Multilocus Sequence Typing System for the EndosymbiontWolbachia pipientis

Abstract

The eubacterial genus Wolbachia comprises one of the most abundant groups of obligate intracellular bacteria, and it has a host range that spans the phyla Arthropoda and Nematoda. Here we developed a multilocus sequence typing (MLST) scheme as a universal genotyping tool for Wolbachia. Internal fragments of five ubiquitous genes (gatB, coxA, hcpA, fbpA, and ftsZ) were chosen, and primers that amplified across the major Wolbachia supergroups found in arthropods, as well as other divergent lineages, were designed. A supplemental typing system using the hypervariable regions of the Wolbachia surface protein (WSP) was also developed. Thirty-seven strains belonging to supergroups A, B, D, and F obtained from singly infected hosts were characterized by using MLST and WSP. The number of alleles per MLST locus ranged from 25 to 31, and the average levels of genetic diversity among alleles were 6.5% to 9.2%. A total of 35 unique allelic profiles were found. The results confirmed that there is a high level of recombination in chromosomal genes. MLST was shown to be effective for detecting diversity among strains within a single host species, as well as for identifying closely related strains found in different arthropod hosts. Identical or similar allelic profiles were obtained for strains harbored by different insect species and causing distinct reproductive phenotypes. Strains with similar WSP sequences can have very different MLST allelic profiles and vice versa, indicating the importance of the MLST approach for strain identification. The MLST system provides a universal and unambiguous tool for strain typing, population genetics, and molecular evolutionary studies. The central database for storing and organizing Wolbachia bacterial and host information can be accessed at http://pubmlst.org/wolbachia/.

Incidence of Wolbachia in aquatic insects

“…Genotyping of Wolbachia strains using methodology from Baldo et al. (2006) should also provide clues of how Wolbachia is spatially distributed.…”

“…PCR was performed on approximately 100 ng of genomic DNA from the samples using wspF (5′ GTCCAATARSTGATGARGAAAC 3′) and wspR primers (5′ CYGCACCAAYAGYRCTRTAAA 3′), yielding a product approximately 600 bp in size. Methods were adapted according to previously established protocols (Baldo et al., 2006). A thermocycler was set for the following conditions: denaturation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 30 s, annealing at 59°C for 45 s, elongation at 72°C for 90 s and a final elongation step at 72°C for 10 min.…”

Genome comparisons indicate recent transfer ofwRi‐likeWolbachiabetween sister speciesDrosophila suzukiiandD. subpulchrella

“…The Wolbachia MLST loci gatB , hcpA , coxA , fbpA , and ftsZ (Baldo et al., 2006) were identified in the assemblies using BLAST. As reported in Hamm et al.…”

“…In particular, Hamm et al. (2014) postulated that because D. suzukii and its sister D. subpulchrella have concordant mitochondrial and nuclear phylogenies and harbor very similar Wolbachia , as indicated by identity at the multilocus sequence typing (MLST) loci used to classify Wolbachia (Baldo et al., 2006), cladogenic Wolbachia acquisition was likely. Here, we use comparative analyses of draft Wolbachia genomes, and extensive nuclear data from Drosophila and other insect hosts, to refute this hypothesis.…”

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

“…Primers and thermocycler protocols for MLST genes (gatB, CoxA, hcpA, fstZ, fbpA) and wsp were as described in Baldo et al. (2006). I further sequenced the gene AAT.…”