“ Endomicrobia ”: Cytoplasmic Symbionts of Termite Gut Protozoa Form a Separate Phylum of Prokaryotes

Abstract

Lignocellulose digestion by wood-feeding termites depends on the mutualistic interaction of unusual, flagellate protists located in their hindgut. Most of the flagellates harbor numerous prokaryotic endosymbionts of so-far-unknown identity and function. Using a full-cycle molecular approach, we show here that the endosymbionts of the larger gut flagellates of Reticulitermes santonensis belong to the so-called termite group 1 (TG-1) bacteria, a group of clones previously obtained exclusively from gut homogenates of Reticulitermes speratus that are only distantly related to other bacteria and are considered a novel bacterial phylum based on their 16S rRNA gene sequences. Fluorescence in situ hybridization with specifically designed oligonucleotide probes confirmed that TG-1 bacteria are indeed located within the flagellate cells and demonstrated that Trichonympha agilis (Hypermastigida) and Pyrsonympha vertens (Oxymonadida) harbor phylogenetically distinct populations of symbionts (<95% sequence similarity). Transmission electron microscopy revealed that the symbionts are small, spindle-shaped cells (0.6 m in length and 0.3 m in diameter) surrounded by two membranes and located within the cytoplasm of their hosts. The symbionts of the two flagellates are described as candidate species in the candidate genus "Endomicrobium." Moreover, we provide evidence that the members of the TG-1 phylum, for which we propose the candidate name "Endomicrobia," are phylogenetically extremely diverse and are present in and also restricted to the guts of all lower termites and wood-feeding cockroaches of the genus Cryptocercus, the only insects that are in an exclusive, obligately mutualistic association with such unique cellulose-fermenting protists.Digestive symbioses are most common among insects feeding on wood or other lignified plant materials. The most prominent example is that of wood-feeding termites (order Isoptera), where the symbiotic digestion of lignocellulose is a complex series of events involving both the host and its gut microbiota. While the events in foregut and midgut are due mainly to host activities, the digestive processes in the hindgut are largely controlled by the symbiotic gut microbiota (5, 7).In the evolutionarily lower termites, the bulk of the hindgut volume is occupied by unicellular eukaryotes which belong to several flagellate taxa that are unique to termites (14, 37). These oxygen-sensitive protozoa, which can make up more than one-third of the body weight of their host, are essential for wood digestion and represent the major source of cellulolytic and xylanolytic activities in the hindgut (14, 16).The gut flagellates are regularly colonized by prokaryotic epibionts attached to the surface and by endosymbionts located in the cytoplasm or in the nucleus (see, e.g., references 1, 8, and 18). Although up to 85% of the total prokaryotes in the termite gut are associated with the gut flagellates (2), only a few of the epibiotic bacteria have been identified by using molecular techniques (23,35,36). Wh...

Bacteroidales ectosymbionts of gut flagellates shape the nitrogen-fixing community in dry-wood termites

“…There is abundant evidence that the bacterial symbionts of most gut flagellates are specific for and even co-speciate with their respective hosts (for example, Stingl et al, 2005;Noda et al, 2006Noda et al, , 2007Noda et al, , 2009Ikeda-Ohtsubo et al, 2007;Ohkuma et al, 2007;Ikeda-Ohtsubo and Brune, 2009;Desai et al, 2010;Strassert et al, 2010). The nitrogenfixing nature of the bacterial symbionts has been documented for at least two of these symbiotic pairs (Hongoh et al, 2008b and this study) and may indeed be the evolutionary driver for many of these associations.…”

Endosymbiotic associations within protists

“…For the digestion of lignocelluloses, the predominant component of woody plants, lower termites rely on anaerobic flagellates of the Parabasalia and Oxymonadida that are located in their hindgut; the majority of which harbour numerous prokaryotic endosymbionts (Ohkuma 2003). Widespread and often predominating in the gut microbial communities are bacteria belonging to the candidate phylum Termite Group 1 (TG1) of the Bacteroidales (Stingl et al 2005;Ohkuma et al 2007). The endosymbiotic bacteria colonize the cytoplasm of their flagellate hosts in large numbers and are surrounded by two membranes; the outermost membrane being either derived from the outer membrane of the Gramnegative bacterium or from the host (Stingl et al 2005).…”

“…Widespread and often predominating in the gut microbial communities are bacteria belonging to the candidate phylum Termite Group 1 (TG1) of the Bacteroidales (Stingl et al 2005;Ohkuma et al 2007). The endosymbiotic bacteria colonize the cytoplasm of their flagellate hosts in large numbers and are surrounded by two membranes; the outermost membrane being either derived from the outer membrane of the Gramnegative bacterium or from the host (Stingl et al 2005). Since neither endosymbiotic bacteria nor the flagellates are cultivable, these symbiotic associations are not readily accessible for research.…”

Developmental microbial ecology of the crop of the folivorous hoatzin

“…Among bacteria that appear late in the succession, Deferribacteres are important in the degradation of amino acids under anaerobic conditions (Godon et al, 1997), suggesting that the redox potential in the chick crop may be too high (not reduced enough) to support such strict anaerobes. Termite group 1 represent a deep branch in the 16S rRNA gene (Hugenholtz et al, 1998) comprising bacteria associated with flagellate protists in woodfeeding insects (Stingl et al, 2005), probably here these bacteria are associated with crop ciliate protozoa contributing to the degradation of the secondary plant cell-wall compounds. Among other bacterial phyla with members involved in fiber degradation, Lentisphaerae and Verrucomicrobia are linked with cellobiose degradation (Zoetendal et al, 2003), which require a low redox potential likely explaining the low abundance of these taxa in the hatchling crop.…”