Dietary inclusion of watermelon rind powder and Lactobacillus plantarum: Effects on Nile tilapia's growth, skin mucus and serum immunities, and disease resistance.

An eight-week investigation was conducted to access the potential impact of dietary watermelon rind powder (WMRP) and L. plantarum CR1T5 (LP) administered individually or in combination on immunity, disease resistance, and growth rate of Nile tilapia fingerlings cultured in a biofloc system. Three hundred twenty fish (average weight 16.57 ± 0.14 g) were distributed into 16 tanks at a rate of 20 fish per tank. The fish were fed different diets: Diet 1 (0 g kg-1 WMRP and 0 CFU g-1 L. plantarum) (control), Diet 2 (40 g kg-1 WMRP), Diet 3 (108 CFU g-1 LP), and Diet 4 (40 g kg-1 WMRP + 108 CFU g-1 LP) for eight weeks. A completely randomized design (CRD) with four replications was applied. Skin mucus, serum immunity, and growth parameters were analyzed every 4 weeks, and a challenge study against S. agalactiae was conducted at the end of the experiment. The findings showed that the inclusion of WMRP + LP, administrated individually or in a mixture, significantly (P<0.05) stimulated growth, skin mucus, and serum immune parameters of Nile tilapia fingerlings compared with the control. The highest values were detected in fish fed the combination of WMRP and LP, as opposed to individual administration of either WMRP or LP, in which no significant differences were detected. Within the challenge study, the relative percent survival (RPS) in Diet 2, Diet 3, and Diet 4 was 48.0%, 52.0%, and 68.0%, respectively. Fish fed 40 g kg-1 WMRP + LP produced significantly higher RPS and protection against S. agalactiae than the other treated groups. Current results suggest that the dual administration of WMRP and LP maybe an effective feed additive for Nile tilapia grown in an indoor biofloc system, capable of improving growth parameters and increasing resistance to S. agalactiae infection.

Size shifting of solid lipid nanoparticle system triggered by alkaline phosphatase for site specific mucosal drug delivery.

We aim to prepare a size-shifting nanocarrier for site-targeting mucosal drug delivery that can penetrate through mucus gel layer and remain close to the absorption membrane. As nanocarriers can be engineered to penetrate mucus but they can also back diffuse into outer mucus regions, a size shifting to micron range once they have reached the absorption membrane would prevent back-diffusion effect and extend drug release over a long period of time. For this purpose, we loaded solid lipid nanoparticles (SLN) with a phosphate ester surfactant and octadecylamine. Alkaline phosphatase (AP), a membrane bound enzyme was for the first time utilized as an in situ partner for triggering the size conversion at epithelial cell surface. Having the size of ~120 nm, SLN with hydrophilic and phosphate-decorated shells were shown to penetrate through mucus gel and form aggregates above cell layer surface. Aggregates of 5-8 µm were formed due to interparticle interactions induced by enzymatic phosphate removal after ~30 min in contact with isolated AP. The developed SLN system could be a potential tool for mucosal drug delivery to AP-expressing tissues like colon, lung, cervix, vagina and some mucus-secreting tumors.

Proteomic profile and protease activity in the skin mucus of greater amberjack (Seriola dumerili) infected with the ectoparasite Neobenedenia girellae - An immunological approach.

Skin mucus is considered the first barrier against diseases in fish. The skin mucus protein profile of the greater amberjack (Seriola dumerili) and its changes due to experimental infection with Neobenedenia girellae were studied by combining 2-DE-MS/MS and gel-free LC-MS/MS proteomic approaches. The 2-DE results led to the identification of 69 and 55 proteins in noninfected and infected fish, respectively, and revealed that keratins were specifically cleaved in parasitized fish. Therefore, the skin mucus of the infected fish showed a higher protease activity due to, at least in part, an increase of metal-dependent protease and serine-type protease activities. Additionally, through a gel-free LC-MS/MS analysis, 1377 and 1251 different proteins were identified in the skin mucus of healthy and parasitized fish, respectively. The functional analysis of these proteins demonstrated a statistical overrepresentation of ribosomal proteins (a well-known source of antimicrobial peptides) in N. girellae-infected fish. In contrast, the components of membranes and protein transport GO categories were underrepresented after infection. Immune system process-related proteins constituted 2.5% of the total skin mucosal proteins. Among these skin mucosal proteins, 14 and 15 proteins exclusive to non-parasitized and parasitized fish were found, respectively, including specific serine-type proteases and metalloproteases in the parasitized fish. Moreover, the finding of tryptic peptides exclusive to some bacterial genera, obtained by gel-free LC-MS/MS, allowed us to construct a preliminary map of the microbiota living in the mucus of S. dumerili, with Pseudomonas and Paracoccus the most represented genera in both noninfected and infected fish.

The effect of Pediococcus acidilactici on mucosal immune responses, growth, and reproductive performance in zebrafish (Danio rerio).

A completely randomized experimental design carried out to investigate the effects of different levels of Pediococcus acidilactici (PA) including 0 (basal diet as a control diet), 1 × 106, 2 × 106, 4 × 106, and 8 × 106 colony-forming unit (CFU) per gram of the diet for 60 days on the mucosal immunity responses, growth, and reproductive performance, in zebrafish, Danio rerio (with mean weigh ± SE: 120 ± 10 mg). The obtained results revealed that the best growth and reproduction indices were related to the concentration of 4 × 106 CFU PA g-1 diet (P < 0.05). The maximum activities of mucosal immune responses including total protein, alternative complement system, IgM, and lysozyme were observed in the fish fed with 4 × 106 CFU PA g-1 diet (P < 0.05). Furthermore, the maximum alkaline phosphatase activity of skin mucus was recorded in the fish fed with 8 × 106 CFU PA g-1 diet (P < 0.05). Fish fed with 4 × 106 CFU PA g-1 diet had the highest villus length and width of the intestine (P < 0.05). Supplementing the diet with 4 × 106 CFU PA g-1 diet more significantly enhanced Cyp19a gene expression in comparison with this in other groups. Hence, PA with a concentration of 4 × 106 CFU g-1 diet can be considered as a proper level of probiotic for improving the health, growth, and reproductive performance of the D. rerio.

Effects of dietary fern (Adiantum capillus-veneris) leaves powder on serum and mucus antioxidant defence, immunological responses, antimicrobial activity and growth performance of common carp (Cyprinus carpio) juveniles.

A 56-day research was performed to examine the influence of graded levels (0 (control), 0.5, 1 and 2%) of Fern (Adiantum capillus-veneris) leaves powder (FLP) in diet on immune competence and growth of common carp (Cyprinus carpio, initial weight = 20 g). The serum total immunoglobulins content and lysozyme activity in the 1 and 2% FLP groups remarkably increased compared to the other groups (P < 0.05). The skin mucosal lysozyme activity enhanced with increasing dietary FLP level in a dose-response manner. Fish fed on the FLP-supplemented diets had higher skin-mucosal superoxide dismutase activity than the control (P < 0.05). However, serum antioxidant enzymes were not affected by dietary fern (P > 0.05). The serum bactericidal activity against human and fish pathogens increased with enhancing the FLP level in diet against Staphylococcus aureus, Escherichia coli (EHEC ATCC 43895), Escherichia coli (CI), Pseudomonas aeruginosa, Klebsiella pneumonia and Aeromonas hydrophila. The serum antibacterial activity against Yersinia ruckeri in the 2% FLP group was higher than the other treatments. Furthermore, the serum bactericidal activity against P. aeruginosa (ATCC 27853) only observed in fish fed on the 1 and 2% FLP-supplemented diets. The skin mucosal bactericidal activity and inhibitory effects increased with enhancing the FLP level in diet against E. coli, K. pneumonia, Y. ruckeri and A. hydrophila in a dose response manner. Moreover, the skin mucosal bactericidal activity against S. aureus only observed in fish fed on 1 and 2% FLP-supplemented diets. The weight gain values in the 1 and 2% FLP groups were higher than the other treatments (P < 0.05). Feed conversion ratio (FCR) improved with increasing FLP level in diet in a dose-response manner (P < 0.05). By considering serum and mucosal bactericidal activities against different pathogenic bacteria, the supplementation of 2% FLP in diet is recommended for C. carpio during grow-out phase.

Exploring epidermal mucus protease activity as an indicator of stress in Atlantic sturgeon (Acipenser oxyrinchus oxyrhinchus).

Atlantic sturgeon are anadromous fish that spend much of their life in near-shore environments. They are designated as "threatened" by the Committee on the Status of Endangered Wildlife in Canada and listed by the IUCN as "near threatened." In Canada, Atlantic sturgeon support small commercial fisheries in the Saint John River, New Brunswick, and the St. Lawrence River, Quebec. While occupying the marine environment, the species is susceptible to various anthropogenic stressors, including by-catch in trawl fisheries and through interactions with coastal engineering projects such as tidal power development. Atlantic sturgeon are also susceptible to implantation of acoustic tags used by researchers to study their movement ecology. These stressors can cause physiological and behavioural changes in the fish that can negatively impact their viability. Because the species are commercially important, and are also of conservation concern, it is important to understand stress responses of Atlantic sturgeon to better mitigate the effects of increased industrial activity in the coastal zone. This study used proteomics to identify and characterize protease activity and identify putative novel protein biomarkers in the epidermal mucus of Atlantic sturgeon. Changes in protein profiles in Atlantic sturgeon epidermal mucus as a result of by-catch and surgery stress were investigated using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. Proteolytic activity was identified and characterized using inhibition zymography, which provided information on the classes of proteases that are associated with stress. Samples were collected from Atlantic sturgeon on the Minas Basin, Nova Scotia, Canada, after capture by brush weir and otter trawl, and after surgical implantation of a V16-69 kHz VEMCO acoustic tag. Significant proteins found in the epidermal mucus include various inflammatory proteins, with calmodulin and complement 9 found ubiquitously, and more rarely lysosome C, identified in a brush weir capture sample. Serum albumin, a blood plasma protein, was another ubiquitous protein and verifies how the sample collection method provides a picture of the internal systems. Protease activity was dominantly exhibited by matrix metalloproteases and serine proteases in all sample collections, with serine proteases more active in otter trawl captures than in brush weir captures. By identifying potential protein biomarkers of stress, this study is an example of a non-invasive method for measuring stress in Atlantic sturgeon. Understanding the defence mechanism and release of non-specific biomarkers can be used to improve conservation regulations, as well as to contribute to the limited scientific knowledge on the stress response of Atlantic sturgeon.

Effect of dietary supplementation with yeast Saccharomyces cerevisiae on skin, serum and liver of gilthead seabream (Sparus aurata L).

The effect of dietary supplementation with Saccharomyces cerevisiae on gilthead seabream (Sparus aurata L.) was studied. Four replicates of fish (n = 6) were fed with a commercial diet containing 0 (control, no yeast added) or 10 mg per kilogram of heat-killed (30 min, 60°C) S. cerevisiae. After 4 weeks, half of the fish (two replicates) were injured and continued with the same diet. At 3 and 7 days post-wounding, samples of blood, skin mucus, skin and liver were obtained from each fish. The results showed that calcium concentrations were significantly higher (with respect to control fish) in the serum from fish sampled at 3 days post-wounding, whereas antioxidant enzymes in the skin mucus were altered after wounding (at both 3 and 7 days). Histological analyses revealed oedema, signs of inflammation and white cell recruitment together with a reduction in the epidermis layer in the wounded regions of fish fed control diet. Yeast supplementation did not change growth performance and helped maintain the normal serum calcium concentrations in wounded fish. Furthermore, a reduction in inflammation around wounds in the animals fed yeast with respect to that fed control diet was evident in the histological study. Furthermore, increased levels of stress-related gene expression in liver and skin from wounded fish were obtained. Overall, yeast supplementation seemed to be a functional and appropriate dietary additive to improve skin recovery reducing the stress resulting from wounds.

Influence of silica particles on mucociliary structure and MUC5B expression in airways of C57BL/6 mice.

Purpose: Impaired mucociliary clearance is an initial characteristic of recurrent cough, respiratory infection and chronic respiratory diseases. It has been demonstrated that prolonged inhalation of respirable silica particles results in a variety of pulmonary diseases, but whether the mucociliary system is involved in this process is unclear. This study aims to evaluate the effects of silica particles on mucociliary structure and MUC5B production in respiratory tract.Materials and Methods: C57BL/6 mice were administered with 2.5 mg silica particles through a single intratracheal instillation. The changes of mucociliary structure and MUC5B expression in trachea was evaluated by HE and AB-PAS staining, transmission electron microscopy and immunohistochemistry on days 1, 7, 28 and 84 post-exposure.Results: The mucociliary structure of airway epithelium was obviously impaired by silica particles, showing disordered, shortened or partially lost cilia on the surface, increased mucus in mucous layer and submucosal glands from day 7 to day 84. A variety of ultrastructural abnormalities were discovered in silica-exposed airway cilia, including absence of central pair microtubules, disorganized microtubules and clusters of axoneme on day 1 and 7. The numbers of ciliary axonemes and basal bodies in ciliated epithelial cells were significantly decreased, whereas the proportion of abnormal axonemes was gradually increased with exposure to silica particles (P < 0.05). In addition, silica particles significantly decreased MUC5B expression on the surface of airway epithelium on day 28 and 84, but obviously increased its production in submucosal glands from day 1 to day 84 (P < 0.01).Conclusions: Silica particles could lead to ultrastructural defects in airway cilia, mucus hypersecretion and altered MUC5B expression in trachea, indicating that impaired mucociliary structure and altered MUC5B production might participate in the development of silica-related respiratory diseases.

Effects of dietary myo-inositol on growth, antioxidative capacity, and nonspecific immunity in skin mucus of taimen Hucho taimen fry.

In this study, the effects of dietary myo-inositol on the skin mucosal immunity and growth of taimen (Hucho taimen) fry were determined. Triplicate groups of 500 fish (initial weight 5.58 ± 0.15 g) were fed different diets containing graded levels of myo-inositol (28.75, 127.83, 343.83, 565.81, and 738.15 mg kg-1) until satiation for 56 days. Thereafter, the nonspecific skin mucus immune parameters, antioxidative capacity, and growth performance were measured. The skin mucus protein and the activities of alkaline phosphatase were significantly higher than those in the control group (P < 0.05). However, there were no significant differences in lysozyme activity among the treatments (P > 0.05). The antimicrobial activity and minimum inhibitory concentration of the skin mucus were increased significantly by myo-inositol supplementation (P < 0.05). The superoxide dismutase, catalase, and glutathione peroxidase activities were significantly elevated in the treatment groups (P < 0.05), whereas the malondialdehyde contents were significantly decreased (P < 0.05). Low-level myo-inositol (28.75 mg kg-1) led to a significantly lower weight gain, feed efficiency, condition factor, and survival rate compared with the other treatments (P < 0.05). In conclusion, dietary myo-inositol deficiency (28.75 mg kg-1) adversely affects the skin mucus immune parameters, antioxidative capacity, and growth performance of Hucho taimen fry.

Colitis susceptibility in mice with reactive oxygen species deficiency is mediated by mucus barrier and immune defense defects.

Reactive oxygen species (ROS) generated by NADPH oxidases (NOX/DUOX) provide antimicrobial defense, redox signaling, and gut barrier maintenance. Inactivating NOX variants are associated with comorbid intestinal inflammation in chronic granulomatous disease (CGD; NOX2) and pediatric inflammatory bowel disease (IBD; NOX1); however Nox-deficient mice do not reflect human disease susceptibility. Here we assessed if a hypomorphic patient-relevant CGD mutation will increase the risk for intestinal inflammation in mice. Cyba (p22phox) mutant mice generated low intestinal ROS, while maintaining Nox4 function. The Cyba variant caused profound mucus layer disruption with bacterial penetration into crypts, dysbiosis, and a compromised innate immune response to invading microbes, leading to mortality. Approaches used in treatment-resistant CGD or pediatric IBD such as bone marrow transplantation or oral antibiotic treatment ameliorated or prevented disease in mice. The Cyba mutant mouse phenotype implicates loss of both mucus barrier and efficient innate immune defense in the pathogenesis of intestinal inflammation due to ROS deficiency, supporting a combined-hit model where a single disease variant compromises different cellular functions in interdependent compartments.

Histochemical distribution of four types of enzymes and mucous cells in the intestine of koi carp (Cyprinus carpio var. koi).

The main purpose of this study was to investigate the distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD), and mucous cells in the intestine of the koi carp Cyprinus carpio var. koi. ACP activity was located in the striated border, enterocytes, and lamina propria of the anterior and middle intestines. The ACP activity in the anterior intestine was higher than that in the middle and posterior intestines. ALP existed in the striated border of enterocytes and lamina propria, serosa, muscular layer, and the junction between muscular layer and submucosa layer of the intestine. The ALP activity in the anterior intestine was higher than that in the middle and posterior intestines. NSE activity was localized in the cytoplasm of enterocytes in the whole intestine, and the middle intestine showed the lower NSE activity than the anterior and posterior intestines. POD activity was localized in the blood cells of the lamina propria and cytoplasm of enterocytes in all intestinal segments. The POD activity among the anterior, middle, and posterior intestines was non-significantly different. Alcian blue periodic acid-Schiff histochemical results revealed three types of mucous cells in the intestine. The total number of mucous cells and percentage of type I cells among the anterior, middle, and posterior intestines were non-significantly different. The percentage of the type II cells was the highest in the posterior intestine, while the lowest in the anterior intestine. The percentage of the type III cells was the highest in the anterior intestine, while the lowest in the posterior intestine.

Comparison of several non-specific skin mucus immune defences in three piscine species of aquaculture interest.

Fish skin mucus is a viscous and semipermeable barrier made mainly of water, glycoproteins and soluble proteins. It represents an important defence against the environment and previous studies have reported the presence of different substances involved in immune defence responses in it. The aim of the present work was to characterize skin mucus protease activity by zymography and esterase activity of the subfamily of carboxylesterases in three species of interest for aquaculture: gilthead sea bream, sea bass and meagre. Mucus antioxidant power was also determined by adapting ferric reducing antioxidant power (FRAP) analysis. As a result of these non-specific immune defence parameters, we compared the antibacterial capacity of skin mucus in these species via in vitro dual bacteria strains-skin mucus co-culture growths. We used Pseudomonas anguilliseptica and Vibrio anguillarum as marine pathogenic bacteria and Escherichia coli as non-pathogenic. For each fish species, in the respective zymograms, we determined a pattern of proteolytic digestion bands. A high-molecular-weight band (around 200 kDa; H-band) was evident in sea bream and sea bass, and showed chymotrypsin activity. One or two intermediate-molecular-weight bands (around 75 kDa; I-bands) with non-trypsin and non-chymotrypsin activity, and putatively with metalloprotease activity, were evident in all species. Finally, low-molecular-weight bands (between 14 and 30 kDa; L-bands) showed distinct patterns for each species and matched trypsin activity. Despite the conservative pattern of digestion bands, the levels of total proteolytic activity (TPA) were 5 and 10 times higher in meagre than in sea bass and sea bream, respectively. In parallel, three carboxylesterase activities were detected in the mucus of the three fish species, using myristate (pNPM-CE activity), butyrate (pNPB-CE activity) and acetate (pNPA-CE activity) as substrates. Both pNPB-CE and pNPA-CE were the most abundant in fish mucus, and meagre was again the species with the highest levels. In contrast, the antioxidant power of meagre skin mucus was the lowest. We established the capacity of skin mucus to block or limit bacterial growth (lytic activity) using 24 h growth curves. The log-growth phase of V. anguillarum was strongly blocked by sea bream and meagre mucus for a few hours; but not by sea bass mucus. However, if mucus was not renewed, log-growth was at the end of 24 h studied period. For its part, P. anguilliseptica growth curve was delayed by the three mucus types during the entire growth period. Only meagre achieved lytic activity against E. coli growth. All parameters studied here will be of a great interest as non-invasive bioindicators of non-specific immune defences in fish skin mucus.

Enrichment of common carp (Cyprinus carpio) fingerlings diet with Psidium guajava: The effects on cutaneous mucosal and serum immune parameters and immune related genes expression.

The present study aimed at evaluation of possible effects of Psidium guajava as immunostimulant on mucosal and serum immunity, as well as related genes expression in common carp (Cyprinus carpio) fingerlings. In an eight weeks feeding trial, fish were fed with experimental diets containing guava leaf powder [GLP] (0, 0.25, 0.5 and 1%). Then, skin mucus and serum immune parameters (total Ig, alkaline phosphatase activity, lysozyme activity), as well as immune related genes expression (TNF-alpha, IL1b, IL8) were measured. All levels of GLP inclusion in the diet brought about the increase in total Ig in the serum, while in the mucus only the highest level of GLP was effective. No significant differences were observed in mucus alkanine phosphatase activity, while fish fed 0.25% of GLP had noticeably higher serum lysozyme activity. The mucus total protein electrophoretic pattern revealed some differences between the control and the treated fish. Two high MW bands were present in the control group but not in the treated groups. Three bands with a low MW comprised between 35 and 17 mw were noticed in GLP fed carps and not in the control. Gene expression studies revealed no noticeable alterations in TNF-alpha gene between treated fish and control. However, the level of expression of IL-8 was strongly upregulated in fish fed 0.5 and 1% of GLP. Also, feeding on 0.5% of GLP significantly upregulated IL1b gene expression. Evaluation of growth performance revealed improved performance parameters in GLP treated groups. These results confirmed possible immunomodulatory and beneficial effects of GLP on common carp fingerlings.

Analysis of enzyme activity, antibacterial activity, antiparasitic activity and physico-chemical stability of skin mucus derived from Amphiprion clarkii.

Recently, mucosal surfaces, especially fish skin and its secreted mucus, have attracted significant interest from immunologists. Amphiprion clarkii, a member of the family Pomacentridae, lives symbiosis with sea anemones and has a good resistance to common seawater bacterial diseases and parasites owing to the protection from its abundant skin mucus. In the present work, the activity of immune-related enzymes (lysozyme, protease, antiprotease, cathepsin B, alkaline phosphatase and peroxidase), the antibacterial activity against two Gram-positive bacteria and five Gram-negative bacteria, the antiparasitic activity against the pathogen of marine white spot disease (Cryptocaryon irritans theronts) and the physico-chemical stability (to pH and heat) of the skin mucus of A. clarkii were analysed. The results showed that the levels of lysozyme and peroxidase were very similar (from 2 to 4 U mg-1 protein). However, cathepsin B was detected of 63.32 U mg-1 protein and alkaline phosphatase was only 0.12 U mg-1 protein. Moreover, protease showed a higher percentage of activity than antiprotease. A. clarkii skin mucus showed a strong antibacterial activity against Gram-negative bacteria, particularly against Aeromonas hydrophila and Vibrio parahaemolyticus but showed no effect on Gram-positive bacteria at the tested concentrations. The bactericidal activity functioned within a short time in a distinct time- and dose-dependent manner. SEM showed that after treated with A. clarkii skin mucus, the V. parahaemolyticus cells distorted and piled together, and the filaments appeared and became into cotton-shaped or quasi-honeycomb texture to adhere cells. Meanwhile, A. clarkii skin mucus showed an apparent antiparasitic activity against C. irritans theronts with a distinct dose- and time-dependent relationship. LM and SEM observation showed that after treated with skin mucus, the theronts quickly stopped their swimming and cilia movement, cells became rounded, cilia shed, small bubbles formed on the surface, cell nucleolus enlarged, cytoskeleton deformed, cell membranes ruptured and cell content leaked out. Antibacterial activity was not affected by 30-90 °C heat treatment but was slightly suppressed by 100 °C. In the pH treatment groups, antibacterial activity was not affected by the moderate pH treatment of 5.0-8.0, but slightly suppressed by weak acid and weak base. Therefore, we speculated that the skin mucus of A. clarkii might be a potential source of novel antibacterial and antiparasitic components for fish or human health-related applications. This study broadened our understanding of the role of skin mucus in the innate immune system and provided a basis for the further isolation and purification of active substances.

Terminal carbohydrates abundance, immune related enzymes, bactericidal activity and physico-chemical parameters of the Senegalese sole (Solea senegalensis, Kaup) skin mucus.

Recently, interest in mucosal surfaces, more specifically fish skin and its secreted mucus, has greatly increased among immunologists. The abundance of terminal carbohydrates, several enzymes (proteases, lysozyme, peroxidase, alkaline phosphatase, esterases and ceruloplasmin), bactericidal activity against fish pathogenic and non-pathogenic bacteria and several physico-chemical parameters (protein concentration, pH, conductivity, redox potential, osmolarity, density and viscosity) in the skin mucus of Senegalese sole (Solea senegalensis, Kaup) have been evaluated. Present results evidence the abundance of N-acetylneuraminic acid, mannose, glucose and N-acetyl-galactosamine in skin mucus. The levels of lysozyme, proteases, esterases and alkaline phosphatase were very similar (from 20 to 30 Units mg-1 protein). However, 93 Units mg-1 protein were detected of ceruloplasmin and only 4'88 Units mg-1 protein of peroxidase. Skin mucus of S. senegalensis showed high bactericidal activity against the tested pathogen bacteria but weak activity against non-pathogenic bacteria. Finally, a clear relationship between mucus density and temperature was detected, while viscosity showed a direct shear- and temperature-dependent behaviour. These results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system, as well as, for elucidating possible relationships between biological and physico-chemical parameters and disease susceptibility.

Physiological response, blood chemistry profile and mucus secretion of red sea bream (Pagrus major) fed diets supplemented with Lactobacillus rhamnosus under low salinity stress.

Environmental stressors caused by inadequate aquaculture management strategies suppress the immune response of fish and make them more susceptible to diseases. Therefore, efforts have been made to relieve stress in fish by using various functional feed additives in the diet, including probiotics. The present work evaluates the effects of Lactobacillus rhamnosus (LR) on physiological stress response, blood chemistry and mucus secretion of red sea bream (Pagrus major) under low salinity stress. Fish were fed four diets supplemented with LR at [0 (LR0), 1 × 102 (LR1), 1 × 104 (LR2) and 1 × 106 (LR3) cells g-1] for 56 days. Before stress, blood cortisol, urea nitrogen (BUN) and total bilirubin (T-BIL) showed no significant difference (P > 0.05), whereas plasma glucose and triglyceride (TG) of fish-fed LR2 and LR3 diets were significantly lower (P < 0.05) than those of the other groups. Plasma total cholesterol (T-CHO) of fish-fed LR3 diet was significantly (P < 0.05) lower than that of the other groups. Furthermore, total plasma protein, mucus myeloperoxidase activity and the amount of mucus secretion were significantly enhanced in LR-supplemented groups when compared with the control group (P < 0.05). After the application of the low salinity stress test, plasma cortisol, glucose, T-CHO and TG contents in all groups showed an increased trend significantly (P < 0.01) compared to the fish before the stress challenge. However, plasma total protein and the amount of secreted mucus showed a decreased trend in all groups. On the other hand, BUN, T-BIL and mucus myeloperoxidase activity showed no significant difference after exposure to the low salinity stress (P > 0.05). In addition, the fish that received LR-supplemented diets showed significantly higher tolerance against low salinity stress than the fish-fed LR-free diet (P < 0.05). The physiological status and the detected immune responses, including total plasma protein and mucus myeloperoxidase activity in red sea bream, will provide a more comprehensive outlook of the effects of probiotics to relieve stress in fish.

Humoral and mucosal defense molecules rhythmically oscillate during a light-dark cycle in permit, Trachinotus falcatus.

Circadian rhythm provides organisms with an internal system to maintain temporal order in a dynamic environment. This is typified by a 24-h cycle for a number of physiological processes, including immunity. The present study characterized the humoral and mucosal defense molecules and their dynamics during a light-dark (LD) cycle in juvenile permit, Trachinotus falcatus. All studied defense molecules were constitutively identified in serum and skin mucus. Serum generally exhibited higher levels of these defenses than skin mucus, with the exception of anti-protease (ANTIPRO). The difference in ANTIPRO, lysozyme (LYZ), esterase (ESA) and catalase (CAT) levels between serum and skin mucus was not affected by the phase of the daily cycle. However, a clear phase-dependent difference was observed in protease (PRO), globulin (GLOB), myeloperoxidase (MPO), alkaline phosphatase (ALP) and glutathione peroxidase (GPX) levels. Activities of ALP and GPX displayed significant daily rhythmicity in both serum and skin mucus. Circadian profile of ALP was identical in both biofluids, but an antiphasic feature was exhibited by GPX. GLOB and MPO levels also exhibited significant daily oscillation but only in serum with acrophases registered at ZT 14.5 and 6.15, respectively. Mucus PRO and serum ANTIPRO demonstrated significant temporal variations during a daily cycle albeit not rhythmic. Cluster analysis of the defense molecules in serum and skin mucus revealed two different daily profiles suggesting a possibility of distinct circadian control between humoral and mucosal immunity. These observations indicate that LD cycle had a remarkable impact in the defense molecules characterizing the humoral and mucosal immunity in permit. Daily rhythmic patterns of these defense molecules contribute to our understanding of the barely explored interplay of immunity and circadian rhythm in teleost fish. Lastly, the results could be useful in developing aquaculture practices aiming at modifying the immune functions of permit for improved health.

Comparative analysis of the humoral immunity of skin mucus from several marine teleost fish.

Fish skin mucus contains several immune substances that provide the first line of defence against a broad spectrum of pathogens although they are poorly studied to date. Terminal carbohydrate composition and levels of total IgM antibodies, several immune-related enzymes (lysozyme, peroxidase, alkaline phosphatase, esterases, proteases and antiproteases) as well as the bactericidal activity (against fish pathogenic Vibrio harveyi, Vibrio angillarum, Photobacterium damselae and non-pathogenic bacteria Escherichia coli, Bacillus subtilis, Shewanella putrefaciens) were identified and measured in the skin mucus of five marine teleosts: gilthead seabream (Sparus aurata), European sea bass (Dicentrarchus labrax), shi drum (Umbrina cirrosa), common dentex (Dentex dentex) and dusky grouper (Epinephelus marginatus). First, lectin binding results suggests that skin mucus contain, in order of abundance, N-acetylneuraminic acid, glucose, N-acetyl-glucosamine, N-acetyl-galactosamine, galactose and fucose residues. Second, results showed that while some immune activities were very similar in the studied fish (e.g. IgM and lysozyme activity) other such as protease, antiprotease, alkaline phosphatase, esterase and peroxidase activities varied depending on the fish species. High levels of peroxidase and protease activity were found in U. cirrosa respect to the values obtained in the other species while E. marginatus and S. aurata showed the highest levels of alkaline phosphatase and esterase activities, respectively. Moreover, skin mucus of S. aurata revealed higher bactericidal activity against pathogenic bacteria, contrarily, to what happened with non-pathogenic bacteria (E. coli, B. subtilis). Thus, study of the variations in the carbohydrate profile and immune-related components of the fish skin mucus could help to understand the fish resistance as well as the presence and distribution of pathogens and magnitude of infections, aspects that are of major importance for the aquaculture industry.

Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata).

Mucosal surfaces of fish, including skin, gill and gut, contain numerous immune substances poorly studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to identify and characterize for the first time different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata). To do this, the levels of total immunoglobulin M, several enzymes and proteins (peroxidase, lysozyme, alkaline phosphatase, esterases, proteases and antiproteases), as well as the bactericidal activity against opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum, Photobacterium damselae) and non-pathogenic bacteria (Escherichia coli, Bacillus subtilis) were measured in the skin mucus and compared with those found in the serum. This study demonstrates that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. In addition, skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, while human bacteria can even grow more in the presence of mucus. The results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system with potential application for the aquaculture.