Uring purification of Buchnera from aphids by use of Percoll-gradient centrifugation to separate the bacterial cells from other organelles or cellular debris [17]. An elegant method was applied to purify TG-1 bacteria from termite flagellates, where a single protozoan cell was manipulated and the posterior part of the protist cell was manually ruptured to collect the specific bacteria [15]. Additional examples of successful endosymbiont isolation from complex environments may facilitate the study of other bacterial symbioses. Almost all cockroaches harbour the bacterial endocytosymbiont Blattabacterium cuenoti (phylum Bacteriodetes, class Flavobacteria) in mycetocytes of their fat bodies. Molecular phylogenetic studies show this bacterium is closely related to other Flavobacterial endosymbionts of insects, in particular Sulcia muelleri from auchenorrhynchan insects and male-kiling symbionts from ladybird beetles [18]. The Blattabacterium/cockroach symbiosis was first discovered in the late 19th century [19]. These bacteria have been co-evolving with cockroaches for at least 130 million years [20,21]. It has been proposed that the symbiosis is mediated by juvenile hormone secreted from corpora allata [22]. On the basis of extensive biochemical, radiochemical, and antibiotic studies using normal and aposymbiotic cockroaches, the relationship has been shown to be one of obligate mutualism. The bacteria contribute to the nitrogen metabolism of their hosts by mobilizing the uric acid stored in the fat bodies when cockroaches feed on nitrogen-poor diets [23,24]. The uricolysis is thought to be mediated by xanthine dehydrogenase rather than uricase [23], although further details for this process have yet to be provided. In addition, the bacteria have been proposed to provide some essential amino acids to cockroaches [25] and to be involved in sulphate assimilation into sulphur amino acids [26]. These studies were conducted based on comparisons between normal and aposymbiotic insects, however, effects of the antimicrobial treatments on the intestinal bacterial diversitywere not well assessed. Overall, the details of the interaction between the two partners have not yet been clarified. Luminespib chemical information genomic or proteomic analyses are likely to greatly enhance our understanding of B. cuenoti biology. Purification of B. cuenoti cells is the first step in this process. Purification of B. cuenoti is challenging for the following reasons. First, the bacterial endosymbionts co-exist with organelles and nuclei that have to be eliminated. Second, in the fat body tissues, the mycetocyte is always surrounded by urocytes and trophocytes [27], which contain a large amount of urates, lipids, and other intracellular extra substances as well as PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27321907 usual organelles and nuclei. In the present paper, we describe the first successful purification of the genomic DNA of the flavobacterial endosymbiont in cockroaches.MethodsHost insects Panesthia angustipennis were collected at Mt. Tsukuba in Ibaraki prefecture, Japan. The cockroaches were reared with wood chips and sliced pieces of fresh carrot at room temperature. Purification of B. cuenoti from cockroaches All procedures were performed at 4 unless otherwise indicated. Two adult female individuals (6.5 g in total) were dissected to remove the fat bodies. The collected fat bodies were homogenized in 6 ml of a solution with the following components: 41.2 mM sodium chloride, 10.2 mM sodium hydrogen carbonate, 5.7 mM trisodium citrate.