In situ hybridization on whole larvae: a novel method for monitoring bivalve larvae

Accurate and efficient identification to the species level of early larval stages has long been a problematic step in the study of marine invertebrates, due to the extremely small size of the larvae and their lack of diagnostic morphological characters. It is nonetheless, a prerequisite for any ecological study. As a consequence, a number of molecular approaches, mostly based on the PCR technique, have been developed over the last decade. We developed a method relying on specific rRNA-targeted oligonucleotide probes for in situ molecular hybridization using whole larvae. A colorimetric reaction following the hybridization allows signal detection with a light microscope. A total of 9 probes have been developed, targeting the species that are major components of benthic communities in several European bays. The method can be applied to roughly sorted wild plankton samples, and some steps could be automated. It is relatively inexpensive to implement and does not require costly equipment or expensive staff training. The overall larval morphology is preserved, allowing visual inspection. The quantitative aspect of the approach is another asset. Field-based studies of larval distribution and behaviour are possible applications of the method, as large numbers of samples can be screened to meet the requirements of adequate spatial and temporal coverage. It is also relevant for routine monitoring of target species, such as species of commercial interest, bio-indicators or invasive species.