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

Abstract

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.

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

“…In situ hybridization protocols typically target 16S (prokaryotes) or 18S (eukaryotes) small subunit rRNA (ssu rRNA), as it is abundant in cells, is variable at genus or species levels for many taxa, and is an appropriate target for FISH probes for bivalves (Pernthaler et al 2001, Le Goff-Vitry et al 2007a and references therein). Probes were designed for 2 bivalve taxa, the Mytilus edulis/galloprovincialis/ trossulus species complex, and Musculista senhousia, targeting the 18S ssu rRNA.…”

“…Our current method targets 18S ssu rRNA, the same target widely used in similar FISH cell-sorting techniques for identifying such taxa as marine bacteria (where the prokaryotic ssu rRNA, 16S, is the target; Pernthaler et al 2001) and other marine invertebrates (Goffredi et al 2006, Le Goff-Vitry et al 2007a. While in many taxa a short oligonucleotide probe targeting 18S can differentiate species, in other groups 18S is not variable enough to allow for more than a single base pair difference between species in a ~20 base pair oligonucleotide probe.…”

“…Becker et al 2007). Thus, there has been a recent focus on developing molecular techniques to improve identification of larvae (Goffredi et al 2006, Livi et al 2006, Vadopalas et al 2006, Le Goff-Vitry et al 2007a, Pradillon et al 2007). However, while these techniques more accurately identify targeted species in the plankton, they either sacrifice the ability to quantify plankton or still require time-consuming sorting.…”

“…Fluorescent-labeled target species can be difficult to distinguish from autofluorescent plankton (C. Henzler pers. obs., Pradillon et al 2007), although this can be addressed by in situ hybridization with a colored dye-labeled probe and sorting using a white-light microscope (Le Goff-Vitry et al 2007a,b, Pradillon et al 2007. Recently, a cell sorter that can handle organisms as large as marine copepods and invertebrate larvae has been developed.…”

An overview of chemosynthetic symbioses in bivalves from the North Atlantic and Mediterranean Sea

“…Investigating dispersal patterns of bivalve hosts is problematic. Early larvae and dispersal stages of chemosymbiotic bivalves are difficult to identify in the water column, despite various methodologies based on diagnostic PCR, or in situ hybridization of larvae using specific probes having been developed Le Goff-Vitry et al, 2007;Pradillon et al, 2007). Consequently, the distributions of larvae and their positioning in the water masses are not known.…”