title: Wolbachia Sequence Typing in Butterflies Using Pyrosequencing.
abstract: Wolbachia is an obligate symbiotic bacteria that is ubiquitous in arthropods, with 25-70% of insect species estimated to be infected. Wolbachia species can interact with their insect hosts in a mutualistic or parasitic manner. Sequence types (ST) of Wolbachia are determined by multilocus sequence typing (MLST) of housekeeping genes. However, there are some limitations to MLST with respect to the generation of clone libraries and the Sanger sequencing method when a host is infected with multiple STs of Wolbachia. To assess the feasibility of massive parallel sequencing, also known as next-generation sequencing, we used pyrosequencing for sequence typing of Wolbachia in butterflies. We collected three species of butterflies (Eurema hecabe, Eurema laeta, and Tongeia fischeri) common to Korea and screened them for Wolbachia STs. We found that T. fischeri was infected with a single ST of Wolbachia, ST41. In contrast, E. hecabe and E. laeta were each infected with two STs of Wolbachia, ST41 and ST40. Our results clearly demonstrate that pyrosequencing-based MLST has a higher sensitivity than cloning and Sanger sequencing methods for the detection of minor alleles. Considering the high prevalence of infection with multiple Wolbachia STs, next-generation sequencing with improved analysis would assist with scaling up approaches to Wolbachia MLST.
authors.affiliation: BK21PLUS Program in Embodiment: Health-Society Interaction, Department of Public Health Sciences, Graduate School, Korea University, Seoul 136-701, Republic of Korea.
authors.forename: Sungmi
authors.initials: S
authors.lastname: Choi
authors.affiliation: BK21PLUS Program in Embodiment: Health-Society Interaction, Department of Public Health Sciences, Graduate School, Korea University, Seoul 136-701, Republic of Korea.
authors.forename: Su-Kyoung
authors.initials: SK
authors.lastname: Shin
authors.affiliation: Department of Ecology and Evolution, National Institute of Ecology, Seocheon-gun, 325-813, Republic of Korea.
authors.forename: Gilsang
authors.initials: G
authors.lastname: Jeong
authors.affiliation: BK21PLUS Program in Embodiment: Health-Society Interaction, Department of Public Health Sciences, Graduate School, Korea University, Seoul 136-701, Republic of Korea.|School of Biosystem and Biomedical Science, Korea University, Seoul 136-703, Republic of Korea.
authors.forename: Hana
authors.initials: H
authors.lastname: Yi
mesh_terms: D000818:Animals; D002080:Butterflies / Q000382:microbiology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D007723:Korea; D058885:Multilocus Sequence Typing / Q000379:methods*; D020577:Wolbachia / Q000145:classification* / Q000235:genetics*
keywords: Eurema; MLST; Tongeia; Wolbachia; pyrosequencing; wsp