Molecular Diversity Of Bacterial Coummunity In The Gut Of Holotrichia Parallela (Coleoptera: Scarabaeidae)

The root-feeding larva of Holotrichia parallela is a important plant pest in china and has caused significant economic damage.Diversity research of the intestinal bacterial community of H.parallela can explore intestinal bacteria on the impact of the physiological function and ecological significance of H.parallela,and understand coevolution between H.parallela,and its symbiotic bacteria,provide new knowledge for species evolution ways.Finally,these bacteria could be a potential way of reseach for new white grub control methods and reduce the damaged caused by it.In this study,the bacterial communities in different larval stages larvae gut,and wild geographic populations of H.parallela were analysed by Denaturing Gradient Gel Electrophoresis(DGGE)of amplified 16S rRNA sequences,and the results were as follows:35 different DGGE profiles of bacteria clones were obtained from the midgut of all three instars larvae of H.parallela.With sequence similarities and phylogenetic tree analyzing,seven major phyla of bacteria were observed.These bacteria respectively belong to Deltaproteobacteria,Actinobacteria, Betaproteobacteri,Alphaproteobacteria,Gammaproteobacteria,Clostridia and Bacteroidetes.In all three instar larvae,Gammaproteobacteria and Clostridia which were the preponderant bacterium in the midgut represented 80%of the total clones.Furthermore,In the midgut of first instar larvae,we have obtained five phyla of bacteria only,which were Betaproteobacteria,Deltaproteobacteria, Gammaproteobacteria,Clostridia and Bacteroidetes,In second instar larvae,Actinobacteria as a new phylum was separated with all the other 5 phyla found in first instar larve,accounted for 3.33%of the total bacteria in midgut.Alphaproteobacteria was isolated in the midgut of the third instar larvae. These results showed that the amount and proportion,as well diversity of bacteria in H.parallel larva were increasing as the insect growthing.57 different DGGE profiles of bacteria clones were obtained from the hindgut of all three instar larvae of H.parallela.With sequenceing similarities and constructing phylogenetic tree analyzing,eight major phyla of bacteria were observed as follow:Deltaproteobacteria,Betaproteobacteria, Alphaproteobacteria,Bacilli,Clostridia,Bacteroidetes,Actinobacteria,Gammaproteobacteria respectively.Among the 8 phyla of bacteria,Alphaproteobacteria was not observed in first instar larvae, and it has been detected till the second instar larvae.So we also can confirm the diversity of bacteria in larva guts would raise with the instar increasing.In addition,Clostridia was the preponderant bacteria in hindgut,no matter which stage it was.Three species of Clostridia have been checked in hindgut of larvae in all stages,take 16.7 percentage.of all Clostridia species These may indicate that the bacterium of Clostridia were crucial to H.parallel larvae.24 diverse species were isolated from midgut of Shandong wild species of H.parallel larvae,and 58 from hindgut.9 bacteria both existed in midgut and hindgut.That's to say 73 bacteria species in total were obtained from Shandong wild species.The result showed how the various of bacteria in H.parallel larva was.Compare with the species both in midgut and hindgut,it can conclude the bacteria live in hindgut was richer than midgut,not only the amount,but also the class belong to. Since 9 species were found both in midgut and hindgut,We can infer that these bacteria have widespread distributed in gut.However,As the rest species of bacteria were not similar with each other between midgut and hindgut,it can reach a conclusion that different part of gut has its unique bacteria type,even in the same larva.