Toward a Wolbachia Multilocus Sequence Typing System: Discrimination of Wolbachia Strains Present in Drosophila Species

Abstract

Among the diverse maternally inherited symbionts in arthropods, Wolbachia are the most common and infect over 20% of all species. In a departure from traditional genotyping or phylogenetic methods relying on single Wolbachia genes, the present study represents an initial Multilocus Sequence Typing (MLST) analysis to discriminate closely related Wolbachia pipientis strains, and additional data on sequence diversity in Wolbachia.We report new phylogenetic characterization of four genes (aspC, atpD, sucB and pdhB), and provide an expanded analysis of markers described in previous studies (16S rDNA, ftsZ, groEL, dnaA and gltA). MLST analysis of the bacterial strains present in sixteen different Drosophila-Wolbachia associations detected four distinct clonal complexes that also corresponded to maximum-likelihood identified phylogenetic clades. Among the sixteen associations analyzed, six could not be assigned to MLST clonal complexes and were also shown to be in conflict with relationships predicted by maximum-likelihood phylogenetic inferences. The results demonstrate the discriminatory power of MLST for identifying strains and clonal lineages of Wolbachia and provide a robust foundation for studying the ecology and evolution of this widespread endosymbiont.

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

“…The Wolbachia strain(s) of the donor and of the transinfected Drosophila hosts were genotyped using a Multi Locus Sequence Typing system developed in Paraskevopoulos et al (2006).…”

Clonal population structure of Legionella pneumophila inferred from allelic profiling

“…Since then, this robust and portable technique (or variations of it) has been applied to the study of genetic diversity and clonal expansion, and to the long-term epidemiological analysis of microbial populations (see http://pubmlst.org/ and http://www.mlst.net/) (Giske et al, 2006;Paraskevopoulos et al, 2006;Vassileva et al, 2006).…”

“…Since then, this robust and portable technique (or variations of it) has been applied to the study of genetic diversity and clonal expansion, and to the long-term epidemiological analysis of microbial populations (see http://pubmlst.org/ and http://www.mlst.net/) (Giske et al, 2006;Paraskevopoulos et al, 2006;Vassileva et al, 2006). (Stone & Abu Kwaik, 1998), aspartate-b-semialdehyde dehydrogenase (Harb & Abu Kwaik, 1998), macrophage infectivity potentiator (Engleberg et al, 1989), a major outer-membrane protein of~29 kDa (Ehret & Ruckdeschel, 1985; accession no.…”

Tracing the history and ecological context of Wolbachia double infection in a specialist host (Urophora cardui)—parasitoid (Eurytoma serratulae) system

“…I further sequenced the gene AAT. AAT has been proposed as an additional MLST gene (Paraskevopoulos, Bordenstein, Wernegreen, Werren, & Bourtzis, 2006), but it is also the primary gene defining the transition zone in U. cardui (Steinmetz et al., 2004). Hence, apart from delimiting Wolbachia strains, AAT sequence analysis also assessed whether the transition zone in U. cardui was influenced by Wolbachia allele diversity rather than by its own, nuclear diversity.…”

“…Hence, apart from delimiting Wolbachia strains, AAT sequence analysis also assessed whether the transition zone in U. cardui was influenced by Wolbachia allele diversity rather than by its own, nuclear diversity. AAT was amplified with the primers aspC49‐F (5′‐ATYGCTGTRACYGATAAGGYAA) and aspC1134‐R (5′‐AGARGTWGCATAAGARATTCTRA) (Paraskevopoulos et al., 2006) using the wsp PCR amplification protocol. Each PCR reaction was performed in 25 μl reaction volume consisting of 1 μl forward and 1 μl reverse primer, 12.5 μl OneTaq 2X Master Mix (NEB) and 9.5 μl H 2 O (sterile) and 1 μl DNA.…”