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Don Anushka Sandaruwan Elvitigala, and Jehee Lee*
Marine Molecular Genetics Lab,
Department of Marine Life Science,
School of Marine Biomedical Sciences,
Jeju National University,
Jeju Special Self Governing Province,
690-756, Republic of Korea.

Black rockfish (Sebastes schlegelii) is one of the highly demanded maricultured finfish delicacies in Northeastern Asia, especially in Korea. However, due to the intensive culturing conditions in farms pathogenic infections in these fish was found to increase drastically over time, severely affecting the crop quality and yield. Therefore, the development of a proper disease management system in black rockfish mariculture farming is becoming a necessity to increase the resistance of these creatures to infections. Thus, the investigation of innate immune mechanisms in this fish on a molecular level, and the identification of ways to increase resistance to infection using modern molecular techniques is one of the productive ways to face the pathogenic threat, successfully.

Cystatins are large group of evolutionary related reversible inhibitors of cysteine proteases, classified into four families; family 1(stefines), family 2 (Cystatins), family 3 (kininogens) and family 4. Cystatin B is categorized under family 1cystatins, some of which were found to induce NO production under IFNγ stimulation and involve in antibacterial defense in macrophages. Herein we molecularly characterized a homologe of cytatin B from black rock fish (RfCytB). The complete open reading frame of RfCytB was 291 bp in length coding for a 97 amino acid protein with a predicted molecular mass of 11 kD. Derived protein sequence of RfCytB resembled typical cystatin family protein signatures, including cysteine protease inhibitory signature harboring penta peptide QxVxG consensus sequence. As expected, RfCytB shares significant sequence consistency with its teleostan counterparts prominently with that of olive flounder. Phylogenetically, RfCytB was positioned with family 1 cystatins and clustered with teleostan counterparts sharing common vertebrate ancestral origin.

RfCytB was detected to express ubiquitously in all the tissues examined at transcriptional level with eminent transcript levels in spleen and gill tissues. Moreover, its expression was modulated in head kidney and spleen tissues in response to Aeromonas salmonocida experimental infection with inductive expression levels. In addition recombinantly expressed RfCytB protein demonstrated the concentration depended papain inhibitory activity and protein was shown to have significant thermal stability. Taken together, our findings in this study hints on the putative role of RbCytB in innate immune responses, plausibly mediated by its cysteine protease inhibitory activity.

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