FISH-CS-A rapid method for counting and sorting species of marine zooplankton

Understanding population dynamics in marine species has long been hindered by the inherent difficulties of studying species in which all or part of the life cycle is planktonic. Plankton sample processing is laborious and, due to morphological similarity between disparate taxa, often identifies zooplankton only to higher taxonomic levels. As a consequence, many scientific issues that require identification to species level are impossible to explore adequately. Several in situ hybridization protocols show promise for identifying marine larvae by color-coding them with taxon-specific, dye-labeled DNA probes. We adapted these protocols and coupled them with recent cell sorting technology to rapidly and accurately identify bivalve larvae from diverse plankton samples. We developed probes for 2 bivalve taxa: Musculista senhousia and the species complex Mytilus edulis/galloprovincialis/trossulus. Coupled fluorescence in situ hybridization and cell sorting (FISHCS) separated M. galloprovincialis larvae from both oyster Crassostrea gigas larvae and from a mixed plankton/ M. galloprovincialis sample. The number of false positives and false negatives was assessed by a PCR assay. Our FISH-CS method is robust to plankton autofluorescence and can be easily adapted to work with nearly any planktonic species or life stage of appropriate size.