Extensive variation in intracellular symbiont community composition among members of a single population of the wood-boring bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)

Abstract

Shipworms (wood-boring bivalves of the family Teredinidae) harbor in their gills intracellular bacterial symbionts thought to produce enzymes that enable the host to consume cellulose as its primary carbon source. Recently, it was demonstrated that multiple genetically distinct symbiont populations coexist within one shipworm species, Lyrodus pedicellatus. Here we explore the extent to which symbiont communities vary among individuals of this species by quantitatively examining the diversity, abundance, and pattern of occurrence of symbiont ribotypes (unique 16S rRNA sequence types) among specimens drawn from a single laboratory-reared population. A total of 18 ribotypes were identified in two clone libraries generated from gill tissue of (i) a single specimen and (ii) four pooled specimens. Phylogenetic analysis assigned all of the ribotypes to a unique clade within the gamma subgroup of proteobacteria which contained at least five well-supported internal clades (phylotypes). By competitive quantitative PCR and constant denaturant capillary electrophoresis, we estimated the number and abundance of symbiont phylotypes in gill samples of 13 individual shipworm specimens. Phylotype composition varied greatly; however, in all specimens the numerically dominant symbiont belonged to one of two nearly mutually exclusive phylotypes, each of which was detected with similar frequencies among specimens. A third phylotype, containing the culturable symbiont Teredinibacter turnerae, was identified in nearly all specimens, and two additional phylotypes were observed more sporadically. Such extensive variation in ribotype and phylotype composition among host specimens adds to a growing body of evidence that microbial endosymbiont populations may be both complex and dynamic and suggests that such genetic variation should be evaluated with regard to physiological and ecological differentiation.

FIG. 1.

Phylogenetic relationships among 18 unique symbiont ribotypes (RTs) identified in the combined SSL and MSL. Brackets (P1 to P5) indicate clades (phylotypes) that can be distinguished by CDCE with the primers 948f and 1063r-GC. Bold values in parentheses indicate the numbers of clones in the combined libraries belonging to a specific ribotype. The tree was inferred by an evolutionary-distance method (PAUP 4.0, with minimum evolution optimality criterion and HKY85 correction; 1,234 bp were considered in the analysis). Bootstrap proportions are expressed as a percentage of 1,000 replicates and are indicated on the tree for nodes with values >50%. The reference sequences were T. turnerae T7902^T AY028398 and “S. degradans” 2-40 AF055269.

FIG. 2.

CDCE profiles obtained from gills of the shipworm L. pedicellatus after PCR amplification with primers 948f and 1063r-GC. (A) Combined gills from four specimens used for the MSL but not included among the 13 individual specimens examined. (B to H) CDCE profiles from gills of seven representative specimens of L. pedicellatus, each showing a different combination of identified peaks. The identities of peaks P1 to P5 were confirmed by comigration with PCR products from sequenced clones spiked into samples after PCR and prior to CDCE. U1 in panel E represents an unidentified peak observed in one specimen only.

FIG. 3.

Phylotype abundance in the gills of 13 individual specimens of L. pedicellatus as determined by QPCR-CDCE expressed as a percentage of the total CDCE peak area for each specimen. (A) Phylotypes are as follows: P1, diagonal stripes; P2, vertical stripes; P3, horizontal stripes; P4, diamonds; P5, checkering; unidentified peak U1, black. (B) Number of phylotypes detected per specimen ranked in order of decreasing frequency. (C) Average abundance of phylotypes expressed as percentages of the total combined CDCE peak area for gills of 13 specimens of L. pedicellatus.