Evaluation of a fluorescent dye assay to assess glochidial health

Abstract

Unionid mussels have the greatest diversity in North America but have suffered high extinction and imperilment rates due to habitat loss, fragmentation, and deterioration. Unionid mussels have a parasitic larval stage (glochidium) that requires a fish host to metamorphose into a juvenile. The current technique of evaluating glochidia viability is known as the salt test, which predicts metamorphosis success based on the shell closure in response to salt (NaCl). However it is only useful on open glochidia. This study evaluated a dual staining assay that involves fluorescein diacetate (FDA) and propidium idodide (PI); FDA can be combined with PI to evaluate membrane integrity and physiological state of open and closed glochidia. Proportion live, questionable, and dead glochidia can be evaluated using fluorescent microscopy; living glochidia will fluoresce green and dead glochidia will fluoresce red. Questionable glochidia (live but heavily damaged) exhibited green and red fluorescence. The dye assay was comparable to that of the salt test when predicting metamorphosis success. It appears to be a useful tool for ecotoxicology testing and could be a key technique in development of successful cryopreservation techniques for glochidia. Cryopreservation could preserve genetic diversity and reduce genetic bottles in future generations of threatened and endangered freshwater mussels.