Cloning And Expression Response Of The Detoxifying Enzyme Genes From Chinese White Pine Beetle(Dendroctonus Armandi)

The Chinese white pine beetle(Dendroctonus armandi)is one of the most arguably destructive forest insect in the Qinling Mountains,Shaanxi,China to Chinese white pine(Pinus armandi).This native pest often reaches epidemic proportions and causes widespread mortality of trees in both natural and managed forest ecosystems.Bark beetles use enzyme metabolism and physiological and biochemical effects of symbiotic fungi and themselves to overcome the protogenesis and induced resistance of host trees,thus completing the survival and life cycle in the trunk tissue of host trees.Bark beetles can not only detoxify the host tree secondary terpenoids through detoxification enzyme genes,but also participate in the synthesis of pheromones and hormones in insects.In this study,D.armandi was taken as the research object.Through the phylogenetic development of the glutathione S-transferase(GSTs)and carboxylesterase(Car Es)genes of D.armandi.Research the expression of GSTs and Car Es gene in different development stages of D.armandi and to study the expression differences of GSTs gene in different tissues;the expression response of GSTs and Car Es gene to the volatile substance of D.armandi;the differences of body mass,energy stores and detoxication enzymes activity among different generations were also studied.The aim is to reveal the gene expression of the main detoxication enzymes and the mechanism of overcoming the resistance of the host.The results are as follows:1.The percentage of male in the early season was 45.79%,and 39.70% in the late season.Furthermore,patterns of body mass,energy stores,and detoxication enzymes were found to differ between the two sexes and two seasons.Compared with the males,the females have a larger body mass and higher amounts of stored lipids.2.Sixteen full-length sequences of the GSTs family were obtained by cloning of D.armandi,belonging to the four GSTs categories of delta,epsilon,sigma and theta.The Da GSTs gene of D.armandi and its GSTs have high homology with those of other insects according to multiple sequence alignment analysis.The clone obtained eight Car Es genes from D.armandi,Da Car E3,Da Car E5 and Da Car E6 belong to the cytosolic Car Es,Da Car E1,Da Car E2,Da Car E4,Da Car E7 and Da Car E8 belong to the secretory proteins.The phylogenetic tree showed that the eight Da Car Es were not closely related to the identified Car Es of the Lepidoptera and Diptera insect.3.The expression levels of GSTs gene was significant differences among the different developmental stages and after feeding on host phloem in sexes.The GSTs gene was upregulated in females and downregulated in males,which may be related to the first invasion of females to the host trees to overcome the terpenes defensive substances of the host trees.All Da GSTs were upregulated in male antennae,indicating that GSTs genes were involved in information transmission and odor metabolism of D.armandi.In addition,GSTs gene was upregulated in females after feeding on host phloem,indicating that GSTs gene was involved in the metabolism of terpenes to the host by D.armandi.4.There are significant differences in transcript expression levels of Car E genes amongst the different development stages of D.armandi.Da Car E1-Da Car E4 present stable transcript levels in different development stages,and Da Car E5-Da Car E8 mainly changed the transcript levels from mature larvae to teneral adults.Statistically significant differences were not found between sexes in emerged adults of Da Car E1-Da Car E7.This may be related to the special functions performed and the specific behaviors involved.The transcriptional level of Car E gene in half of the females and most of the males was significantly different after feeding on the phloem of the host.5.Differential transcript levels of the detoxification enzyme genes were observed in adult beetles after feeding on different types of feed.Significant differences were observed between two types of hemi-natural feeds with the phloem of pines,and pure artificial feed containing five stimuli,(-)-?-pinene,(-)-?-pinene,(+)-3-carene,(±)-limonene and turpentine oil,also caused differential transcript levels in the detoxification enzyme genes.This indicates that the main detoxifying enzyme gene of D.armandi has a specific expression response mechanism to the P.armandi.