The Bacterial Composition And Diversity In Honeydew Of Cinara Tujafilina

Aphids are commonly found having mutualistic interactions with ants. Their honeydewcan be a significant food resource for some ants. Previous research has found a kind ofsecondary symbiotic bacteria of aphid “Candidatus Serratia symbiotica” in the digestive tractof this ant. In order to reveal that whether this kind of symbiotic bacterium comes from thehoneydew of aphids, we collected the honeydew of Cinara tujafilina in different seasons(from June to October in2011and April to June in2012); three methods includingculture-dependent method,16S rRNA-RFLP and PCR-DGGE were adopted to investigate thebacterial composition and diversity in the honeydew of C. tujafilina. The results are asfollows:1. The quantity and diversity dynamics of culturable bacteria in honeydew is closelyrelated to sampling seasons. Overall, the bacterial quantity in summer and fall of2011produced great fluctuation, which reached maximum in July. The bacterial diversity index ofJuly sample also reached maximum in this year, followed by October, August and September.The bacterial quantity in spring of2012was relatively steady, which reached minimum inJune. The bacterial diversity indexes were increased with the passage of time. Bacteria strainsbelonging to25genera were isolated from all honeydew samples collected in differentseasons, which means the honeydew of C. tujafilina harbored abundant culturable bacteria,among which, Bacillus, Curtobacterium and Agrobacterium showed obvious colonyquantitative superiority. For seasonal distribution, Bacillus occurred most frequently inhoneydew, then Curtobacterium, Acinetobacter, Sphingomonas and Streptomyces.Phylogenetic analysis showed, Proteobacteria harbored three lineages (Alphaproteobacteria,Gammaproteobacteria and Betaproteobacteria), which ocuppied71.2%in all sequences. Thenext were Firmicutes and Actinobacteria, only one strain belonged to Bacteroidetes.2. Bacteria belonging to35differrent groups were detected by16S rRNA-RFLP method.The restriction endonuclease patterns of16S rRNA gene clone libraries of bacteria inhoneydew collected in July, October in2011and April in2012were more than other months,the diversity level were higher as well. The dominant bacteria species in honeydew collectedin summer (from June to August) of2011was Pseudomonas aeruginosa, while P. aeruginosa and Enterobacter spp. were the dominant bacteria in honeydew collected in autumn (fromSeptermber to October) of2011and spring (from April to May) in2012. Phylogeneticanalysis showed that bacteria belonging to the phylum Proteobacteria were clustered intothree lineages: Betaproteobacteria, Gammaproteobacteria and Alphaproteobacteria. Clonesbelonging to Gammaproteobacteria occupied the most proportion, accouting for90.9%of allclones in these seven clone libraries. In addition, a small number of clones belonging toActinobacteria, Firmicutes, Verrucomicrobia and Bacteroidetes were detected as well.Interestingly, only one clone of Pantoea, a kind of symbiotic bacteria widely existed in insects,was found in bacterial16S rRNA gene clone libraries of honeydew collected in September,2011, one and three clones of “Ca. Serratia symbiotica” were found in libraries of October,2011and April,2012, respectively. Additionally, two clones of Buchnera, the primarysymbiotic bacteria of aphids, were found in April.3. A total of40bands of different positions were excised from the DGGE gel and54sequences were obtained. The blasting results in the GenBank database showed that thesebacteria belong to17different groups.90.7%of all the sequences belong to Proteobacteria,among which bacteria belonging to Pseudomonas and Enterobacteriaceae were dominant.Phylogenetic analysis showed that bacteria belonging to the phylum Proteobacteria wereclustered into three lineages: Alphaproteobacteria, Betaproteobacteria andGammaproteobacteria. In addition, a small amont of bacteria belonging to Firmicutes andActinobacteria were detected as well. The diversity analysis result showed that the richness ofhoneydew samples collected in April,2012and September,2011was higher than others, aswell as diversity index. Cluster analysis of DGGE banding patterns showed that the similaritybetween the honeydew samples collected in June and the beginning of August was higher thanothers, while the similarity index of the honeydew samples collected in the end of August andOctober was highest. Both of these two similarity analysis methods reveal that honeydewsample collected in September had the lowest similarity with other months. Additionally,“Ca.Serratia symbiotica” was found in six samples with the low content.