Recombinant antimicrobials for feed based delivery in aquaculture

Two antimicrobial peptides of marine origin, tachyplesin from the horseshoe crab (Tachyplesus tridentatus) and pleurocidin from the winter flounder (Pseudopleuronectes americanus), were evaluated for potential use in feed-based delivery in oyster aquaculture. Tachyplesin, a beta sheet structure was more effective against a range of gram-positive and gram-negative pathogens, as well as against the protozoan oyster parasite Perkinsus marinus. In addition, tachyplesin's structure made it more resistant to the effects of proteases from the oyster and the parasite. The two peptides also had different effects on the host bacteria of the oyster digestive system, with tachyplesin selectively killing more gram-negatives and pleurocidin more gram-positives, thereby suggesting a role based on species evolutionary origin. Attempts to express tachyplesin in the yeast secretion system Pichia pastoris were unsuccessful due to the difficulty of visualizing such a small protein and the lack of an available antibody. A suppression subtraction hybridization library was constructed to discover what genes were upregulated by eastern oyster hemocytes in response to P. marinus infection. An expressed sequence tag (EST) fragment showed remarkable homology to human histone H4, a member of a group of proteins with a suggested secondary role in antimicrobial defense. The full sequence for oyster histone H4 was obtained by RACE PCR from oyster cDNA. The protein was antimicrobial to many bacteria including Staphylococcus aureus and Escherichia coli at concentrations <5 muM. In addition, western blotting showed the histone was present in higher levels in the hemocytes and serum of oysters infected with P. marinus suggesting a possible defense role. The oyster histone H4 sequence was then successfully cloned for intracellular expression in Saccharomyces cerevisiae yeast. Yeast lysate from the histone clone was found to be more antimicrobial against marine Vibrio anguillarum bacteria than lysate from control yeast. Feeding experiments resulted in significantly reduced Vibrio load in Artemia fed histone-containing yeast compared with controls. This validates the use of an oyster antimicrobial for use in a yeast feed-based delivery system in shellfish aquaculture.