Gut-associated Microbiota Diversity Of The White Pine Beetle (dendroctonus Armandi) And Cellulolytic Microbial Community In Its Larval Gut

The Chinese white pine beetle, Dendroctonus armandi Tsai and Li, is arguably the mostdestructive forest insect in the Qinling Mountains in Northern China. Little is known aboutthe structure of the microbial communities associated with D. armandi, even though thiswood-boring insect plays important roles in ecosystem and biological invasion processes thatresulted in huge economic losses in pine forests. In this work, nested PCR-DGGE, clonesequencing, ARDRA, SDS-PAGE and native-PAGE were employed to investigate thecomposition and the roles of the microbial communities present in the guts of D. armandi atdifferent developmental stages. The main points of this dissertation are listed as follows:1. Analysis of PCR-amplified16S rRNA gene fragments of bacteria from the guts of D.armandi larvae, pupae, as well as male and female adults revealed low complexity of bacterialcommunities which differed according to the developmental stage. Citrobacter spp. andPantoea spp. predominated in larvae and adults, whereas Methylobacterium was the dominantgenus at the pupal stage (56%). The main difference between the guts of male and femaleadults was the greater dominance of Citrobacter in females (80%). These results suggestedthat the bacterial community associated with D. armandi guts may influence the developmentof insect.2. Analysis of PCR-amplified18S rRNA gene fragments of fungi from the guts of D.armandi larvae, pupae, male and female adults and their galleries in Pinus armandi revealedlow complexity of fungal communities which differed according to the developmental stage.Yeast of the genus Candida and filamentous fungal Ophiostoma predominated in guts andgalleries of D. armandi. In particular, Candida accounted for56%in the pupal stage, whichwas related to lipolysis and might support pupae physiological activities. The fungalcommunity in D. armandi guts represented one of the factors that may interfere with insectdevelopment.3. A total of91cellulolytic bacteria were isolated and assigned to11genotypes usingamplified ribosomal DNA restriction analysis (ARDRA) in the intestine of D. armandi larvae.Partial16S rDNA sequence analysis and morphological tests were used to assign the11 representative isolates. The results showed that the isolates belonged to α-Proteobacteria,γ-Proteobacteria and Firmicutes. Members of γ-Proteobacteria were the most frequentlyrepresented species and accounted for73.6%of all the cellulolytic bacteria. The cellulolyticbacteria in D. armandi larva gut were mainly identified as Serratia, accounted for49.5%,followed by Pseudomonas, which accounted for22%. In addition, members of Bacillus,Brevundimonas, Paenibacillus, Pseudoxanthomonas, Methylobacterium and Sphingomonaswere found in the D. armandi larva gut. Brevundimonas kwangchunensis, B. vesicularis, M.populi and P. mexicana were firstly reported to be cellulolytic in this study.4. Only one type of cellulolytic fungus was isolated and identified as Leptographiumqinlingensis by Partial ITS rDNA sequence analysis and morphological tests.5. The laccase secreted by the most important symbiotic fungus of D. armandi, L.qinlingensis, are involved in lignin degradation to provide utilizable nutrition for D. armandi,and catalyze some biochemical reactions, causing the damages of tree tissue. In present study,the purity of the laccase was confirmed by SDS-PAGE and native-PAGE and the apparentmolecular mass was61.0kDa. The catalytic efficiencies Kmand Vmaxdetermined by substrateguaiacol were15.4μM and372.9U/mg, respectively. Moreover, it was found that1mM and10mM Cl-, SO42-, NO3-showed no significant influence (p>0.05) on the laccase activity,while,100mM acid radical ions resulted in significant inhibition (p<0.05) on the enzymeactivity except for100mM SO42-(p>0.05).The data obtained in this study, regarding the phylogenetic relationships and thecommunity structure of intestinal microbia at different developmental stages of the D.armandi life cycle, will contribute to our understanding on D. armandi and the biology role ofthe intestinal microbia, which might provide an opportunity and theoretical basis to promoteintegrated pest management (IPM) of D. armandi.