Single-step species identification of bivalve larvae using multiplex polymerase chain reaction

One of the biggest obstacles to studying recruitment variation in marine bivalves is the need to collect and process large numbers of plankton samples. Larval bivalves are notoriously difficult, if not impossible, to identify to species using morphological criteria alone. Remote time-series collections could satisfy the sampling challenge, but efficient identification techniques must be developed to obtain species-specific data. Thus, we have developed a multiplex polymerase chain reaction (PCR) identification assay in which a single reaction is capable of accurate and efficient discrimination of five target bivalve species based on the size of cytochrome oxidase I products. The assay was tested with cultured and field-sampled larvae as well as adult genomic DNAs. Using a single whole larva as template, multiplex PCR reactions were capable of discriminating among the commercially important bivalves: Mercenaria mercenaria, Argopecten irradians, Mulinia lateralis, Spisula solidissima and Mya arenaria. Overall accuracy was 92%, including very few false positives. The efficiency of this assay stems from its ability to discriminate multiple target species with a single molecular step that ultimately can be automated to process large numbers of larvae.