Two types of endosymbiotic bacteria were identified in the gastrodermis of the marine invertebrate Xenoturbella bocki (Xenoturbellida, Bilateria). While previously described Chlamydia-like endosymbionts were rare, Gammaproteobacteria distantly related to other endosymbionts and pathogens were abundant. The endosymbionts should be considered when interpreting the poorly understood ecology and evolution of Xenoturbella.Xenoturbella bocki is a benthic marine worm first described in 1949 and only found at a few geographical locations at apparently low population densities. It has a very simple body plan ( Fig. 1A) with a blind gut (cul-de-sac) and lacks coelomic cavities, a brain, and reproductive and excretory organs (35). Xenoturbella was originally classified as a flatworm, but molecular analyses have placed it within its own animal phylum, with various affiliations (reviewed in reference 34). The most recent phylogenetic analyses using mitochondrial genome and phylogenomic data indicate a position either within Deuterostomia (5) or at the base of Bilateria (14). Thus, the evolutionary history of Xenoturbella remains elusive and it is unclear whether its simple body plan represents a plesiomorphic character or, alternatively, is the result of secondary loss (34). In addition, the ecology of Xenoturbella is not well understood, leaving, e.g., its food source unresolved (4, 18).Symbiotic bacteria are found in a remarkable number of diverse marine invertebrates (11), where they affect both the ecology and the evolution of their hosts. The first indications of intracellular endosymbionts in Xenoturbella were published by Israelsson (17) and confirmed by electron microscopy of a Xenoturbella spermatid cluster (Fig. 1B); however, our first molecular analyses identified the putative endosymbionts as Gammaproteobacteria, whereas Israelsson (17) had reported a group of endosymbiotic Chlamydiae. The objectives of this study were therefore (i) to identify the putative endosymbionts by 16S rRNA gene analysis, (ii) to localize them inside the host by fluorescence in situ hybridization (FISH), and (iii) to specifically test for the prevalence of the gammaproteobacterial endosymbionts in Xenoturbella.Sample collection and preparation. Seven specimens of X. bocki were collected at two different sites on six different dates in 2005 (GPS position: 58°16ЈN, 11°26ЈE) and 2006 (GPS position: 58°17ЈN, 11°31ЈE) by dredging and sieving of surface sediment at a 100-m water depth in the Gullmar Fjord, Sweden (4). Whole animals were fixed in 4% (wt/vol) paraformaldehyde in sterile seawater and stored in 70% ethanol. Two spermatid clusters (i.e., aggregations of male gametes at the stage of maturation just before becoming fully mature spermatozoa) could be dissected from two whole animals and were preserved in 98% ethanol; since these dissected animals could not be utilized, a total of five whole animals and two spermatid clusters were available for further analysis. The identity of the X. bocki specimens and spermatid cluster was confirmed...