Characterization of serine proteases in the monogenean Neobenedenia girellae

Abstract

Proteases of Neobenedenia girellae were examined by using zymographic analysis. Zymography of N. girellae homogenate revealed proteases at approximately 88, 107, 149 and 167 kDa for the adult worms and approximately 147 and 166 kDa for the oncomiracidia, respectively. The enzyme activities were inhibited by Pefabloc SC but were not inhibited by E-64 and Pepstatin A, indicating that these proteases were serine proteases. The gelatinolytic activities of these proteases were more active at pH 8.5, when pH range of 5.5–11.5 was examined, and increased with the increase of temperature within the range of 15–37 °C. Further, the effect of proteinase inhibitors, pHs and temperatures on N. girellae were examined. Egg laying of mature N. girellae was suppressed by Pefabloc SC and seawater of pH 5.5 to 6.5 and at pH 9.5. Additionally, egg laying of mature N. girellae was suppressed by low incubation temperatures (10, 15, 20 °C). The oncomiracidia treated with Pefabloc SC and seawater of pH 5.5 and pH 9.5 were also affected and cilia of the treated parasites ceased moving. These results suggest that serine proteases of the parasite could be important for their survival, and their physiological active substances such as proteases, may have adapted to higher water temperature and tend to be suppressed at pH 5.5–6.5 and pH 9.5. The results of this study could be informative to develop an effective control method against outbreaks of the parasites.