Stingl, U.

ABSTRACT Uncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of the Mollicutes and are only distantly related (<82% sequence identity) to members of the Mycoplasmatales and Entomoplasmatales . Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name “ Candidatus Hepatoplasma crinochetorum” is proposed for this new taxon of Mollicutes colonizing the hepatopancreas of P. scaber .

The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genusStaurojoenina. By using scanning electron microscopy, it was shown that the whole surface ofStaurojoeninasp. from the termiteNeotermes cubanusis densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among theBacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellateMixotricha paradoxa. The closest cultivated relative wasTannerella forsythensis(<85 % sequence identity). Fluorescencein situhybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts ofStaurojoeninasp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface ofStaurojoeninasp., we propose to classify this new taxon ofBacteroidalesunder the provisional name ‘CandidatusVestibaculum illigatum’.