Environmental transmission of a sulfur-oxidizing bacterial gill endosymbiont in the tropical lucinid bivalve Codakia orbicularis

Abstract

Codakia orbicularis is a large tropical member of the bivalve mollusk family Lucinidae which inhabits shallow-water sea-grass beds (Thalassia testudinum environment) and harbors sulfur-oxidizing endosymbiotic bacteria within bacteriocytes of its gill filaments. When a C. orbicularis-specific 16S rDNA (DNA encoding rRNA) primer is used with a bacterium-specific 16S rDNA reverse primer in amplifications by PCR, the primer set was unsuccessful in amplifying symbiont DNA targets from ovaries, eggs, veligers, and metamorphosed juveniles (600 m to 1 mm in shell length) cultivated in sterile sand, whereas successful amplifications were obtained from gill tissue of adult specimens and from metamorphosed juveniles (600 m to 1 mm in shell length) cultivated in unsterilized sea-grass bed sand. To ascertain the presence of the symbiont target in juveniles, restriction fragment length polymorphism analysis, Southern blotting, and transmission electron microscopy were used. Specific hybridizations and observation of endosymbiotic bacteria in the gills of numerous juveniles cultivated in unsterilized sea-grass bed sand showed that the sulfur-oxidizing endosymbionts of C. orbicularis are environmentally transmitted to the new generation after larval metamorphosis.

Putative environmental transmission of sulfur-oxidizing bacterial symbionts in tropical lucinid bivalves inhabiting various environments

“…Comparable PCR experiments have been performed with vertically transmitted symbionts and no problem of detection occurred in the bivalve gonads with template concentration identical or below those we used [17,19]. Moreover, the detection levels of the primer sets used in this study are of the same order of magnitude as those previously described [17,20]. In the case of C. orbicularis, juveniles reared in sterile conditions did not host symbiont although juveniles issued from the same spawn but cultivated in unsterilized sea-grass sand were infected [20].…”

“…The genomic DNA of a bacterium is about 10 fg, and we worked with a mixed genomic DNA for which the ratio prokaryotic DNA/total (eukaryotic and prokaryotic) DNA was unknown, but undoubtedly decreases the detection level under 10 Q cells. For the C. orbicularis primer set, this detection limit was not improved even after Southern blotting and hybridization with a speci¢c probe [20].…”

“…Total DNA was extracted from symbiont-containing gill tissues and Vibrio alginolyticus as described elsewhere [10,21]. Following the recent study on the gill endosymbiont transmission mode in C. orbicularis [20], DNA was extracted from gonad tissues by using the Isoquick Nucleic Acid Extraction kit (ORCA Research Inc., Bothell, USA) according to the manufacturer's instructions. Samples of DNA were independently obtained from eight specimens (four males and four females) for each lucinid species with the exception of L. pensylvanica, of which only two specimens of each sex were available.…”

“…This sequence was used as PCR reverse primer SymLp (5P-TCGCAGGTTCGCTACCGT-3P; Escherichia coli 1252 to 1269 [23]) with the bacterial-speci¢c primer 27f [24]. The primer set Symco1-1492r described elsewhere [20] was used for PCR detection of C. orbicularis symbiont and consequently of C. orbiculata, C. pectinella and L. pensylvanica symbiont.…”

“…Environmental transmission mode is strongly suggested in two oligochaetes [15] and in two vestimentiferans [16]. In bivalves, the eight species studied harbor gill endosymbionts which are vertically transmitted [16^19], except the tropical lucinid Codakia orbicularis in which symbionts are environmentally transmitted to the new host generation [20].…”