Studies on the acorn barnacle Balanus amphitrite and its associated bacteria

Abstract

Despite being a model organism to study settlement in marine invertebrates, little is known about the genetics of the barnacle Balanus amphitrite. To fill this gap, cDNA libraries representative of different developmental stages were generated and sequenced. Nearly 14,000 genes were annotated, which may represent 2/3 of the species’ total protein coding regions. The database that was created to allow public access to this genetic information will profoundly benefit future research aiming to understand the molecular regulation of development and settlement in this species. Furthermore, a quantitative real-time PCR assay to study gene expression in B. amphitrite was designed and validated. Eleven genes were studied for their ability to normalize qRT-PCR data. Total RNA extracted from seven developmental stages was reverse transcribed and the expression stability of the selected genes was compared. It was found that transcripts encoding cytochrome b and NADH dehydrogenase subunit 1 were expressed most stably, and their use to normalize gene expression data is recommended. Conflicting evidence exists on the role of bacteria in B. amphitrite settlement. However, there is a paucity of information on the microbial community naturally associated with this barnacle. In order to reveal the existence of stable associations, a 16S rRNA-based, taxon-specific qPCR assay was developed to monitor the preponderance of 5 bacterial phyla and classes. Furthermore, attempts to profile these qPCR products by DGGE were made. This new method was applied to characterise the bacterial communities associated with different B. amphitrite developmental stages and body parts. It was found that the structure of these communities changed throughout the barnacle life cycle in a highly reproducible manner. Furthermore, bacteria isolated from the barnacle shell were capable of inducing settlement of conspecific larvae. The analysis of these communities at a lower taxonomic level should confirm if any of these ecologically important bacteria are vertically transmitted.