Species identification of marine invertebrate early stages by whole-larvae in situ hybridisation of 18S ribosomal RNA

The ability to identify early life-history stages of organisms is essential for a better understanding of population dynamics and for attempts to inventory biodiversity. The morphological identification of larvae is time consuming and often not possible in those species with early life-history stages that are radically different from their adult counterparts. Molecular methods have been successful in identifying marine larvae; however, to date these methods have been destructive. We describe here an in situ hybridisation (ISH) technique that uses oligonucleotide probes specific for the 18S ribosomal RNA gene to identify marine larvae. Our technique leaves the larvae intact, thus allowing the description of larvae whose morphology was not previously known. Only 1 mismatch between the rRNA sequences of target and non-target species is sufficient to discriminate species, with nearly 100% efficiency. We developed a colourimetric assay that can be detected with a dissecting microscope, and is thus suitable for autofluorescent or large eggs and larvae that cannot be sorted under a microscope. Probe binding is revealed by an enzymatic reaction catalysed by either a horseradish peroxidase or an alkaline phosphatase. ISH was broadly applicable: it was effective in identifying eggs, larvae and adult tissues, soft-bodied larvae (polychaetes) and larvae with hard shells (bivalves), larvae belonging to different phyla and from different environments. Further advantages of this method are its relatively low cost, that only a minimal amount of equipment is needed, and that 100s of specimens can be processed quickly and simultaneously