Microbial population analysis of tick salivary glands; a possible strategy for the surveillance of bacterial pathogens.. organismal metagenomes

Tick is one of the most important blood sucking vectors for infectious agents in human and animals. When ticks are taking blood meals, they inject saliva containing microbes to facilitate blood uptake. Thus, microbial populations in salivary glands should be useful to evaluate vectorial capacity of ticks. Three tick species, Ixodes ovatus I. persulcatus and Haemaphysalis flava, were collected in Shizuoka prefecture, Japan from 2008 to 2011. Each collected tick was dissected under a stereomicroscope and salivary glands were removed. A 16S tag-encoded amplicon pyrosequencing was performed on individual salivary gland, using a 454 GS junior next generation sequencer, in order to characterize a bacterial community in each salivary gland. A Ribosomal Database Project (RDP) classifier was employed to classify sequence reads at a genus level. To compare microbial population of each tick species, principal component analysis (PCA) was done using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Conventional PCR and cloning methods were further used for phylogenetic characterization of Rickettsia species and unclassified bacteria sequences. The total numbers of bacterial genera identified were 71, 127, and 59 in I. ovatus, I. persulcatus, and H. flava, respectively. The predominant genus observed in all tick species was Coxiella. Spiroplasma was only detected in Ixodes but not in Haemaphysalis ticks. The result of PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role for determining of microbial population. Four female I. persulcatus samples contained several sequences belonging to alphaproteobacteria symbionts at high abundance. This study revealed microbial populations in salivary gland of three tick species. The results will be useful to predication of emerging tick-borne diseases.