Proteomic profile and proteases characterisation of greater amberjack skin mucus after Neobenedenia girellae infection

Abstract

Skin mucus is known for being the first physical and immunological barrier in fish. Skin mucus is composed by a wide range of proteins, like glycoproteins, structural proteins, metabolic proteins and immune related protein-components. Likewise, proteome changes in Atlantic salmon (Salmo salar) have been observed due to sea lice infections, demonstrating the importance of protein-immune defenses against ectoparasite infections. Nowadays, greater amberjack (Seriola dumerili) pass-through a biosanitary bottleneck on its on-growing period related with monogenean ectoparasites, which could cause a 90% of mortality (Ogawa et al.,1998). For that reason, this study aimed to compare skin mucus proteome of non-infected and experimentally infected greater amberjack juveniles with Neobenedenia girellae, as well as to characterize proteases of this skin mucus. Thirty greater amberjack juveniles of 150 ±12 g were randomly distributed in 3 cylindroconical tanks of 500 liters. After 10 days of acclimation, skin mucus of non-infected fish was obtained, pooled and immediately freeze in liquid nitrogen. Cohabitation with N.girellae was conducted with 3 previously infected fish stored in cages for 15 days, when all the experimental fish were infected, skin mucus was sampled. The integrative proteomic approach was conducted using a label-free procedure as LC-MS/MS with a 2-DE-PMF-MS/MS. Protease activity was conducted using azocasein hydrolysis assay, while protease characterization was determined combining azocasein hydrolysis with inhibitors of metalloproteases and serine proteases. Results obtained with LC-MS/MS showed the first microbiota analyses in greater amberjack skin mucus, were the most abundant species belonged to gamma-proteobacteria group, and infected and non-infected fish bacterial presence only differed in 6 genus of bacteria.2-DE-PMF-MS/MS analyses showed differences in proteome profile at a qualitative level. Proteins of p/ 5 and molecular weight ranging between 36-66 KD, typically identified as structure proteins, were clearly affected by degradation for N.girellae infected fish. Protease activity analysis showed no difference among infected and non-infected fish, however proteases populations differed in metalloproteases and serine proteases when comparing infected and noninfected fish.