Use of the fluorochrome calcein as an in situ growth marker in the brown mussel Perna perna

Abstract

This study assesses the potential of the fluorochrome calcein for use as a growth marker in bivalve shell growth studies. Calcein solutions were administered in situ to the brown mussel Perna perna (Linneaus), both by injection and immersion, and the effect of calcein concentrations on fluorescent mark deposition and mussel mortality was investigated in the laboratory. Field investigations showed that, 1 month after administration, calcein injection (125 mg l^−l) into the mantle cavity produced superior results to the immersion treatments (150 and 500 mg l^−l). Both methods resulted in fluorescent mark incorporation at the growing edge, but during immersion general calcein deposition associated with endolith activity resulted in fluorescence that made identification of a distinct datum point difficult. In contrast, the injection method produced clearly defined growth marks, which were easily distinguished from autofluorescence and persisted without visible degradation for a minimum of 9 months. Shell growth rates estimated using the fluorescent mark as a datum point were similar to those from earlier studies using different methods. Laboratory investigations revealed that at␣calcein concentrations of 80 mg l^−l and above, 100% of juvenile (20 to 30 mm) and adult mussels (60 to 70 mm) retained a visible growth mark, while at concentrations >160 mg l^−l all marks were bright and clearly defined. No mussel mortality was exhibited at any time, even at calcein concentrations of 640 mg l^−l, eight times higher than those required for mark deposition. These results suggest that, compared to traditional methods of bivalve growth determination, the use of fluorochromes presents a relatively inexpensive, non-invasive and rapid alternative. When using calcein as a growth marker, problems associated with some other fluorochromes (e.g. inconsistent mark incorporation, high post-treatment mortality) were not exhibited.