Superinfection of five Wolbachia in the alnus ambrosia beetle, Xylosandrus germanus (Blandford) (Coleoptera: Curuculionidae).

Wolbachia bacteria are among the most common endosymbionts in insects. In Wolbachia research, the Wolbachia surface protein (wsp) gene has been used as a phylogenetic tool, but relationships inferred by single-locus analysis can be unreliable because of the extensive genome recombination among Wolbachia strains. Therefore, a multilocus sequence typing (MLST) method for Wolbachia, which relies upon a set of five conserved genes, is recommended. In this study, we examined whether the alnus ambrosia beetle, Xylosandrus germanus (Blandford), is infected with Wolbachia using wsp and MLST genes. Wolbachia was detected from all tested specimens of X. germanus (n=120) by wsp amplification. Five distinct sequences (i.e. five alleles) for wsp were found, and labeled as wXge1-5. MLST analysis and molecular phylogeny of concatenated sequences of MLST genes identified wXge3 and wXge5 as closely-related strains. The detection rate of wXge4 and wXge1 was 100% and 63.3%, respectively; wXge2, wXge3 and wXge5 were detected from less than 15% of specimens. We performed mitochondrial haplotype analyses that identified three genetic types of X. germanus, i.e. Clades A, B and C. Wsp alleles wXge1, wXge2 and wXge4 were detected in all clade A beetles; wXge2 allele was absent from Clades B and C. We concluded that (i) five wsp alleles were found from X. germanus, (ii) use of MLST genes, rather than the wsp gene, are more suited to construct Wolbachia phylogenies and (iii) wsp alleles wXge2 and wXge3/wXge5 would infect clade A and clade B/C of X. germanus, respectively.