Alcalase treatment for removal of adhesiveness in greenfin horse-faced filefish (Thamnaconus septentrionalis) eggs

Abstract

The greenfin horse-faced filefish Thamnaconus septentrionalis is a new marine culture species in East Asia in recent years. It has demersal and adhesive eggs. The artificial fertilization technique of T. septentrionalis has not been established, and commercial hatcheries currently rely on the collection of naturally fertilized eggs. It is impracticable to separate dead eggs from the egg mass, and a large number of waste and faecal matter adhere to the surface of fertilized eggs, causing excess bacterial and fungal loading in eggs incubation tanks. This study aimed to develop an effective method to remove the adhesiveness of fertilized eggs of T. septentrionalis. The effectiveness of tannic acid (0.5%, 0.2% and 0.1%), sodium hypochlorite (0.05%, 0.025% and 0.01%) and alcalase enzyme (4%, 2%, 1% and 0.5%) to eliminate the adhesiveness of T. septentrionalis eggs was evaluated. Eggs were exposed to different deadhesion solutions for up to 20 min, and the number of deadhesive eggs was counted. The results showed that only the alcalase enzyme could effectively remove the adhesiveness of eggs, and the deadhesion efficiency was positively associated with enzyme concentration. The deadhesion rate of the 0.5% alcalase enzyme treatment was more than 70%, while that of the 4%, 2% and 1% alcalase enzyme treatments were more than 90%. The hatching rates were not affected by alcalase enzyme treatment. The hatching rates of the 2% and 1% treatments were higher than those of the control groups. Ultrastructural analysis showed that the surface of eggs had a pore-canal structure, and the zona radiata externa was gradually degraded and finally peeled off during the deadhesion process. Similar to freshwater fishes with adhesive eggs, biochemical analysis showed that the adhesiveness of T. septentrionalis eggs was related to the presence of glycoproteins and mucopolysaccharides in the zona radiata externa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of zona radiata externa proteins showed that the nonwater-activated eggs, water-activated eggs and adhesive fertilized eggs had same protein pattern, and only small peptide fragments were detected in the deadhesive fertilized eggs. This study provides valuble theoretical and technical support for improvement of hatching technology of filefish.