PhD Defense Seminar of Mark Ciesielski

Adapting and Developing Quantitative Molecular Tools to Characterize Environmental Pathogen Dynamics Relevant to Shellfish Aquaculture

Oysters are an important food source and economic asset for coastal communities. Though there have been efforts to expand the shellfish industry in coastal North Carolina, an increase in massive oyster mortality events have hindered anticipated growth in the industry. The recurring nature and growing concern regarding mortality events has prompted the development of rapid and sensitive molecular workflows for the detection of environmental pathogens. The accurate quantification of the causative agents of mortality combined with histological assessments of oyster pathology provides valuable insight into the mortality events that are impacting shellfish aquaculture markets. The aims of this work were to 1. identify molecular platforms that are best equipped to detect and quantify pathogens across a range of concentrations in complex matrices, 2. develop and optimize novel assays that would allow for the absolute quantification of shellfish-pertinent pathogens that can be readily adapted by the aquaculture industry, and 3. combine the developed suite of molecular tools with assessments of organism and ecosystem health in dynamic systems to characterize main drivers of mortality events. Aim 1 was tackled by conducting a comparative study between two molecular workflows, qPCR and ddPCR, commonly used for the quantification of molecular targets. ddPCR proved to be the more sensitive of the two platforms and was superior in detecting low concentration signals. For aim 2, novel assays were developed using compiled sequence data for gene targets relating to oyster-specific pathogens. The approach used for target design also made it possible to distinguish and quantify multiple Vibrio species using a single set of primers and probes within a single PCR reaction which has not been successfully done before. The progress made in the first two aims allowed for the success of aim 3 by integrating the knowledge and tools into a holistic and adaptable framework. Longitudinal studies of oyster mortality were conducted providing high-resolution monitoring coupled with novel molecular assays to implicate bacterial etiology in the development of disease. These findings will provide predictive capacity and insight for shellfish management to implement mitigation strategies at the farm level with the hope of preserving resilience in an economically relevant industry.