Whole-mount fluorescence in situ hybridization to visualize symbiotic bacteria in the gills of deep‑sea mussels

Fluorescence in situ hybridization (FISH) is a technique used to visualize the distribution of specific nucleotide sequences. For the purpose of detecting symbiotic bacteria in the tissues of host organisms, FISH is usually performed on paraffin or frozen sections of host tissues. However, the sectioning process requires lengthy procedures and sectioning restricts observations to a particular plane. To solve these problems, we attempted FISH on whole gill filaments of deep-sea mussels of the genus Bathymodiolus. Isolated gill filaments of B. septemdierum and B. platifrons were fixed with paraformaldehyde, hybridized in microcentrifuge tubes containing labeled probes specific to either thiotrophic or methanotrophic symbionts, mounted on glass slides and then observed under a fluorescent microscope. The distribution of the symbionts was clearly visualized; fluorescence signals, packed in the bacteriocytes, occupied most parts of the gill surface. However, fluorescence signals were not observed in the ciliary cells lining the ventral edge of the filaments nor in ciliary junctions. Differences in the morphology of the bacteriocytes harbored by the 2 species were observed under a confocal laser scanning microscope: attached polygonal bacteriocytes in B. septemdierum and detached round particles in B.