muscular cells of poisoned larvae in weak, deep paralysis stages. Organelles and inner membranes were disrupted. Z-line dispersed and sarcomeres disordered in myofibril, mitochondria became swollen, vacuolated and cristae disordered and disintegrated. The sarcoplasmic reticulum expended. The results suggested the sarcolemma and inner membrane of the muscular cells might be influenced by the total alkaloid from T. wilfordii.5. Midgut cells and their organelles of poisoned larvae exhibited distinct pathological changes. The microvilli of column cells were disordered and decreased markedly. The mitochondria had irregularly enlarged, the bilayer membrane was not intact, the cisternae of rough endoplasmic reticulum were distended; the cavity of goblet cell was enlarged, in which the microvilli came off. The results of biochemical analysis showed the activities of protease, amylase and lipase of the poisoned larval midgut had no significant changes compared with that of the control. It was suggested that the total alkaloid from T. wilfordii might act on the midgut cells and make the toxin easily enter the hemocoel and finally come to the target.6. In order to study its insecticidal mechanism and judge its prospects, the effects of the total alkaloid from T. wilfordii on the activities of esterase and its isozyme, carboxyl esterase(CarE), acid phosphatase (ACP) and alkaline phosphatase (ALP), glutathione S-transferase (GSTs) and the O-demethylase activity of cytochrome P450 in fifth instar larvae of M. separate were assayed. The results showed that esterase and CarE in poisoned insects were distinctly activated. The changes of esterase isozyme are the same as that of esterase. The activities of ACP and ALP were remarkably inhibited in tested insects by the total alkaloid expect ALP in 3 h. The activities of GSTs had distinctly activated in 3, 12, 24 h, and were equal to that of the controll in 36 h and 48 h, but was inhibited in 48 h by the total alkaloid. The O-demethylase activity of cytochrome P450 had no distinct change in 3 h, was activated in 12, 24 h, but did not differ from the control in 36, 48 h. The results showed its inhibition on phosphatase activities was probably related to the toxicity of the total alkaloid from T. wilfordii to tested larvae and it was a defending action for insects that some metabolic enzymes were activated by the total alkaloid from T. wilfordii.7. The extracting technology of the total alkaloid in the root bark of T. wilfordii. with ethanol was studied. The extraction rate of the total alkaloid was observed by method of orthogonal design. Combination artificial neural network with traditional orthogonal experiment, a new method for analyzing, processing and optimizing the test data was applied By using this new method, the optimum extraction condition of the total alkaloid from root bark of T. wilfordii was obtained as follows: root bark powder 30 mesh, pH value 2.5, ethanol concentration 65%, solid-liquid ratio 1:10, duration of extraction 2 h and extracting 2 times. Applying this extracting technology, the costs of production reduced, the energy consuming decreased, and total alkaloid content increased. The satisfactory results showed that this new method was reasonable and practicable.8. The total alkaloid in the root bark of T. wilfordii had been formulated to emulsifier concentration (EC) by the way of combining researches in physical and chemical characters with laboratory bioassay and field trial. The solvent and emulsifier which suited 1.0% T. wilfordii alkaloid EC had been screened out and successfully developed. Its quality index met the standard of the commercial pesticide. The LC50 values of the sample were 69.62 and 20.35 mg·L-1(48 h) against third instar larvae of P. xylostella and 93.18 and 44.62 mg·L-1(72 h) against fifth instar larvae of P. rapae, respectively. The control efficacy of the formulation diluted 250 and 500 times against P. xylostella and P. rapae were 82.93% and 82.71% after 7 d trial, respectively. It was safe to crops at the tested dosage. 1.0% T. wilfordii alkaloid EC possesses excellent insecticidal activities to two species of vegetable pests, so it would have good development and application prospects.
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