Phylogenetic Relationships Between The Aphids And Their Endosymbiotic Bacterium Buchnera

Almost all aphids maintain an endosymbiotic association with Buchnera aphidicola, aγ-proteobacterium closely related to Escherichia coll. The endosymbiont, harbored in host-derived vesicles within specialized cells known as bacteriocytes, is transmitted maternally, from mother aphid to progeny. The relationship between Buchnera and the aphid host is mutualistic because aphid needs B. aphidicola for its normal growth and reproduction whereas B. aphidicola cannot live outside the aphid. Molecular study found that Buchnera's genome is lessen, but the most amino acids synthetic genes are preserving.Because of the importance of Buchnera, many authors studied this symbiosis. Previous studies about Buchnera of some aphids by foreign authors only conferred some limited aphids, such as the genus Uroleucon sp., Acryrthosiphon pisum, Diuraphis noxia (Kurdj), Schizaphis graminum etc, but no Myzus persicae, Aphis craccivora and Aphis gossypii. And domestic studies about Buchnera of the three aphids were also few, and this study only conferred the two genes (groEL and 16srDNA) of Buchnera in M. persicae. The three aphids, which are abundant throughout China as well as worldwide, are polyphagous herbivores attacking many vegetables and crops such as cabbages, cucumber, eggplant, tomato, cucurbits, bean and so on. Because of the special relationships between aphids and the Buchnera, the studies on the Buchnera of the three aphids are necessary. Firstly, we explored the phylogenies of the three aphids, M. persieae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera respectively. Then we synthetically analysed phylogenetic relationships between those three aphids and their endosymbiotic bacterium Buchnera.1. The collection and rearing of experimental materialWe collected three aphids M. persicae, A. craccivora and A. gossypii, which are important pests. Those three aphids were then reared in the laboratory on radish, bean, cucumber respectively. For each population, at least two double-deck gauze cages (around 50cm×50cm×50cm) were setto rear the three aphids. And they were kept in a laboratory under controlled environmental conditions (20±2℃, 60-70% R.H., 16L-8D).2. The sequencing of mtDNA COⅠ/COⅡ, 12S/16SrRNA and nuclear gene EFalpha in the three aphids, and the phylogenetic analysis of the three aphidsIn this part of work, we amplified COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes in four populations of M. persicae, Nanjing population A. craccivora and A. gossypii. Three to five individual specimens per population were cloned and sequenced and the standard sequences were their identical sequences.DNA sequences were submitted to the GenBank database and compared online with the published sequences by similarity search engines such as BLAST in NCBI Web, then calculated in GeneDoc and aligned using Clustal X computer program. Analysis of genetic and phylogenetic relationship was performed using MEGA2.0. Genetic relationships among every geographical population were estimated based on the pair-wise matrix of sequence divergences by Kimura-2 Parameter method. Phylogenefic trees were constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ tree were assessed by bootstrapping from 1000 pseudo-replications.Comparing the obtained sequences, we found that the COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes sequences from the four populations M. persicae had very minor differences, and the similarities between the different two populations reached to 99%. And the similarities Of the COⅠ/COⅡ, EFalpha genes between M. persicae and A. gossypii were about 90%, those of the two genes between M. persicae and A. craccivora were near 85%,near 90% respectively, while the similarities of those genes between A. gossypii and A. craccivora were hyper-85%,near 93% respectively. But the similarities of the 12S/16SrRNA genes sequences in different two aphids reached to 95%.Through the phylogenetic trees constructed by the COⅠ/COⅡ, 12S/16SrRNA and EFalpha genes in M. persicae, we found that the COⅠ/COⅡand 12S/16SrRNA gene sequences in M. persicae were more closely related to those in D. noxia, while the EFalpha genes of M. persicae and D. noxia located in two neighboring branches, and the these genes sequences in A. gossypii and A. craccivora were all in the same clades. So we concluded that M. persicae and D. noxia were more close, and A. gossypii and A. craccivora were more close. 3. The sequencing of trpB, dnaN, trpEG in Buchnera, and the phylogenetic analysis of Buchnera in the three aphidsFirstly, we designed primers based on the nucleotide sequences from the currently known trpB, dnaN and trpEG sequences in Buchnera (isolated from Uroleucon. sp, A. pisum, D. noxia, S. graminum and R. padi), and amplified and sequenced these genes sequences of Buchnera from the three aphids, M. persicae, A. craccivora and A. gossypii. Three to five indiyidual specimens per population were cloned and sequenced and the standard sequences were their identical sequences.DNA sequences were submitted to the GenBank database and compared online with the published sequences by similarity search engines such as BLAST in NCBI Web, then calculated in GeneDoc and aligned using Clustal X computer program. Analysis of genetic and phylogenetic relationship, was performed using MEGA2.0. Genetic relationships among every geographical population were estimated based on the pair-wise matrix of sequence divergences by Kimura-2-Parameter method. Phylogenetic trees were constructed by the Neighbor-Joining (NJ) method. Confidence levels for NJ tree were assessed by bootstrapping from 1000 pseudo-replications.Comparing the obtained sequences, we found that the trpB, dnaN and trpEG sequences in Buchnera from the four populations M. persicae had very minor differences, but these sequences between M. persicae and A. craccivora, M. persicae and A. gossypii had major differences, and these differences were little minor between A. craccivora and A. gossypii.Through the phylogenetic trees constructed by this genes (trpB, dnaN and trpEG) in Buchnera, we found that the trpB, dnaN gene sequences in M. persicae were in the same clade with those in D. noxia, that is to say the Buchnera in M. persicae is more similar to the endosymbionts of D. noxia. Besides, the phylogenies of the trpB, dnaN genes of the Buchnera in M. persicae were accordant with that of the trpEG genes, so the plasmid genes trpEG genes of the Buchnera in M. persicae may be vertical transmission with Buchnera. And these genes sequences in A. gossypii and A. craccivora were all in the same clades, but the near clade to the sub-clade of the two aphids was variational. SO we only confirmed that the Buchnera in A. gossypii and A. craccivora were more close, but the conclusions that the Buchnera in these two aphids was close to that in which aphids and the transmission of the the plasmid genes trpEG of the Buchnera in these aphids could not be affirmed. 4. Analysis of phylogenetic relationships between the aphids and their endosymbiotic bacterium BuchneraStudies about the phylogenetic relationships between aphids and their endosymbiotic bacterium Buchnera are few. In our present study, we explore phylogenetic relationships between the three aphids, M. persicae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera, by comparing the characteristics of the phylogenies of those aphids and their Buchnera. We found that M. persicae and D. noxia were coevolutional, but we cannot conclude the coevolutional relationships between the two aphids, A. gossypii and A. craccivora, and their Buchnera. So we conclude that phylogenetic relationships between different aphids and their endosymbiotic bacterium Buchnera may be different, and this conclusion is accordant with previous study. This work not only fill a gap on phylogenetic relationships between the three aphids, M. persicae, A. craccivora and A. gossypii and their endosymbiotic bacterium Buchnera, but also help us detailedly know the three aphids' Buchnera.As described above, these results first exposed the phylogeny of Buchnera in the three aphids, M. persicae, A. craccivora and A. gossypii. And these works also explored phylogenetic relationships between those three aphids and their endosymbiotic bacterium Buchnera. These works will be helpful for exploring other characteristics of those aphids' Buchnera and studying those aphids'secondary endosymbiosis.