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The effects of the oral administration of heat-inactivated Lactobacillus delbrüeckii ssp. lactis and Bacillus subtilis, individually or combined, on gilthead seabream immune responses were investigated both systemically and locally in the gut. In a first experiment, seabream (65 g) were fed for 3 weeks different diets supplemented with 1 x 10(7)CFU g(-1)Lactobacillus, 1 x 10(7)CFU g(-1)Bacillus, or 0.5 x 10(7)CFU g(-1)Lactobacillus plus 0.5 x 10(7)CFU g(-1)Bacillus. Controls were fed non-supplemented diet. Six fish per group were sampled at the end of the trial and some humoral and cellular systemic innate immune parameters were evaluated. Feeding the mixture of the two killed bacteria species significantly increased natural complement, serum peroxidase and phagocytic activities compared with controls. In a second experiment, juvenile seabream (13 g) were fed for 3 weeks the same experimental diets and total serum IgM and numbers of gut IgM(+) cells and acidophilic granulocytes were evaluated. All these parameters were significantly higher in the multispecies probiotic group compared to monospecies and control fed groups. The advantages provided by administration of killed probiotic bacteria as well as multispecies versus monospecies formulations are discussed in light of the results obtained and for their possible application in aquacultural practices.

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Biocides are chemical substances that can deter or kill the microorganisms responsible for biofouling. The rapid expansion of the aquaculture industry is having a significant impact on the marine ecosystems. As the industry expands, it requires the use of more drugs, disinfectants and antifoulant compounds (biocides) to eliminate the microorganisms in the aquaculture facilities. The use of biocides in the aquatic environment, however, has proved to be harmful as it has toxic effects on the marine environment. Organic booster biocides were recently introduced as alternatives to the organotin compounds found in antifouling products after restrictions were imposed on the use of tributyltin (TBT). The replacement products are generally based on copper metal oxides and organic biocides. The biocides that are most commonly used in antifouling paints include chlorothalonil, dichlofluanid, DCOIT (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one, Sea-nine 211®), Diuron, Irgarol 1051, TCMS pyridine (2,3,3,6-tetrachloro-4-methylsulfonyl pyridine), zinc pyrithione and Zineb. There are two types of risks associated with the use of biocides in aquaculture: (i) predators and humans may ingest the fish and shellfish that have accumulated in these contaminants and (ii) the development of antibiotic resistance in bacteria. This paper provides an overview of the effects of antifouling (AF) biocides on aquatic organisms. It also provides some insights into the effects and risks of these compounds on non-target organisms.

Abbreviations: AFP, antifreeze proteins; APOA1, Apolipoprotein A1; ASC, apoptosisassociated speck-like protein containing a caspase recruitment domain; C3, complement component 3; CALM, calmodulin; CALR, calreticulin; CASP, caspase; cDNA, complementary DNA; ERp57,endoplasmic reticulum protein 57; FBL, fucose-binding lectin; H1/4, histone H1/H4; IL1B, interleukin 1-beta; LEI, leukocyte elastase inhibitor; LYZ, lysozyme; qPCR, real-time PCR; PRDX, peroxiredoxin; SOD superoxide dismutase; TF, transferrin; WAP65, warm temperature acclimation related 65 kDa protein; www.proteomics-journal.com Page 2 ProteomicsThis article is protected by copyright. All rights reserved.Keywords: European sea bass (Dicentrarchus labrax), mucosal immunity, proteome, skin mucus, transcript profile. AbstractSkin mucus is the first barrier of fish defence. Proteins from skin mucus of European sea bass (Dicentrarchus labrax) were identified by 2DE followed by LC-MS/MS. From all the identified proteins in the proteome map, we focus on the proteins associated with several immune pathways in fish. Furthermore, the qPCR transcript levels in skin are shown. Proteins found include apolipoprotein A1, calmodulin, complement C3, fucose-binding lectin, lysozyme and several caspases. To our knowledge, this is the first skin mucus proteome study and further transcriptional profiling of the identified proteins done on this bony fish species.This not only contributes knowledge on the routes involved in mucosal innate immunity, but also establishes a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.www.proteomics-journal.com Page 3 ProteomicsThis article is protected by copyright. All rights reserved. Statement of significance of the studyThe skin mucus is the first defence barrier of teleost fish; together with the skin, it protects the animal from pathogens, potential harmful chemicals and physical factors in the water where it is constantly submerged. This study identifies for the first time the main proteins in the skin mucus of European sea bass (Dicentrarchus labrax). In particular, we identify immune relevant proteins. The presence of RNA of the immune relevant genes in skin indicates that the proteins could be synthesised in the skin itself.

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