Date of Award

2020

Degree Type

Thesis

Degree Name

Master of Science in Biological and Environmental Sciences (MSBES)

Specialization

Cell & Molecular Biology

Department

Cell & Molecular Biology

First Advisor

David R. Nelson

Abstract

Acute Hepatopancreatic Necrosis Disease (AHPND) causes more than a billion dollars of losses to the shrimp farming industry annually. In 2013, strains of the marine pathogen Vibrio parahaemolyticus containing genes for the PirAvp/Bvp toxins, encoded on a 70 Kb plasmid, were identified as the causative agent of AHPND. Antibiotics have been used to attempt to reduce losses, however their use in aquaculture is controversial. Additionally, the pathogen has demonstrated the ability to rapidly acquire antibiotic resistances through genetic mutation and lateral gene transfer. Probiotics, often used to prevent disease in aquaculture, may offer the opportunity to mitigate AHPND infections without the risks to consumers and the environment posed by antibiotic use. The goal of this study was to determine if the marine probiont Phaeobacter inhibens S4 protects Litopenaeus vannamei post-larvae (PLs) against fatal V. parahaemolyticus infections and to elucidate its protective mechanisms. A further goal of this study was to isolate and identify other potential probionts, including “Bdellovibrio and Like Organisms” (BALO), whose prey range includes V. parahaemolyticus.

P. inhibens S4 was tested in challenge assays using Artemia nauplii and L. vannamei for its ability to protect these organisms from V. parahaemolyticus-induced mortality. Zhao et al previously identified three key probiotic activities of P. inhibens S4: 1) the production of the antibiotic compound tropodithietic acid (TDA), 2) the formation of a copious biofilm, and 3) quorum sensing/quorum quenching activity through N-acyl homoserine lactone (AHL) signaling. Mutant strains of P. inhibens S4 with one or a combination of these three mechanisms altered were used in PL challenge assays to asses which probiotic functions were key to protecting shrimp. Competition assays, to assess in vitro growth of V. parahaemolyticus PSU5579 when co-cultured with P. inhibens S4 or one of the mutant strains, were also performed to determine the ability of S4 to inhibit the growth of the pathogen in biofilms and planktonic states. Results showed that P. inhibens S4SmKm protects L. vannamei PLs from AHPND, increasing survival from 45% in the infected control to 80% when the environment is pre-treated with P. inhibens S4SmKm. Assays with wild type and mutant strains of P. inhibens show protection whether or not TDA is produced; however, S4 no longer protects when biofilm production is reduced or quorum sensing/quorum quenching is disrupted. In support of these results, it was shown that P. inhibens S4 does not inhibit the growth of V. parahaemolyticus when cocultured, nor is V. parahaemolyticus sensitive to the antibiotic TDA, even up to concentrations of 100 μg/ml.

To screen for other potential probionts and a BALO organism, bacteria isolated from seawater and shrimp tank water were screened using a zones of inhibition (ZOI) assay and for plaque forming capabilities in lawns of V. parahaemolyticus. A candidate BALO was isolated from Narragansett Bay that showed predatory activity against V. parahaemolyticus. Genomic DNA was extracted, and the genome was sequenced. The organism appears to be a previously unidentified species of Halobacteriovorax, given the strain name GB3. While Halobacteriovorax GB3 does not harm L. vannamei PLs, we did not test its ability to protect shrimp from AHPND because it does not reduce V. parahaemolyticus concentrations below 1x106 CFU/ml, which is an infectious dose.

Available for download on Friday, December 03, 2021

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