Spirochetes in crystalline styles of marine bivalves: group-specific PCR detection and 16s RRNA sequence analysis.
Article Type: Report
Subject: Spirochetes (Research)
Spirochetes (Physiological aspects)
Spirochetes (Genetic aspects)
Bivalvia (Research)
Bivalvia (Physiological aspects)
Authors: Husmann, Gunnar
Gerdts, Gunnar
Wichels, Antje
Pub Date: 12/01/2010
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2010 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: Dec, 2010 Source Volume: 29 Source Issue: 4
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Germany Geographic Code: 4EUGE Germany
Accession Number: 247523228
Full Text: 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

INTRODUCTION

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 causative agents of syphilis (Treponema pallidum), swine dysentery (Serpulina hyodysenteriae), and Lyme disease (tick-borne symbionts Borrelia burgdorferi) (Harwood & Canale-Parola 1984, Loesche 1988, Stanton et al. 1996).

As early as 1882, Certes observed helical-shaped organisms in the digestive tracts of bivalves by microscopy (Certes 1882). Later they were characterized by morphological features and described as the spirochete bacteria Cristispira (Noguchi 1921). The genus Cristispira is named after its crest, a bundle of more than 100 periplasmic flagellae (Lawry et al. 1981, Tall & Nauman 1981). These spirochetes were microscopically observed in the digestive tracts of several marine bivalves (especially oysters) and some freshwater species. They seem to be limited to the crystalline styles.

The ecology of the adaptation of spirochetes to this particular morphological structure of bivalves remains a puzzle. Even the physiology and function of the crystalline style itself is still poorly understood. Styles are noncellular cylindrical rods of a gelatinous texture. They consist of a mixture of water, proteins, carbohydrates, and inorganic matter. The composition and dimensions depend on bivalve species and environmental conditions. The tip of the style extends into the stomach of the bivalve. The main function seems to be digestive (Alyakrinskaya 2001). Rotated by cilia, in the style diverticulum the style generates friction against the solid gastric shield of the bivalve. In this way, it promotes grinding of detritus or plankton by acting like a pestle (Yonge 1923, Bernard 1973).

The hypothesis that style-associated spirochetes might play a major role in extracellular enzymatic digestion processes of bivalve hosts has never been verified in this context. Crystalline style spirochetes have been detected by light microscopy, but for further identification of the bacteria, morphological criteria are far too vague (Amann et al. 1995). Because of their unknown physiology and complex in vitro conditions, spirochetes from crystalline styles have not been cultivated as of yet. The recent discovery of spirochetes in different environments was achieved mainly with culture-independent molecular methods. The phylogenetic positioning of the genus Cristispira (Paster et al. 1996) was determined by 16S recombinant RNA (rRNA) gene sequence analysis from Cristispira-like organisms present in material from crystalline styles. Based on all these previous findings, the current study aims to detect and identify crystalline style-associated spirochetes in bivalves from different habitats and geographical regions using spirochete-specific ribosomal polymerase chain reaction (PCR) primers.

METHODS

Study Site and Sample Collection

Specimens were collected in summer 2006 (June and July) from 4 sites in the German Bight, North Sea. Mya arenaria and Cerastoderma edule were collected in sandy tidal flat areas off the coastline of the German island of Sylt and in nearshore waters in the area of the Weser estuary. Arctica islandica was sampled offshore near the island of Helgoland by dredging. Crassostrea gigas was collected in sandy tidal flat areas of Sylt. Antarctic Laternula elliptica were collected by divers during an expedition to King George Island (62[degrees]14' S, 58[degrees]40' W), western Antarctic Peninsula (October to December 2005) at a depth of 10-15 m. Samples of crystalline styles were frozen (-80[degrees]C) until analysis.

Preparation of Crystalline Styles and Genomic DNA Extraction

Crystalline styles were prepared and the gut, digestive glands, and covering tissue of the bivalves were removed. In total 170 DNA extracts of crystalline styles were obtained. Each tissue sample was stored separately at -20[degrees]C until DNA extraction.

For DNA extraction of crystalline styles with a more solid texture (like those from M. arenaria), the FastDNA kit (Qbiogene, Heidelberg, Germany) and the Fastprep FP 120 instrument (Qbiogene) were used to disrupt the tissue mechanically. Up to 200 mg of the sample was applied according to the manufacturer's recommendation. After disruption of the style matrix and cells (Fastprep FP 120 instrument, level 4, 30 sec), the homogenate was centrifuged (12,000g, 10 min). The supernatant was subjected to proteinase K (1 mg) for 90 min at 56[degrees]C and afterward was added to a spin column (Qiaquick, Qiagen, Hilden, Germany) for DNA purification. Binding buffer and ethanol were added, and DNA was isolated by a selective binding step on the column matrix, followed by an elution step, as described by the manufacturer (Qiagen). DNA extracts were frozen at 20[degrees]C until further analysis.

Softer and more viscous crystalline styles (like those from A. islandica, C. edule, and L. elliptica) were crushed and mechanically homogenized in STE buffer by a tissue mill, which was composed of a small aseptic pestle used in combination with a spinning motor (Kontes, Vineland, NJ). Cell lysis was performed by adding lysozyme (1 mg) and SDS (1%) for 60 min at 37[degrees]C. DNA extraction was carried out using phenol chloroform. After precipitation of the DNA with isopropanol and cleaning with ethanol (75 %), all DNA extracts were kept in TE buffer solution. The DNA extracts from the crystalline styles showed varying DNA quality and concentration as revealed by agarose gel electrophoresis (0.8%, w/v). After ethidium bromide staining of the gels, the PCR products were visualized with the ChemiDoc XRS System (BioRad, Munchen, Germany).

Detection of Spirochetes by Polymerase Chain Reaction

Purified DNA extracts from crystalline styles were used with different sets of PCR primers targeting eubacteria or spirochetes (Table 1). One primer pair (341f-GC and 907r) was used for general detection of bacteria by amplification of a 550-bp fragment from the region V3 of eubacterial 16S rRNA (Muyzer et al. 1995). A 100-[micro]L PCR mixture contained 10 [micro]L 10x Taq buffer (Eppendorf, Hamburg, Germany), 15 [micro]L 5X Master Enhancer (Eppendorf), 300 [micro]M each dNTP (Promega, Mannheim, Germany), 0.3 [micro]M each primer, and 2 U Taq DNA polymerase (Eppendorf). Because of the varying nucleic acid concentrations of the extracts, different volumes of template DNA were applied (0.2-5 [micro]L undiluted or 1 [micro]L of dilutions 1:10, 1:100, 1:250). PCR was performed as described by Sapp et al. (2007).

For amplification of the entire spirochete 16S rRNA (~1,500 bp), the spirochete-specific reverse primer C90 (Dewhirst et al. 2000) in combination with the universal primer C75 was used (Table 1). In addition, another, shorter specific fragment (550 bp) was amplified using C90 in combination with primer 907f (Table 1), because the amplification of the shorter fragment was successful for more samples. The PCR thermal profiles were carried out using the method described by Paster et al. (1996): initial melting at 95[degrees]C for 3 min, followed by 30 cycles of melting (95[degrees]C, 45 sec), annealing (55[degrees]C, 45 sec), and extension (68[degrees]C, 90 sec). The final extension was programmed at 68[degrees]C (15 min).

Both spirochete-specific primer sets were used for PCR with each DNA sample. The reaction mixture with a total amount of 50 [micro]L for each PCR vial contained 5 [micro]L 10x Taq buffer, 10 [micro]L 5x Master Enhancer, 300 [micro]M each dNTP, 1 [micro]M each primer (907f/ C90 or C75/C90), 2 U Taq DNA polymerase, and 0.2-3 [micro]L template. For additional cloning, either the long or the short spirochete-specific fragment was used.

Cloning and Sequencing of Spirochete-Specific Ribosomal Genes

Cloning of selected 16S ribosomal gene fragments from crystalline styles (1,500 bp or 550 bp) was performed using the TOPO-cloning kit (Invitrogen, Darmstadt, Germany) following the manufacturer's protocol. In total, 6 spirochete-specific PCR products from crystalline styles of A. islandica (907f/C90 and C75/C90), C. edule (907f/C90 and C75/C90), C. gigas (907f/C90), and L. elliptica (907f/C90) were cloned. Transformed Escherichia coli were plated onto LB agar plates (added Kanamycin 50 mg/ mL). After incubation (overnight, 37[degrees]C) at least 50 colonies for each of the 6 cloning experiments were transferred to a "master plate." For a rapid screening to distinguish unique from duplicate clones, single base sequencing was performed with 1 dideoxynucleotide; ddG was used in this case (Schmidt et al. 1991). DNA profiling was performed with an automatic DNA sequencer (Licor Biosciences, Bad Homburg, Germany). The clones from each cloning experiment were grouped into clone types. A single clone from each clone type was subsequently analyzed by DNA sequencing. For sequencing, the inserts of the respective clone types were amplified using T3 and T7 primers, and the DNA was purified via the Qiaquick PCR purification kit (QIAGEN, Hilden, Germany) following the instructions of the manufacturer's protocol. Products were checked by electrophoresis on 1.2% (w/v) agarose gels. Sequencing was carried out by QIAGEN GmbH (Hilden, Germany) using an ABI PRISM 3700 DNA Analyzer (Applied Biosystems, Carlsbad, CA). The sequencing primers used were T3, T7, and 907r. Nearest relatives were searched by BLAST (http://www.ncbi.nlm.nih.gov).

Phylogenetic Analysis

Sequence data were checked for the presence of PCR-amplified chimeric sequences using the CHECK_CHIMERA program (Cole et al. 2003). The ARB software package (http://www.arb-home.de) was used for phylogenetic analysis (Ludwig et al. 2004). After addition of sequences to the ARB 16S ribosomal sequences database (release May 2005), alignment was carried out with the Fast Aligner integrated in the program and refined by comparison of closest relatives retrieved by BLAST. Sequences with more than 1,300 nucleotides were used to calculate phylogenetic trees. The ARB "parsimony interactive" tool was used to add partial sequences (shorter than 1,300 bp) to respective trees. Phylogenetic relationships were deduced by the neighbor joining method, including the correction algorithm devised by Felsenstein (1993).

Nucleotide Sequence Accession Numbers The sequences obtained in this study are available from GenBank under accession numbers EU857733-EU857763.

RESULTS

By prescreening for the presence of bacterial DNA by PCR using universal eubacterial primers, eubacteria were detected in 130 of 170 crystalline styles (Table 2). From these 130 DNA extracts, 62 samples revealed a spirochete-specific PCR fragment, amplified with the primers 907f/C90 (Table 1). Although bacteria were identified, on average, in 77% of the investigated samples, the spirochetes were present in 48 % of all investigated crystalline styles (Table 2). Styles from M. arenaria, C. edule, and C. gigas also harbored spirochetes (14%, 67%, and 28%, respectively; Table 2), although the PCR fragment of M. arenaria did not show the appropriate size. The crystalline styles of A. islandica and L. elliptica revealed only a few positive bacterial signals (Table 2), but, interestingly, all of them were also identified as spirochete-specific DNA.

Each DNA extract revealing a positive spirochete signal was again amplified with a second set of specific primers: C90/C75. The aim was to obtain the full sequence of the spirochete ribosomal gene (1,500 bp). This larger fragment was successfully amplified in only 5 of 62 samples: 2 of which came from A. islandica, 1 from C. edule, and 2 from C. gigas. The PCR fragments obtained for M. arenaria did not show the appropriate size and were therefore excluded from further cloning experiments.

Phylogenetic Analysis

The spirochete-specific PCR fragments obtained from 1 crystalline style of A. islandica (PCR fragments 907f/C90 and C75/ C90), C. edule (PCR fragments 907f/C90 and C75/C90), C. gigas (PCR fragments 907f/C90), and L. elliptica (PCR fragment 907f/C90) were cloned, resulting in a total of 300 clones. After screening for the correct amplicon, positive clones were collected (29 for A. islandica, 33 for C. edule, 13 for C. gigas, and 20 for L. elliptica). In total, 31 different amplicons were identified (Table 3) by single base DNA profiling (Schmidt et al. 1991) and subsequently sequenced. Clones with a similarity greater than 98% were assumed to be identical and were ultimately combined to 7 clone types of spirochetes (A. islandica, 2; C. edule, 2; C. gigas, 2; L. elliptica, 1; Table 3).

As determined by BLAST search, all clone types were generally related to previously published 16S rRNA sequences from spirochetes. The closest relatives found in the BLAST search are listed in Table 3. The results revealed some close matches to known spirochete sequences; clone types from crystalline styles of C. gigas displayed 97% similarity to 16S rRNA gene sequences in GenBank. All other clones were more distantly related to spirochete 16S rRNA gene sequences in GenBank (83%-92%).

Each investigated bivalve harbored distinct spirochete groups that were again only distantly related to groups from the other investigated bivalve species. All clones from A. islandica (29 in total) were related to spirochete clones from estuarine microbial mats from the Ebro Delta (83%-91% similarity, AY605168). In this study, 2 clone types (I1 and I14) were placed in this particular cluster. From a crystalline style of C. edule, 2 similar clone types were retrieved (from a total of 33 clones), all of which were related to spirochete clones from deep-sea coral samples (e.g., DQ395980, DQ395500), showing similarities of 91% and 83%, respectively. All but 1 clone from C. gigas was related to "Cristispira sp. CP1" (U42638), showing 97% sequence identity. This sequence has previously been identified in crystalline styles from the same bivalve species (Paster et al. 1996). One clone showed a lower similarity to the sequence of Spirochaeta asiatica (88%, X93926). For the Antarctic bivalve L. elliptica, all clones were identical and were related to a spirochete clone sequence AY499871 from the sediments of an Australian fish farm showing low similarity using BLAST search (91%).

A neighbor-joining tree of the spirochetes revealed a more detailed view of the relationship of the spirochete sequences. Each representative of a bivalve species harbored specific spirochetes that fell clearly into different clusters (Fig. 1). The representative clones were placed next to their nearest neighbor. One clone type from C. gigas clustered with a Cristispira sp. CP1 sequence from a crystalline style of C. gigas (Paster et al. 1996). One single clone found in C. gigas (CS3) did not branch into this cluster, but grouped together with the spirochete isolate Spirochaeta aurantia (M57740) as well as a DNA sequence retrieved from the sediments of an Australian fish farm (AY499871). All clones of C. edule clustered together with a number of DNA sequences retrieved from the gills of M. arenaria and from deep-sea octacoral samples (e.g., DQ395500). All clones found in L. elliptica were best matched with spirochete clones from microbial mats from the Ebro Delta (AY605175). In the neighbor-joining tree, these clones were only distantly related to those that matched best by BLAST algorithm. All clones from A. islandica fell together with spirochete clones from microbial mats from the Ebro Delta (AY605168) and were distantly related to Brachyspira innocence (M57744) in the genus Brachyspira.

DISCUSSION

Since the discovery of spiral-shaped organisms in oysters by Certes (1882), who first assigned them to the phylum protozoa according to their complex structure and dimensions (Trypanosoma balbianii), these bacteria and their proposed habitat, the crystalline style of bivalves, have rarely been investigated. This study aims to deliver phylogenetic details of spirochete groups present in crystalline styles of selected bivalve species from different habitats. Cristispira sp.-like spirochetes were micro scopically identified in early 1921 (Noguchi 1921) in 3 of 10 investigated bivalve species from North America (including oysters, Venus and Modiolus spp.). Later, they were found in several other bivalves (Margulis & Hinkle 2006). The 16S rRNA sequence of Cristispira sp. was first obtained from C. gigas styles in 1996 (Paster et al. 1996).

Our survey on crystalline style-associated spirochetes is based on culture-independent molecular tools and PCR techniques, because "culturability" of spirochetes could not be postulated per se. From most of the style samples, we successfully isolated DNA of sufficient quality for the following molecular analysis. We applied a sensitive and specific PCR method to detect spirochetes in crystalline styles of bivalve species. Bacterial 16S rRNA genes were identified in most of the investigated crystalline styles using universal bacterial ribosomal primers. The presence of spirochete DNA in the crystalline styles of the investigated bivalve species was indicated by PCR products using the respective specific spirochete primers. Although the spirochete PCR signal did not occur in each sample, the results suggest that spirochetes are widespread in all the bivalve species tested. However, because spirochetes were only present in a part of all investigated crystalline styles (48%), they are presumably not essential symbionts of these bivalves.

The clone libraries of the spirochete-specific PCR products showed that clones retrieved from all investigated bivalve species matched sequences of the spirochetal group. Clones related to Cristispira sp. CP1 were only found in C. gigas (similarity, 97%). The phylogenetic analysis of sequences from styles of C. edule, A. islandica, and L. elliptica also revealed the presence of spirochetes. Only the results for M. arenaria were ambiguous, and this species was not used for further analysis. Different spirochete clones from 1 bivalve species formed distinct clusters. In addition, there were clear differences between spirochetes of different bivalve species (Fig. 1), indicating specific spirochete populations in each of those bivalve species. Collectively, all identified spirochete clones fell into 2 families: the Spirochaetaceae with 2 genera, Cristispira and Spirochaeta, and the Brachyspiraceae, with the genus Brachyspira (Paster & Dewhirst 2000, Leschine et al. 2006, Stanton 2006). Identification of Cristispira sp. CP1 confirms the microscopy observations of previous investigators (Noguchi 1921, Lawry et al. 1981, Tall & Nauman 1981), who already discussed a specific association of Cristispira with crystalline styles of oysters. The genus Cristispira of the Spirochaetaceae has not been found outside of bivalve species or in other bivalves than in Crassostrea sp. so far. This was also confirmed in this study. Identification of Cristispira sp. in C. gigas collected in geographically distinct areas and repeatedly over a long period of time support the hypothesis of a close relationship between these specific spirochetes and bivalve hosts, or even coevolution of the organisms. Localization of Cristispira in the crystalline style matrix of C. gigas (Tall & Nauman 1981), which is a component of the digestive system, may prevent them from digestion and exposure to absorptive epithelial cells and phagocytosis (Bernard 1973). Host-specific physiologies, such as low concentrations of enzymes or rates of phagocytosis (Bernard 1973) in the alimentary system, for example, could explain the exclusive occurrence of Cristispira in oysters. For Cristispira, the crystalline style itself may serve as a food source. Up to 30% of the crystalline style matrix consists of carbohydrates (Alyakrinskaya 2001), which may serve as source of energy. Excretion of proteolytic enzymes, discussed by Lawry et al. (1981) and Harwood and Canale-Parola (1984), could promote the digestion of the style protein components needed for amino acid metabolism of some spirochetes (Canale-Parola 1977).

[FIGURE 1 OMITTED]

Bacteria of the genus Spirochaeta were found in three of the four investigated bivalves: C. edule, L. elliptica, and C. gigas. In a previous study (unpublished data), Spirochaeta were also identified in gill samples of two other bivalve species (Scrobucilaria sp., M. arenaria) and in the gills of L. elliptica. Several DNA sequences analyzed by DGGE, which were obtained from bivalve gills, were related to the genus Spirochaeta. The gill might be a reservoir for spirochetes that are recruited from the environment and somehow transferred to the styles. Nevertheless, the genus Spirochaeta contains a huge number of different spirochete species that were retrieved from different environments, such as microbial mats or sediments. Symbiotic Spirochaeta from gutless worms, including Olavius algarvensis, Olavius crassitunicatus, and Olavius loisae (Blazejak et al. 2005, Ruehland et al. 2008), also belong to this genus. Our DNA sequences, however, are only distantly related to these endosymbiotic spirochetes. It is well known that many species of the genus Spirochaeta are also common in sediments (Blazejak et al. 2005, Leschine et al. 2006). Spirochaeta were often described as anaerobic fermenters of carbohydrates and show a strong chemotaxis toward high concentrations of cellobiose or degraded cellulose compounds (Harwood & Canale-Parola 1984, Leschine et al. 2006). Hence, it is most likely that these organisms can be observed in sediments and crystalline styles as a result of the physiochemical conditions providing them with special niches.

The sequences of spirochetes from the crystalline style of A. islandica all belong to a distinct cluster branching with sequences affiliated with the Brachyspiraceae. The closest related sequence was a spirochete clone from sediment of the Ebro Delta (Mediterranean Sea). Next relatives within the genus Brachyspira are species of Brachyspira sp., which are potentially pathogenic for vertebrates (Pettersson et al. 1996). Whether these spirochetes are pathogenic for the investigated bivalves is a question that needs to be examined in the future. Concerning the increasing importance of aquaculture for human nutrition, the microbiomes of bivalves have to be examined in the future more briefly. In this context, the scientific focus that is currently on well-known pathogens (e.g., Vibrio spp.) must be expanded to other bacterial groups like spirochetes, because we know that they are present in several bivalve species, but the character of the association and its function remains enigmatic until now.

ACKNOWLEDGMENTS

We thank Birgit Hussel (Alfred Wegener Institute Sylt) for assistance in collecting samples and Doris Abele (Alfred Wegener Institute Bremerhaven) for providing crystalline styles of Laternula elliptica from King Georg Island, Antarctica. We thank Katherina Schoo for improving the manuscript.

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GUNNAR HUSMANN, (1) GUNNAR GERDTS (2) AND ANTJE WICHELS (2), *

(1) Alfred Wegener Institute Foundation for Polar and Marine Research, Am Handelshafen 12 27570 Bremerhaven, Germany; (2) Alfred Wegener Institute Foundation for Polar and Marine Research, Biologische Anstalt Helgoland, PO Box 180, 27483 Helgoland, Germany

* Corresponding author. E-mail: Antje.Wichels@awi.de
TABLE 1.
Otigonucleotides used in this study.

Primer   Target/Target sequence          Primer sequence (5'-3')

341f     Bacterial V3-region 16S-rDNA    CCTACGGGAGGCAGCAG
907r     Bacterial V3-region 16S-rDNA    CCGTCAATTCMTTTGRTT

907f     Bacterial V3-region 16S-rDNA    AACSAAAMGAATTGACGC
C75      Bacterial 16S-rDNA              GAGAGTTTGATMCTGGCTCAG
C90      Spirochete 16S-rDNA             GTTACGACTTCACCCTCCT

Primer   Reference

341f     Muyzer et al. (1993)
907r     Modified, Wichels
           et al. (2006)
907f     Modified, this article
C75      Paster et al. (1996)
C90      Paster et al. (1996)

TABLE 2.
Amplification of bacterial and spirochete-specific 16S rDNA
with specific primers as listed in Table 1.

                                             PCR       Products

                      No. of Crystalline   Bacteria   Spirochete
Bivalve Species             Styles         Specific    Specific

Mya arenaria                  43              43       6 (14% *)
Arctica islandica             20               4       4 (20%)
Cerastoderma edule            46              45      31 (67%)
Crassostrea gigas             43              29      12 (28%)
Laternula elliptica           18               9       9 (50%)

* Percentage of all investigated crystalline styles.
Templates: DNA from crystalline styles of marine bivalves.

TABLE 3.
Phylogenetic affiliation of bacterial 16S rRNA gene sequences
isolated from crystalline styles of marine bivalves.

                     Clone   No. of
Species              Type    Clones        Best Match

Arctica islandica    I1        20     Uncultured spirochete
                                        clone LH020
                     I14        9     Uncultured spirochete
                                        clone LH020
Cerastoderma edule   IV2        3     Uncultured spirochete
                                        clone CGOAB15
                     IV5       30     Uncultured spirochete
                                        clone CGOGA33
Crassostrea gigas    V5        12     Cristispira sp. CPI
                     V3         1     Spirochaeta asiatica
Laternula elliptica  V12       20     Uncultured spirochete
                                        clone Dover 173

                     Clone   Phylogenetic   Similarity
Species              Type       Group          (%)        bp

Arctica islandica    I1      Spirochaetes       83         601
                     I14     Spirochaetes       91       1,425
Cerastoderma edule   IV2     Spirochaetes       91       1,430
                     IV5     Spirochaetes       92       1,473
Crassostrea gigas    V5      Spirochaetes       97         594
                     V3      Spirochaetes       88         580
Laternula elliptica  V12     Spirochaetes       91         594

                     Clone
Species              Type    Ace. No.

Arctica islandica    I1      AY605168
                     I14     AY605168
Cerastoderma edule   IV2     DQ395500
                     IV5     DQ395980
Crassostrea gigas    V5      U42638
                     V3      X93926
Laternula elliptica  V12     AY499871

The number of clones of each clone type is given in column 3.
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