Study On The Relationship Between γ-aminobutyric Acid And The Acaricidal Activity Of Abamectin In Tetranychus Cinnabarinus(Boisduval)

The carmine spider mite Tetranychus cinnabarinus is one of the most serious pests in economical crops including cottons and vegetables. Control of the mites mainly relies on chemicals spray. As an acaricide, abamectin shows excellent efficacy on the pests because of broad pesticidal spectrum, high efficacy, low residue and being safe to humans and animals. Previous studies showed the γ-aminobutyric acid（GABA） content was significantly higher in abamectin resistant strains（AbR） than that in susceptive strains（SS）. GABA is the most important inhibitory neurotransmitters in the central nervous system of vertebrates and invertebrates.A change of GABA amount in vivo is the results of a balance between GABA biosynthesis and degradation, in which glutamic acid decarboxylase（GAD） and GABA-transaminase（GABA-T） are involved, respectively. Another study showed the acitivity of GABA-T in AbR was weaker than that in SS. However, the GAD activity had no difference between two strains. Based on the previous results, the relationship between GABA content and the acaricidal activity of abamectin in T. cinnabarinus was studied.The main results were as follows:1. Two GAD genes and two GABA-T genes were cloned by RT-PCR and PCR. Genes were then named as GAD^TC1, GAD^TC2, GABA-T^TC1 and GABA-T^TC2, with GenBank accession numbers of JQ942360, JQ798396, JQ942361, and JQ942362 respectively. Amino acids of above genes showed high identity with Tetranychus urticae. The expression characteristics of these genes were detected via the comparative quantitative real-time PCR（qPCR）. Results showed there were no differences between SS and AbR regarding the expression levels of GAD genes. But the mRNA expression of two GABA-T genes were significantly down regulated in the AbR compared with that in SS, which was consistant with the results of enzyme activities tests. This indicates that the molecular mechanism of high level GABA in the AbR was the result of the down-regulated expression of GABA-T genes.2. As the specific inhibitor of GABA-T, antiepileptic drug, vigabatrin, was used to improve the GABA concentration endogenously to study the relationship between GABA and the acaricidal activity of abamectin in T. cinnabarinus. In vitro vigabatrin inhibiting experiments showed that activity of GABA-T in T. cinnabarinus was decreased with the increase of vigabatrin concentrations, with an inhibition concentration（IC50） of 0.443 mM. In vivo, the activity of GABA-T could also be inhibited by vigabatrin.3. The results of HPLC quantification showed that the GABA amount in T. cinnabarinus significantly increased after treated with vigabatrin for 4h in vivo, compared with the contrast. While the amount recovered to almost the normal level 8h after treatment. Additionally, with abamectin spray on mites bodies, the GABA content in SS also increased after 4h of treatment. But it remained in high level during the next 4h. This showed that abamectin could stimulate GABA release in T. cinnabarinus and the release of GABA last longer than vigabatrin treatment. On the other hand, if the GABA content was pre-induced with vigabatrin for 4h, the subsequent treatment of abamectin could not increase the content of GABA any more. Bioassay results showed that when the endogenous GABA was increased in SS with vigabatrin exposure, the resistance of SS against abamectin increased 4.1-folds, which further demonstrated high GABA amount could reduce the acaricidal effect of abamectin.4. The RNA interference method by feeding dsRNA of GABA-T gene for T. cinnabarinus was used to increase the GABA content endogenously. Results showed that the expression of GABA-T genes decreased about 33% after feeding with GABA-T-dsRNA and the GABA content increased with significance accordingly, which revealed that inhibiting the expression of GABA-T by RNAi method could also result in higher GABA amounts in T. cinnabarinus. Besides, behavioral changes were observed that those mites treated with GABA-T silencing moved slower than the control mites. Additionally, when the expression of GABA-T genes were suppressed via RNAi, the mortality of T. cinnabarinus decreased about 12% and 18% with exposure of LC30 and LC50 of abamectin, respectively compared with control. These results revealed that the tolerance of T. cinnabarinus against abamectin could be improved after increasing the GABA level by RNAi.5. The exogenetic increase of GABA by spray also made the SS mites move more slowly than control. Moreover, when doses of abamectin were 6.25, 25 and 100μM, the mortality was decreased compared with control and the tolerance of T. cinnabarinus treated by exogenetic GABA against abamectin was significantly increased within a certain range of doses and durations.