| The turbot (Scophthalmus maximus), belonging to the family Bothidae, is economically important in wild fisheries and has stimulated the development of aquaculture for several species (Tilseth 1990). Since the introduction of turbot to China in 1992, breeding and cultivating technology have been developed and commercialised. However, the flatfish farming industry were limited to certain areas because of their temperature requirement. Skin mucus covering the epidermal surface, being the first line of defense against invading pathogens, can be extremely sensitive to abrupt changes in the environment. For it comprising a number of immune components such as lysozyme, immunoglobulin complement, lectins, proteolytic enzyme and antimicrobial peptides and so on. The subject in the context of this study, take turbot juveniles as the research object. Using histochemistry, biochemistry, molecular biology, protemics, carry out a series of studies to heat stress on the body surface mucus. Observed the changes of mucous cell in physiological and biochemical, determined the temperature-related functions of mucous proteins, and positioned indicator protein of temperature. This information could be useful in better understanding the role of mucus as a component of the innate immune system, and identify genotypes that are best suited to the environments in which the fish are to be farmed.Studies were carried out on the effect of high temperature (20,23,25,27 and 28℃) on mucous cells of juvenile turbot (Scophthalmus maximus) by method of AB-PAS (AB pH25, pH1.0) combined staining. Results showed that significant changes in the distribution, numbers and type of skin mucus cells occurred following high temperature treatment. Compared to the control group (16℃), the number of typeⅡmucus cells had the maximum increasing, type IV seconded, type I showed no significant change, and typeⅢpresented no regularity change. The skin mucus comprising a number of hydrolase such as lysozyme, acid phosphatase, alkaline phosphatase and so on, which create an acidic environment to protect the fish from damage. With the temperature increasing, pathogenic microorganisms become more active and have a greater threat to the health of the fish. In order to prevent pathogen invasion, the acidic mucus has a significant increased at high temperature. TypeⅡand IV mucus cells were AB-positive and containing acidic mucoitin, and the increase in the number may create mucus for the skin to defend pathogenic intrusion.By RT-PCR and spectrophotometric methods, determined the immune-related factors including immunoglobulin M (IgM), IL-1β, hepcidins, transferrin, lysozyme, acid/alkaline phosphatase and superoxide dismutase. The results suggested that there was a significant variation in mucus immunity. This information could be useful in better understanding the role of mucus as a component of the innate immune system, and this study was unique in its scope because it was the first assessment of the effect of high temperature on the mucus immune function from the skin of turbot.In order to exploring the skin mucous of juvenile turbot (Scophthalmus maximus) protein which related with thermo-treatment, the proteome maps of different temperature (20,23,25 and 27℃) were made by two dimensional gel electrophoresis (2-DE), and then, analyzed by ImageMaster 2D Software. The peptide mass map is obtained with MALDI-TOF-TOF, a novel protein of polypeptide is identified by database retrieval. The results showed that the proteome maps were obviously different in the different temperature, it represent that the increase and decrease of the protein spot. Totally,209 protein spots were matched for the five maps (To,T1,T2,T3,T4) using Imagemaster 2D Platinum 6.0. Six protein spots (S1, S2, S3, S4, S5, S6) which have significant deferent were selected, and identified by MALDI-TOF-TOF. Then, search in the data base for protein identification and function prediction. Five protein were confimed, two protein in which named lectin and cytokeratin have the high value for further research.According to the result of the histology, molecular biology, biochemistry and proteomics, the conclusions of the author are as follows:The regular changed of type II mucus cell at high temperature stress. Determined several proteins include IgM, IL-1β, hepcidins, lysozyme, acid phosphatase, superoxide dismutase, lectin and cytokeratin, which related with thermo-treatment, moreover, superoxide dismutase, lectin and cytokeratin have the potential as the marker-protein. These information could be useful in better understanding the role of mucus as a component of the innate immune system, and studying the functional protein, and identify genotypes that are best suited to the environments in which the fish are to be farmed. |
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