Molecular Detection And Phylogenetic Analysis Of Secondary Endosymbiont Wolbachia From Nine Gall Aphid Species

Gall aphid can stimulate the expansion of plant tissues to form galls,which is important group of gall insects and most of them are agricultural and forestry pests.Howerver,some species have no obvious damage to host plants,such as gallnut aphids.They parasize in Rhus to form galls.Gallnuts are mainly composed of gallnut tannins,which are widely used in medicine,chemical industry,mining and environmental protection.Wolbachia(Proteobacteria,Rickettsiales,Anaplasmataceaeis)is an important secondary endosymbiont that exists widely in insects,which regulate the reproduction of the host insect inducing male killing,feminization,cytoplasmic incompatibility,parthenogenesis.In addition,it also can regulate the host and affect its species formation and evolution.Previous studies on aphids Wolbachia were mainly focused on a few species of free-living aphids,but less on the species living in the closed galls.Therefore,this study on the strains and phylogenetic relationships of Wolbachia in galling aphids is helpful to understand the distribution and phylogenetic relationship of Wolbachia among different hosts,and to provide references for further study of its function and coevolution with the host.In this study,the primer sequences of the 16S rRNA,gro EL,glt A and wsp genes of Wolbachia were selected to detect the Wolbachia infection in 14 populations of 9 species of gall aphids,such as gallnut aphids,poplar gall aphids and Pistacia chinensis gall aphids.Then the specific genes of infected Wolbachia were sequenced,and the phylogenetic tree was constructed with the same gene series of Wolbachia of other aphids,the difference and relationship between the strains infected with Wolbachia and other aphids were analyzed.The main results were listed as follows:1.The low infection rate of Wolbachia in gall aphidsA total of 3 species of gall aphids were infected with Wolbachia from 14 populations of 9 species of gall aphids by specific gene detection.They are Kaburagia rhusicola Takagi of Kunming population,Schlechtendalia chinensis of Emei population and Pemphigus yunnanensis of Kunming population.The infection rate of Wolbachia in the three species of gall aphids was 66.7%,11.1% and 11.1%,respectively.The results showed that the infection of Wolbachia in the 9 species of gall aphids was less common and the infection rate was lower than that of free-living insects.2.Sequencing of Wolbachia specific genes in Gall aphidsThe population of gall Aphids infected with Wolbachia was amplified and sequenced by specific genes,the corresponding gene sequences were obtained from six genes.The length of 16S rRNA gene sequences of Wolbachia in Kaburagia rhusicola Takagi of Kunming population,Schlechtendalia chinensis of Emei population and Pemphigus yunnanensis of Kunming population were 1077 bp,1061 bp,1054 bp(using 16 S 281F/1372 R primer)and 440 bp,440bp,442bp(using Wspec F/Wspec R primer),The fragment lengths of gat B and fbp A genes are 497 bp,492bp,497bpand476 bp,479bp,480 bp.In addition,three gene fragments(cox A,hcp A,fts Z)were obtained from Pemphigus yunnanensis of Kunming population,and the length of the fragments are 488 bp,509bp and 518 bp,respectively.The specific gene sequences of three species of gall aphids were compared with those of other free-living aphids by NCBI-Blast,and the two kinds of gallnut aphids is less similar to that of other aphids free-living.The specific gene sequences of Pemphigus yunnanensis were similar to those of other free-living aphids and whitefly.It suggests that the Wolbachia infected by these two types of gall aphids may have different origins.3.The Wolbachia strains infected by gallnut aphids and poplar chinensis gall aphids belonging to different strainsThe 16S rRNA gene sequences and five multi-locus typing gene sequences of Wolbachia were used to homology comparison and construct phylogenetic tree combined with each supergroup representative strains of Wolbachia download from NCBI.The results showed that The strains from Pemphigus yunnanensis of Kunming population are closely related to B supergroup strains of infection in other free-living aphids,belonging to B supergroup.However,the strains from Kaburagia rhusicola of Kunming population and Schlechtendalia chinensis of Emei population were far with free-living aphids that belong to O supergroup,which suggested that the Wolbachia infected by gallnut aphids and the Wolbachia infected by gall aphis and other aphids with free-living may have different origins.4.In this study,Wolbachia infection was detected in two species of gallnut aphid,and it was initially identified as O supergroup,and Wolbachia infection was detected for the first time from Kaburagia rhusicola.Therefore,the Wolbachia strains infected by aphids may exist other supergroups other than supgroups A,B,M and N.