Critical aspects of fluorescent age-pigment methodologies: Modification for accurate analysis and age assessments in aquatic organisms

Fluorescent age-pigment (FAP) quantification has recently been proposed as a means of estimating age in aquatic vertebrates and invertebrates. However, various aspects related to currently adopted procedures remain untested and a distinct relationship between FAP accumulation and chronological age has yet to be established. The present study was undertaken to critically evaluate the effects of specimen and extract handling procedures (including temperature, time, ultrasonication, and solvent systems) on the expression of native FAP fluorescence using the post-mitotic tissues of laboratoryreared specimens of the teleostOreochromis mossambicus as model systems. FAP-like fluorophores increased in vitro in brain, heart, and muscle tissues and their extracts with increased storage temperature (–20°C and above) and time. Sonification of homogenates greatly enhanced this effect and generated other non-native fluorophores in sample solution. Fluorescence assay temperature also affected expression of results, and aqueous-phase extracts, used in previous studies, were found to contain large amounts of fluorescent flavin contaminants. Using modifications of the above procedures, age-related patterns of accumulation were subsequently examined inO. mossambicus brain and, for the first time, positive correlations between chronological age and whole organ as well as weight-specific FAP concentration were validated. The significance of these findings are discussed in relation to previous FAP studies.