Date of Award

2017

Degree Type

Thesis

Degree Name

Master of Science in Biological and Environmental Sciences (MSBES)

Specialization

Cell and Molecular Biology (CMB)

Department

Cell & Molecular Biology

First Advisor

David R. Nelson

Abstract

Acute hepatopancreatic necrosis disease (AHPND) causes mass mortalities in farmed penaeid shrimp and has proven difficult to control using typical disease control measures. The causative agent of AHPND has been identified as Vibrio parahaemolyticus strains possessing the 69 kbp plasmid pVPA3-1 containing genes homologous with Photorhabdus insect-related (Pir) toxin-like genes (pirA- and pirB-like). Probiotics have been used successfully in shrimp aquaculture to control disease outbreaks caused by pathogenic Vibrio, but there are currently no probiotics available that have been proven to control AHPND. The goal of this study was to screen and characterize marine bacterial isolates as potential agents to prevent Artemia nauplii and Litopenaeus vannamei post-larvae (PL) mortality by the pathogen Vibrio parahaemolyticus. Twelve candidate probiotic organisms were tested in an Artemia sp. model. Phaeobacter inhibens was the only candidate probiont that significantly increased the survival of Artemia nauplii challenged with AHPND V. parahaemolyticus (p<0.001). Candidate probionts Pseudoalteromonas piscicida, Pseudoalteromonas flavipulchra, and Pseudoalteromonas arabiensis were lethal to Artemia nauplii (p<0.001). Six species of candidate probiotic organisms were tested in L. vannamei. P. inhibens was the only candidate probiont tested which was not harmful to L. vannamei PLs and significantly increased the survival of PLs challenged with AHPND V. parahaemolyticus (p<0.001). Genome analysis of V. parahaemolyticus PSU5579 revealed the presence of the multiple putative virulence genes including nine hemolysins, six secreted proteases, and six secretion systems including one T3SS and two T6SS. The genome also contains the 69 kbp pVPA3-1 plasmid encoding the pirA- and pirB-like toxin genes. Genome analysis of Bowmanella denitrificans JL63 revealed several gene clusters potentially involved in the production of the following antibacterial compounds: colicin V (or bacteriocin), lanthionine, the broad-spectrum antibacterial protein marinocine encoded by the lodAB operon, a secreted hemolysin-type calcium-binding bacteriocin, lantipeptide, bacteriocin, and a nonribosomal peptide.

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