riparius endosymbiont (Fig. 2a–d). The granular layer was found on all electron-microscopically investigated eggs (n=20), which had been oviposited Alpelisib by P. riparius. In order to check whether this granular layer is already applied to the eggshell in the ovaries during oogenesis or somewhere else in the internal female genitalia

during egg passage, several eggs (n=9) were prepared out of the common oviduct and analysed by electron microscopy. These eggs always exhibited a strongly folded, smooth surface, indicating that a granular layer was absent (Fig. 3c and d). In order to approve these findings molecularly, two eggs from the common oviduct (cf. Fig. 3e) and five already oviposited eggs from different female beetles (n=10) were analysed by pks PCR. pks gene fragments indicating Paederus endosymbionts were amplified from all oviposited eggs, but not from eggs originating from the common oviduct, indicating that the endosymbionts are applied to the egg shell inside the efferent duct (cf. Fig. 3e). Many endosymbiotic bacteria are still unable to grow in vitro, potentially because of specific nutrients present exclusively within the source/host

habitat and are not available in conventional culture media (Lewis, 2007; Davey, 2008). FISH allows the visualization of prokaryotic cells in their natural environment regardless of their culturability. The FISH method targets rRNA, which is essential to basic cellular metabolism and is thought to degrade soon after cell death (Nocker & Camper, 2009). Thus, this method is a very powerful tool for the detection and localization CP 868596 of unknown bacterial communities from a range of different habitats (Amann et al., 1995, 2001; Berchtold et al., 1999; Darby Ribonucleotide reductase et al., 2005;

Davidson & Stahl, 2006; Ferrari et al., 2006, 2008; Vartoukian et al., 2009), such as endosymbiotic bacteria that reside in invertebrates like insects within specific cells or symbiotic organs. Consequently FISH may facilitate isolation and potential cultivation of newly detected or previously uncultivable bacteria, as could be demonstrated recently (Vartoukian et al., 2010). A FISH approach using novel oligonucleotide probes was developed and demonstrated that essentially a ‘pure culture’ of the Pseudomonas-like pederin-producing endosymbionts of P. riparius covers the whole surface of P. riparius eggs, which extends previous reports suggesting that the endosymbiont is transmitted to the offspring via the egg (Kellner, 2001a, b, 2002a, b, 2003; Piel, 2002, 2004, 2005). Most bacteria appear to form biofilms, including P. aeruginosa, and such a multicellular mode of growth likely predominates in nature as a protective mechanism against hostile environmental conditions (Costerton et al., 1995; Costerton & Stewart, 2000). Consequently, this ability could also be existent for the Paederus endosymbiont because of its close relationship to P. aeruginosa (Kellner, 2002a; Piel, 2002; Piel et al., 2004).