ABSTRACT Although spirochetes were first detected in crystalline styles of bivalves more than 100 years ago, little is known about the characteristics of these consortia (commensalism or parasitism). The presence of spirochetes in bivalves can so far not be generalized. The purpose of this study was the detection and phylogenetic identification of spirochetes associated with crystalline styles of different marine bivalve species collected in temperate regions and Antarctica. Polymerase chain reaction amplification of spirochete 16S ribosomal gene sequences was performed. 16S ribosomal gene clones were identified by phylogenetic analysis, and the variability within each bivalve species was determined. The spirochetes were mainly related to yet uncultured or potentially pathogenic spirochetes from the marine environment. All identified spirochete clones fell into 2 families: the Spirochaetaceae with 2 genera, Cristispira and Spirochaeta, and the Brachyspiraceae, with the genus Brachyspira. The diversity of spirochetes in the crystalline style of each bivalve species was low. All clone sequences from crystalline styles of the oyster Crassostrea gigas clustered into the group of Cristispira species. Interestingly, these Cristispira spirochetes were previously found in Crassostrea virginica, another oyster species. The spirochete clones of each bivalve species formed distinct clusters. We therefore assume that the investigated bivalve
species harbor distinct populations of spirochetes. Although spirochetes were not found in all the investigated samples, the occurrence of spirochetes was not random and implies a closer association between the bivalve species and the specific spirochete cluster.
KEY WORDS: crystalline style, bivalve, spirochetes, digestive system
Spirochetes are a group of helically shaped motile bacteria with an enormous phenotypic diversity and spectrum of different habitats. In principle they consist of a typical coiled "protoplasmic cylinder," which contains cytoplasm and genomic regions, enfolded by a cytoplasmic membrane and a peptidoglycan layer (Johnson 1977). Like "rotating screws," spirochetes are able to generate friction in environments of high viscosity in which other flagellated bacteria are immobilized (Jarosch 1967, Greenberg & Canale-Parola 1977). Aerobic, facultative aerobic, or obligatory anaerobic bacteria of this group have been found in a wide range of habitats. Free-living spirochetes occur in sediments, microbial mats, and the water column of aquatic habitats. Furthermore, they have been reported from extremely selective habitats like salt marsh sediments, saline solar lakes, or deep-sea hydrothermal vents (Harwood & Canale-Parola 1984).
Host-associated spirochetes have so far been found mainly in the digestive tract of eukaryotes such as arthropods (Berlanga et al. 2007), molluscs (Romero & Espejo 2001), or different vertebrates, including humans (Dewhirst et al. 2000). Recently, spirochetes were also found in/on gills of Mediterranean cold seep clams (Duperron et al. 2007) and as symbionts in gutless worms (Dubilier et al. 1999, Blazejak et al. 2005, Ruehland et al. 2008). Most of these associated spirochetes are described as being part of the benign microflora, with proposed symbiotic relationships (Harwood & Canale-Parola 1984). Spirochete-host interactions are manifold and only a few have been well explored.
Some spirochetes are presumably involved in human gingivitis, whereas others are well known pathogens to both animals and humans. These include the...