Control of microbial contamination during in vitro culture of larval unionid mussels

Abstract

In vitro metamorphosis of freshwater mussel glochidia allows researchers to acquire juvenile mussels in the absence of a fish host. One major impediment to the success of in vitro mussel culture is the lack of control over fungal contamination brought in from the larvae and/or parent mussel. Eight antimycotics were investigated for controlling fungal contamination during in vitro culture with a unionid bivalve: amphotericin B, hygromycin B, nystatin, benomyl, dichloran, econazole, ketoconazole and thiabendazole. While all performed well on unionid fungal isolates on agar testing, no antimycotics were successful at controlling fungal growth at a concentration not cytotoxic to glochidia under culture conditions. The imidazole class antimycotics (econazole and ketoconazole) best controlled fungi commonly associated with mussels, but depressed larval development. Modifications to the culture protocol, including reduced concentrations of amphotericin B combined with daily media changes, closure of larvae before extraction, elimination of dead and contaminated larvae and the use of fish serum tested were also investigated. The resultant modified protocol was the best method for reducing fungal growth and increasing the efficacy of in vitro mussel culture.

Key words:

• Unionid
• in vitro metamorphosis
• mussels
• antimycotic
• contamination