Abstract
Wolbachia are wide-spread, endogenous α-Proteobacteria of arthropods and filarial nematodes. 15–75% of all insect species are infected with these endosymbionts that alter their host's reproduction to facilitate their spread. In recent years, many insect species infected with multiple Wolbachia strains have been identified. As the endosymbionts are not cultivable outside living cells, strain typing relies on molecular methods. A Multi Locus Sequence Typing (MLST) system was established for standardizing Wolbachia strain identification. However, MLST requires hosts to harbour individual and not multiple strains of supergroups without recombination. This study revisits the applicability of the current MLST protocols and introduces Allele Intersection Analysis (AIA) as a novel approach. AIA utilizes natural variations in infection patterns and allows correct strain assignment of MLST alleles in multiply infected host species without the need of artificial strain segregation. AIA identifies pairs of multiply infected individuals that share Wolbachia and differ in only one strain. In such pairs, the shared MLST sequences can be used to assign alleles to distinct strains. Furthermore, AIA is a powerful tool to detect recombination events. The underlying principle of AIA may easily be adopted for MLST approaches in other uncultivable bacterial genera that occur as multiple strain infections and the concept may find application in metagenomic high-throughput parallel sequencing projects.
Highlights
Wolbachia are obligatory endosymbiotic a-Proteobacteria found in 15–75% of all insect species worldwide [1,2,3,4], in many other arthropods and filarial nematodes [5,6]
We present a novel method, Allele Intersection Analysis (AIA), enabling correct assignment of Multi Locus Sequence Typing (MLST) alleles retrieved from multiply infected individuals
Hosts can be infected by a multitude of Wolbachia strains rendering the assignment of MLST alleles a challenge
Summary
Wolbachia are obligatory endosymbiotic a-Proteobacteria found in 15–75% of all insect species worldwide [1,2,3,4], in many other arthropods and filarial nematodes [5,6]. The wide range of infected species suggests an ability of Wolbachia to spread horizontally to new hosts [12,13] Their potential for horizontal transmission, combined with high maternal transmission efficiencies and low levels of endosymbiont loss by environmental curing [14], as well as the emergence of novel strains due to recombination [15,16,17,18] are expected to contribute to an accumulation of Wolbachia strains in individual hosts. AIA requires (a) the identification of the Wolbachia strains infecting a single individual (further referred to as ‘infection type’) using a highly variable marker gene and (b) the cloning and sequencing of MLST alleles from a pair of multiply infected individuals that share or differ in only one Wolbachia strain. Combinations of infection types that allow complete allele assignment are termed ‘informative’
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