Biomedical Sciences ETDs

Publication Date

7-1-2011

Abstract

Infectious diseases are an increasing threat to shrimp farming industries worldwide and account for nearly $3 billion of annual economic loss. The need to devise novel approaches against these infectious agents is pressing, as traditional methods are insufficient to ward off infections. The main aim of this study was to apply paratransgenesis to control infectious agents mediating shrimp diseases. As proof-of-concept we showed that Artemia, a food source in shrimp aquaculture, internalized bacteria expressing recombinant proteins. Stable expression and retention of these marker molecules up to 10 hours after feeding with the transgenic bacteria were evident within the gut of Artemia, coincident with the time period of highest density of internalized microorganisms. Uptake of recombinant proteins by Artemia occurred during active feeding, with their rapid depletion during a non-feeding washout phase. Bioamplification of recombinant proteins through increasing trophic levels via the paratransgenic approach was then tested. For this trials were conducted aimed at delivery of recombinant proteins to larval stages of the commercial white shrimp, Litopenaeus vannamei, via direct feeding of transgenic bacteria or via feeding transgenic bacteria-engorged Artemia. In both trials, shrimp extracts showed recombinant protein accumulation during the active feeding phase and decrease during the washout period. From these studies we concluded that transgenic expression of proteins in bacteria can be detected through their paratransgenic expression in Artemia and shrimp, suggesting that this commonly employed feed organism could be applied to target infectious agents in shrimp mariculture. For this purpose we identified cecropin and melittin as effective antimicrobial peptides against vibrio species, and their combination was particularly potent without toxicity toward probiotic bacteria or algal feed organisms. Furthermore, unlike mellitin alone antibiotic resistance did not develop in Vibrio strains exposed to the cecropin/melittin mixture. Finally, the melittin gene was transduced into B.subtilis, and expression of significant levels of melittin was detected, although this was insufficient to cause detectable anti-vibrio activity. Optimization of B.subtilis expression for higher antimicrobial peptide production or employment of algal strains for anti-infectious molecule expression are key future directions for developing a paratransgenic approach as an environmentally sustainable disease mitigation strategy in marine aquaculture.

Keywords

"Paratransgenesis, Shrimp diseases, Aquaculture"

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Advisor

Rubin, Robert

First Committee Member (Chair)

O'Hair, Kevin

Second Committee Member

Peabody, David

Third Committee Member

Wilmer, Sibbitt

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