Defense Date


Document Type


Degree Name

Master of Science


Environmental Studies

First Advisor

Dr. James Vonesh

Second Advisor

Dr. Rima Franklin

Third Advisor

Jennifer Ryan

Fourth Advisor

Rachel Mair


In vitro propagation efforts play an essential role in conserving and restoring threatened freshwater mussel populations by circumventing the need for a fish host. Across a broad range of taxa, transformation is induced with an artificial M199 medium and rabbit serum. However, such formulation may not be sufficient in culturing critical species with more specific physiological requirements. In this study, multiple serum mixtures were tested to improve in vitro transformation of two freshwater mussel species: yellow lampmussel (Lampsilis cariosa) and tidewater mucket (Atlantaconcha ochracea). These species were selected because they parasitize similar fish host species but have different rates of transformation in previous propagation trials. Juvenile transformation on rabbit serum only treatments was tested against juvenile transformation from treatments using rabbit serum supplemented with fish extract, blue catfish (Ictalurus furcatus) serum, or grass carp (Ctenopharyngodon idella) serum. L. cariosa showed an aptitude for a wide variety of serum types except for blue catfish, which showed signs of toxicity during early glochidia development. A. ochracea increased in transformation when cultured in full or partial carp serum compared to treatments utilizing only rabbit serum or rabbit serum with gill extract. Given the availability of local grass carp and the ability to mix with rabbit serum, it may be a preferred sera alternative for species like A. ochracea which exhibit poor transformation with serum.

In vitro propagation allows for transformation of freshwater mussel juveniles without a fish host using modified cell culture techniques. However, microbial contamination can greatly decrease the likelihood of successful transformation. A broad-spectrum antimicrobial mixture of rifampicin, carbenicillin, gentamycin, and amphotericin b (RCGA) is used to curb the proliferation of microbes, but this may not be suitable for all types of contamination. Additionally, some antimicrobial compounds such as amphotericin b can negatively impact juvenile transformation at higher concentrations. In this study, an alternative antimicrobial mixture, Primocin™ (InvivoGen, San Diego, California, Cat. #ant-pm-2), was considered for in vitro propagation of Elliptio complanata. Primocin™ was assessed against the original RCGA mixture to determine its efficacy and test for toxicity to transforming juveniles. Antimycotic components were also tested at lower concentrations to determine if microbial contamination can still be controlled without impact on glochidia development. Contaminated replicates underwent DNA extraction and analysis to identify bacterial and fungal pathogens. While Primocin™ successfully curbed microbial proliferation, Elliptio complanata transformers showed no signs of tissue development. In RCGA treatments, there was no significant difference between replicates with or without amphotericin b. Results of DNA analysis identified unique contamination for each replicate without antimicrobials. Contamination could be attributed to known pathogens that were ubiquitous across a range of environments or common in shellfish and aquaculture production.


© Raquel Wetzell

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