Prevailing triple infection with Wolbachia in Callosobruchus chinensis (Coleoptera: Bruchidae).

Prevailing triple infection with three distinct Wolbachia strains was identified in Japanese populations of the adzuki bean beetle, Callosobruchus chinensis. When a polymerase chain reaction (PCR) assay was conducted using universal primers for ftsZ and wsp, Wolbachia was detected in all the individuals examined, 288 males and 334 females from nine Japanese populations. PCR-restriction fragment length polymorphism (RFLP) analysis of cloned wsp gene fragments from single insects revealed that three types of wsp sequences coexist in the insects. Molecular phylogenetic analysis of the wsp sequences unequivocally demonstrated that C. chinensis harbours three phylogenetically distinct Wolbachia, tentatively designated as wBruCon, wBruOri and wBruAus, respectively. Diagnostic PCR analysis using specific primers demonstrated that, of 175 males and 235 females from nine local populations, infection frequencies with wBruCon, wBruOri and wBruAus were 100%, 96.3% and 97.0%, respectively. As for the infection status of individuals, triple infection (93.7%) dominated over double infection (6.1%) and single infection (0.2%). The amounts of wBruCon, wBruOri and wBruAus in field-collected adult insects were analysed by using a quantitative PCR technique in terms of wsp gene copies per individual insect. Irrespective of original populations, wBruCon and wBruOri (107 -108 wsp copies/insect) were consistently greater in amount than wBruAus (106 -107 wsp copies/insect), suggesting that the population sizes of the three Wolbachia strains are controlled, although the mechanism is unknown. Mating experiments suggested that the three Wolbachia cause cytoplasmic incompatibility at different levels of intensity.

Genome fragment of Wolbachia endosymbiont transferred to X chromosome of host insect.

The adzuki bean beetle, Callosobruchus chinensis, is triple-infected with distinct lineages of Wolbachia endosymbiont, wBruCon, wBruOri, and wBruAus, which were identified by their wsp (Wolbachia surface protein) gene sequences. Whereas wBruCon and wBruOri caused cytoplasmic incompatibility of the host insect, wBruAus did not. Although wBruCon and wBruOri were easily eliminated by antibiotic treatments, wBruAus persisted over five treated generations and could not be eliminated. The inheritance pattern of wBruAus was, surprisingly, explained by sex-linked inheritance in male-heterozygotic organisms, which agreed with the karyotype of C. chinensis (2n = 20, XY). Quantitative PCR analysis demonstrated that females contain around twice as much wsp titer as males, which is concordant with an X chromosome linkage. Specific PCR and Southern blot analyses indicated that the wBruAus-bearing strain of C. chinensis contains only a fraction of the Wolbachia gene repertoire. Several genome fragments of wBruAus were isolated using an inverse PCR technique. The fragments exhibited a bacterial genome structure containing a number of ORFs typical of the alpha-proteobacteria, although some of the ORFs contained disruptive mutations. In the flanking region of ftsZ gene, a non-long terminal repeat (non-LTR) retrotransposon sequence, which is typical of insects but not found from bacteria, was present. These results strongly suggest that wBruAus has no microbial entity but is a genome fragment of Wolbachia endosymbiont transferred to the X chromosome of the host insect.

Internal spatiotemporal population dynamics of infection with three Wolbachia strains in the adzuki bean beetle, Callosobruchus chinensis (Coleoptera: Bruchidae).

The adzuki bean beetle, Callosobruchus chinensis, is infected with three distinct lineages of endosymbiotic bacteria belonging to the genus Wolbachia, which were designated wBruCon, wBruOri, and wBruAus. In an attempt to understand the mechanisms underlying the infection with these three organisms, the spatiotemporal infection dynamics of the three Wolbachia strains was investigated in detail by using a quantitative PCR technique. During the development of C. chinensis, the wBruCon, wBruOri, and wBruAus infection levels consistently increased but the growth patterns were different. The levels of infection plateaued at the pupal stage at approximately 3 x 10(8), 2 x 10(8), and 5 x 10(7) wsp copy equivalents per insect for wBruCon, wBruOri, and wBruAus, respectively. At the whole-insect level, the population densities of the three Wolbachia types did not show remarkable differences between adult males and females. At the tissue level, however, the total densities and relative levels of the three Wolbachia types varied significantly when different tissues and organs were compared and when the same tissues derived from males and females were compared. The histological data obtained by in situ hybridization and electron microscopy were concordant with the results of quantitative PCR analyses. Based on the histological data and the peculiar Wolbachia composition commonly found in nurse tissues and oocytes, we suggest that the Wolbachia strains are vertically transmitted to oocytes not directly, but by way of nurse tissue. On the basis of our results, we discuss interactions among the three coinfecting Wolbachia types, reproductive strategies of Wolbachia, and factors involved in the different cytoplasmic incompatibility phenotypes.