Identification of Balanus amphitrite larvae from field zooplankton using species-specific primers

Abstract

Identification of marine invertebrate larvae using morphological characters is laborious and complicated by phenotypic plasticity. Balanus amphitrite is a dominant barnacle, important in the context of intertidal ecology and biofouling of manmade structures. Morphological identification of barnacle larval forms in a mixed population is difficult because of their intricacy and similarity in size, shape and developmental stages. We report the development and application of a nucleic acid-based Polymerase Chain Reaction (PCR) method for the specific identification of the barnacle, B. amphitrite, from the heterogeneous zooplankton sample. This method is reliable and accurate thereby overcoming taxonomic ambiguity. Sequence alignment of the 18S rRNA gene region of selected species of barnacles allowed the design of B. amphitrite-specific PCR primers. Assay specificity was evaluated by screening DNA obtained from selected species of barnacles. The oligonucleotide primers used in the study flanked a 1600 bp region within the 18S rRNA gene. The primer is specific and can detect as few as 10 individuals of B. amphitrite larvae spiked in a background of ~186 mg of zooplankton. This technique facilitates accurate identification and the primer can be used as a marker for enumeration of B. amphitrite larvae in the plankton.