Molecular assessment of raphidophyte species and their association with Vibrio in Delaware's inland bays

Main, Christopher Robin
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University of Delaware
There is little information on the dynamics between bacteria and phytoplankton within the marine environment. One genus of bacteria, Vibrio is often found in association with eukaryotic organisms, forming relationships that range from mutualistic to pathogenic in nature. In this dissertation, I investigated community-level and species-specific associations between Vibrio and three algal classes (diatoms, dinoflagellates, and raphidophytes) that form frequent blooms in Delaware's inland bays. Results demonstrated a significant correlation between particle-associated Vibrio abundance and all three algal classes. Further species-specific interactions were examined over the course of four days during a mixed bloom of the harmful algal bloom species (HABs) Heterosigma akashiwo (3.0-20 μm size fraction) and Fibrocapsa japonica (>20 μm size fraction). Results indicated a closer relationship for particle-associated Vibrio abundance with H. akashiwo abundance. Changes in the Vibrio community during the bloom were examined using automated ribosomal intergenic spacer analysis (ARISA) and showed differences between size fractions but not over time. Grazing experiments during a separate mixed raphidophyte bloom demonstrated that associations with different size fractions may provide refuge for certain members of the Vibrio community despite an increased grazing pressure on the total population. Species-specific interactions were then examined showing that V. cholerae was the major species associated with H. akashiwo . The transcriptional response of H. akashiwo to the presence of V. cholerae was evaluated using next generation sequencing, representing the first reported transcriptome of a raphidophyte species. This transcriptome was then used to examine differential expression of genes from H. akashiwo in response to the presence of V. cholerae . Genes related to antioxidant scavenging, amino acid transport, post-translation modification and translation were significantly up regulated, while those associated with energy production and conversion, and lipid transport and metabolism were down regulated. Finally, direct comparison of quantitative real-time PCR (qPCR) and sandwich hybridization assay (SHA) was carried out using H. akashiwo as a model organism. The effects of diel cycle, growth stage, and macronutrient stress on enumeration of H. akashiwo was examined on both methods. A singular cellular homogenate was generated from each culture and split for analysis by qPCR and SHA. Results showed significantly greater rRNA content during lag and exponential phases compared to stationary phase for SHA, and a significant decrease in rRNA during the light cycle compared to the dark cycle. In contrast, there were no significant differences for qPCR over a diel cycle or during the different growth stages. However, under N stress both rRNA and DNA content per cell were significantly lower when compared to nutrient replete conditions. This work increases our understanding of dynamics between harmful algal species and Vibrio spp. in Delaware's inland bays, as well as an understanding on the effects of diel cycle, growth stage, and macronutrient stress on enumeration of H. akashiwo using two molecular methods. Finally, results of this work are broadly applicable to the research and monitoring of HABs and Vibrio in the Mid-Atlantic region.