caliginosus DNA; therefore the LAU1F-CB2 primer pair was used for species identification. The latter amplified all Macrolophus-DNA, although the LAU1-primer was designed

to specifically amplify M. caliginosus-DNA [35]. Results are summarized in Table 1. 16S rRNA gene sequencing A PCR assay was carried out on a pool of adult M. pygmaeus males and females of the laboratory strain using general primers targeting the bacterial 16S rRNA gene. A total of 23 clones were sequenced, VE-822 mouse varying in length depending on the use of primer pair 27F-806R or 27F-1525R (Table 2). These sequences were compared with the non-redundant (nr) nucleotide database at the National Center for Biotechnology (NCBI) using BLASTN. Three of the cloned bacteria can be considered as endosymbionts, namely Wolbachia and two Rickettsia species (Table 3). The two Rickettsia species were BMN 673 cost identified using the primer pair 27F-806R. In order to obtain approximately 1500 base pairs of their

16S rRNA gene, a PCR using a forward SN-38 supplier primer based on the partially known sequences of the two Rickettsia species was designed and combined with the general bacterial 1492R primer (Rick1F-1492R, Table 2). One of these Rickettsia species exhibited a 99% similarity to Rickettsia limoniae and the Rickettsia endosymbiont of the water beetle Deronectes platynotus. The second one was 99% similar to Rickettsia bellii and the Rickettsia endosymbiont of the pea aphid Acyrthosiphon pisum. Other cloned bacteria are not regarded as endosymbiotic bacteria, but rather as environmental or gut bacteria

(Table 3). Table 3 Partial 16S rDNA sequences isolated in this study by cloning and PCR-DGGE. The accession number of the closest relative is indicated between brackets. Closest known relative Phylogenetically related class Sequenced length (bp) Identity (%) Accession no. 16S rRNA PCR cloning of M. pygmaeus         Rickettsia limoniae strain Brugge (AF322443) Alpha-proteobacteria 1422 99 HE583202 Rickettsia GPX6 bellii (L36103) Alpha-proteobacteria 1422 99 HE583203 Wolbachia endosymbiont of Culex quinquefasciatus (AM999887) Alpha-proteobacteria 1461 98 HE583204 Uncultured bacterium (GQ360069) Gamma-proteobacteria 1496 99 HE583205 Uncultured bacterium (HM812162) Firmicutes 767 100 HE583206 Uncultured bacterium (FJ512272) Firmicutes 764 99 HE583207 Uncultured bacterium (GU118480) Beta-proteobacteria 743 99 HE583208 PCR-DGGE*         1) Wolbachia endosymbiont of Polydrusus pilifer (JF304463) Alpha-proteobacteria 135 100 HE583209 2) Rickettsia bellii (L36103) Alpha-proteobacteria 135 99 HE583210 3) Uncultured bacterium (JF011887) Gamma-proteobacteria 160 100 HE583211 4) Uncultured bacterium (JF011887) Gamma-proteobacteria 160 99 HE583212 5) Rickettsia limoniae strain Brugge (AF322443) Alpha-proteobacteria 137 100 HE583213 6) Uncultured Streptococcus sp.