Diversity Of Intestinal Bacteria And Cellulose Degradation Function In The Larvae Of Alternaria Sylvestris

Monochamus alternatus Hope is not only the main vector of pine wood nematode disease,but also can cause pines to weaken and even die by feeding on the phloem and xylem during larval stages.The composition of lignocellulose from food sources has changed during different M.alternatus larval instars.Is there any effect on the intestinal bacterial diversity and cellulose degradation function?Little is known about it,and which is worth to study in depth.In this paper,we used metagenomics,aseptic culture,enzymatic determination and bacterial genomics techniques to study the intestinal bacterial diversity and the enzymatic hydrolysis function of larvae of M.alternatus in different stages.The main conclusions are as follows:1.The intestine bacterial community diversity of early larvae feeding on phloem was significantly higher than that of later larvae feeding on xylem;These gut bacterias belonged to several families,among which the relative abundance of Enterobacteriaceae was the highest;The relative abundances of the dominant genus of gut were different in five instars,and Erwinia was the most abundant bacterium in the of 1st instar(58.3%±3.7%)and 5th instar(82.0%±16.9%),and Enterobacteriaceae is the dominant dominance in the intestines of 2nd instar(31.7%± 20.1%),3rd instar(97.1%±2.3%)and 4th instar(49.2%±14.2%).In this study,Actinomycetes were found in M.alternatus gut for the first time.Cellulomonadaceae(Actinomyces)is the dominant bacterium only to the most abundant bacteria family in the 2nd instar larvae(10.6%).2.A total of 51 strains of cellulose-degrading bacteria were isolated from the intestinal cellulose degrading bacteria of the M.alternatus larvae,belonging to the four phyla(including Proteobacteria,Mycobacteria,Bacteroides and Actinomycetes),nine families,ten genus and eleven species.Among them,strain BS16 put up the highest cellulase activity in the qualitative determination of CMC(the ratio of colony clear circle diameter to colony diameter was 2.72).The degradation rate of filter paper on BS16 for each was 10.71%,17.45%and 24.31%at 2 d,5 d and 7 d.BS16 produced more enzymes and the cellulase activity was better.Cellulase activity results showed that the endoglucanase activity was highest(57.48 U/mL).the exocellulase activity was 49.62 U/mL.And the(3-glucosidase activity was 27.02 U/mL.So,the B.cereus BS16 strain had an intact cellulase living system.3.The whole genome size of B.cereus BS16 is 5,347,914 bp and the total number of genes is 5,494.The total GC content of all gene sequences is 35.87%in the genome.The average length of the gene is 820 bp.The number of tRNA encoding genes is 102.The number of rRNA encoding genes is 11.And the number of sRNA encoding genes is 17.There are 25 functional categories by comparing in the GO database.Compared with the COG database,25 functional categories were predcted.And the number of including functional genes is 4402(80.16%).The number of coding genes about predicted function,amino acid transport and metabolism,transcription and carbohydrate transport and metabolism is 437,411,373 and 276.Through classifing and comparing in the CAZy database,the whole genome sequence of B.cereus BS16 was predicted to contain encoding gene of glycosyl hydrolyzed family(5 GHs),auxiliary activities family(one AAs)and carbohydrate-binding module(8 CBMs)for cellulose degradation of the complete enzyme system.These families includes one beta-glucosidase GH4(EC 3.2.1.86)gene,one exoglucanase GH5(EC 3.2.1.91)gene,one endoglucanase GH8(EC 3.2.1.4)gene,one xylanase GH10(EC 3.2.1.8)gene,4 chitinase GH18(EC 3.2.1.14)gene and 3 lytic polysaccharide monooxygenases AA10(EC 1.14.99.54)gene and 38 CBMs genes.This result reveals the cellulose degradation function and mechanism of action of B.cereus BS16 from the genetic level.