Induction Of Acetylcholinesterase By Sublethal Doses Of Anticholinesterase Agents In Helicoverpa Armigera (Hübner)

Induction of acetylcholinesterase (AChE) by sublethal doses of anticholinesterase agents in susceptible strain (SS-H) of cotton bollworm Helicoverpa armigera (HUbner) was investigated in lab. And AChE was partial purified from the heads of the SS-H strain by affinity cliromatography followed by gel filtraton. Southern blot analysis of genomic DNA of cotton bollworm was done using a 296-bp AChE cDNA fragment generated by RT-PCR as a homologous probe from susceptible strains, RR-Y population and three strains treated by sublethal doses of anticholinesterase agents. The details were as following:1. Effects of induction by sublethal doses of anticholinesterase agents (LD10) on toxicity of 5 insecticides had studied at 24 hours after treatments in the SS-H strain. The changes of susceptibility of cotton bollworm larvae to phoxim, malathion, methomyl, deltamethrin and p-cypermethrin was significantly difference with treatments of three anticholinesterase agents.2. The time-course study revealed that AChE specific activity of SS-H strain was moderately inhibited in 144 hours after treatment with LDioof phoxim and increased with induction of sublethal doses of malathion and methomyl. The sublethal dose of phoxim had no effect on AChE affinity to the substrate analogue, ATCh, in 48 hours after treatment, however AChE affinities were significantly reduced by induction with LD10 of malathion and methomyl. There was no obvious difference among Km values of AChE in different times after treatments, while the Km was significantly increased during 72-120 hours in the cotton bollworm untreated.3. The dosage-effect of induction confirmed that AChE specific activity of larvae was inhibited by different doses of phoxim. But AChE activity was increased after firstly and then decreased depended on the increasing doses of malathion and methomyl. The effect of induction by methomyl was stronger than by malathion with the same dosage.4. The specific activity and the Fmax value of AChE were increased after firstly and then decreased with induction of sublethal dose of phoxim over 4 generations. There were not significant changes in the specific activity and Km value of AChE after induction by LD10 of malathion and methomyl over two generational. All treatments of sublethal doses of anticholinesterase agents depressed the growth and development of cotton bollworm larvae.5. The toxicity of cotton bollworm to phoxim, malathion and methomyl were compared by topical application between the susceptible strains (SS-N, SS-H) and resistant population (RR-Y) collected from field. The results showed that the level of resistance of RR-Y to three anticholinesterase agents was 7-, 13-and 20-fold respectively comparison with SS-N and SS-N strain was more sensitive to three anticholinesterase agents than SS-H strain.There was no significant difference in the specific activity and the Vmax value of AChE between resistant and susceptible strains, whereas Km of the enzyme from the RR-Y strain was 3-fold higher than that from susceptible strains using ATCh as substrate. A cDNA encoding part of AChE gene was clonedfrom three strains by the method of RT-PCR amplification and there was one nucleotide acid different between RR-Y and susceptible strains, which resulted in no amino acid mutation. This part of AChE gene was used as a probe to perform Southern blot, results showed that there was no gene amplification in resistant cotton bollworm. Southern blot also indicated that there was no gene amplification in cotton bollwortn treated by three sublethal doses of anticholinesterase agents and other induction mechanisms may be in existence.6. Sucrose density gradient centrifugation showed that there was significant different distribution of AChE activity in sample gradient, 5% gradient and 20% gradient with the treated by phoxim, methomyl and malathion respectively at 24 h after induction of sublethal doses of anticholinesterase agents. The AChE sensitivity of different isofonns was investigated from...