Effects of Terpinen-4-ol on Four Metabolic Enzymes and Polyphenol Oxidase (PPO) in Mythimna separta Walker

To study insecticidal mechanism of terpinen-4-ol, a main insecticidal composition in the essential oil of Sabina vulgaris, the 5th instar larvae of Mythimna separta, were investigated with terpinen-4-ol by topical application. The activities of phosphatase, glutathione S-transferase （GSTs）, cytochrome P450 （P450）, and polyphenol oxidase （PPO） of tested insects were determined in all poisoning stages, including exciting stage, convulsing stage, paralysis stage, and recover stage. The result showed that the activities of both acid phosphatase （ACP） and alkaline phosphatase （AKP） in treated insects were induced by terpinen-4-ol, but ACP was inhibited in paralysis stage. The activities of GSTs were inhibited in exciting stage, convulsing stage, and paralysis stage, but gradually recovered in recover stage. O-demethylase activity of cytochrome P450 was inhibited by terpinen-4-ol, and the inhibition rate in all poisoning stages were 26.27, 46.03, 80.24, and 90.22%, respectively. PPO activities were strongly inhibited by terpinen-4-ol both in vitro and in vivo. In conclusion, the activities of P450, GSTs, and PPO could have relation with toxicity of terpinen-4-ol against larvae of the Mythimna separta, but recover stage of the poisoning insects might be related to GSTs induced. As a new insecticide or synergist, terpinen- 4-ol has a potential value in field of insecticide resistance management.

Bacterial Expression and Purification of an Active ω-Atracotoxin-Ar1b from Spider Atrax robustus

The ω-atracotoxin-Arlb toxin （ω-ACTX-Arl b） is one of the arthropod-selective peptide neurotoxins from the venom of Australian funnel-web spider Atrax robustus. The gene of Arlb was synthesized and cloned into pET-32a（＋） vector to allow expression of Arlb as a fusion protein with thioredoxin and the His-tag （rTrx-Arlb） in E. coli BL21 （DE3）. The optimal condition for inducing the expression ofrTrx-Arl b was 1.0 mmol L-1 IPTG for 6 h at 28℃. The fusion protein rTrx- Arlb was expressed in soluble form and was purified effectively by HisTrap HP affinity column and rpHLPC and a final yield of purified rTrx-Arlb was 95 mg from 1 000 mL E. coli culture. The LD50 values for Mythimna separate and Tenebrio molitor were 111.66 and 11.04 ug g-1 determined by injection of the purified rTrx-Arlb. The results indicated that the recombinant Arlb protein was successfully expressed in E. coli and it was high toxicity against tested insects.