Kondo Natsuko, Ijichi Nobuyuki, Shimada Masakazu, Fukatsu Takema
Department of Systems Sciences (Biology), University of Tokyo, Meguro, Tokyo 153-8902, Japan.
Mol Ecol. 2002 Feb;11(2):167-80. doi: 10.1046/j.0962-1083.2001.01432.x.
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.
在日本的小豆象（Callosobruchus chinensis）种群中，发现了三种不同的沃尔巴克氏体（Wolbachia）菌株同时感染的情况。当使用ftsZ和wsp的通用引物进行聚合酶链反应（PCR）检测时，在所有检测的个体中都检测到了沃尔巴克氏体，这些个体包括来自日本九个种群的288只雄性和334只雌性。对单个昆虫克隆的wsp基因片段进行PCR-限制性片段长度多态性（RFLP）分析表明，三种类型的wsp序列在昆虫中共存。对wsp序列的分子系统发育分析明确表明，小豆象携带三种系统发育上不同的沃尔巴克氏体，暂定为wBruCon、wBruOri和wBruAus。使用特异性引物的诊断PCR分析表明，在来自九个当地种群的175只雄性和235只雌性中，wBruCon、wBruOri和wBruAus的感染频率分别为100%、96.3%和97.0%。至于个体的感染状态，三重感染（93.7%）占主导地位，双重感染（6.1%）和单一感染（0.2%）较少。利用定量PCR技术，从每个昆虫个体的wsp基因拷贝数方面，分析了野外采集的成年昆虫中wBruCon、wBruOri和wBruAus的含量。无论原始种群如何，wBruCon和wBruOri（107 - 108个wsp拷贝/昆虫）的含量始终高于wBruAus（106 - 107个wsp拷贝/昆虫），这表明三种沃尔巴克氏体菌株的种群大小受到控制，尽管其机制尚不清楚。交配实验表明，这三种沃尔巴克氏体在不同强度水平上导致细胞质不亲和。
