Different Fish Meal and Fish Oil Dietary Levels in European Sea Bass: Welfare Implications After Acute Confinement Stress

To provide practical feeding management guidelines preceding a stressful episode during farming practices, European sea bass juveniles (initial weight: 72.3 g) were fed for 60-days different fish meal (FM) and fish oil (FO) dietary levels [high (30% FM, 15% FO, FM30/FO15), intermediate (20% FM, 7% FO, FM20/FO7), and low (10% FM, 3% FO, FM10/FO3)] in triplicate conditions. Fish were then fasted for 36 h and exposed to a 2-h acute crowding (80 kg m–3 biomass). Plasma biochemistry, skin mucus parameters and gene expression of stress and immune-related genes were performed before, at 2 and 24 h after crowding. At the end of the trial, the FM10/FO3 group showed lower final body weight, weight gain, and specific growth rate compared to the other treatments. Most of the plasma parameters were mainly affected by crowding condition rather than diet; however, after stress, lactate was higher in the FM30/FO15 group compared to the other treatments. Similarly, protease, antiprotease, peroxidase and lysozyme in skin mucus were mostly affected by crowding conditions, while fish fed FM10/FO3 displayed higher skin mucosal IgM and bactericidal activity against Vibrio anguillarum and V. harveyi. Most of the stress-related genes considered (hsp70 and gr-1 in the brain; hsp70, gr-1 and gr-2 in the head kidney), showed an overall expression pattern that increased over time after stress, in addition, hsp70 in the head kidney was also up-regulated in fish fed FM30/FO15 after stress. Higher plasmatic lactate together with the up-regulation of some stress-related transcripts suggest a higher reactivity to acute crowding of the stress-response mechanism in fish fed high FM and FO dietary levels. Otherwise, the higher skin mucosal IgM and bactericidal activity observed in fish fed FM10/FO3 dietary levels seems to indicate that acute crowding was able to activate a higher pro-inflammatory response in this treatment. Overall, the results of the present study seem to indicate that 10% FM and 3% FO dietary levels might affect stress and immune responses.

Identification of Antimicrobial Peptides Isolated From the Skin Mucus of African Catfish, Clarias gariepinus (Burchell, 1822)

Antimicrobial peptides (AMPs) constitute a broad range of bioactive compounds in diverse organisms, including fish. They are effector molecules for the innate immune response, against pathogens, tissue damage and infections. Still, AMPs from African Catfish, Clarias gariepinus, skin mucus are largely unexplored despite their possible therapeutic role in combating antimicrobial resistance. In this study, African Catfish Antimicrobial peptides (ACAPs) were identified from the skin mucus of African Catfish, C. gariepinus. Native peptides were extracted from fish mucus scrapings in 10% acetic acid (v/v) and ultra-filtered using 5 kDa molecular weight cut-off membrane. The extract was purified using C18 Solid-Phase Extraction. The antibacterial activity was determined using the Agar Well Diffusion method and broth-dilution method utilizing Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). Thereafter, Sephadex G-25 gel filtration was further utilized in bio-guided isolation of the most active fractions prior to peptide identification using Orbitrap Fusion Lumos Tribrid Mass Spectrometry. The skin mucus extracted from African Catfish from all the three major lakes of Uganda exhibited antimicrobial activity on E. coli and S. aureus. Lake Albert’s C. gariepinus demonstrated the best activity with the lowest MIC of 2.84 and 0.71 μg/ml on S. aureus and E. coli, respectively. Sephadex G-25 peak I mass spectrometry analysis (Data are available via ProteomeXchange with identifier PXD029193) alongside in silico analysis revealed seven short peptides (11–16 amino acid residues) of high antimicrobial scores (0.561–0.905 units). In addition, these peptides had a low molecular weight (1005.57–1622.05 Da) and had percentage hydrophobicity above 54%. Up to four of these AMPs demonstrated α-helix structure conformation, rendering them amphipathic. The findings of this study indicate that novel AMPs can be sourced from the skin mucus of C. gariepinus. Such AMPs are potential alternatives to the traditional antibiotics and can be of great application to food and pharmaceutical industries; however, further studies are still needed to establish their drug-likeness and safety profiles.

Untargeted Metabolomics on Skin Mucus Extract of Channa argus against Staphylococcus aureus: Antimicrobial Activity and Mechanism

Microbial contamination is one of the most common food safety issues that lead to food spoilage and foodborne illness, which readily affects the health of the masses as well as gives rise to huge economic losses. In this study, Channa argus was used as a source of antimicrobial agent that was then analyzed by untargeted metabolomics for its antibacterial mechanism against Staphylococcus aureus. The results indicated that the skin mucus extract of C. argus had great inhibitory action on the growth of S. aureus, and the morphology of S. aureus cells treated with the skin mucus extract exhibited severe morphological damage under scanning electron microscopy. In addition, metabolomics analysis revealed that skin mucus extract stress inhibited the primary metabolic pathways of S. aureus by inducing the tricarboxylic acid cycle and amino acid biosynthesis, which further affected the normal physiological functions of biofilms. In conclusion, the antimicrobial effect of the skin mucus extract is achieved by disrupting cell membrane functions to induce an intracellular metabolic imbalance. Hence, these results conduce to amass novel insights into the antimicrobial mechanism of the skin mucus extract of C. argus against S. aureus.

Effects of Dietary Glutathione on the Growth Performance, Skin Mucus Antioxidant Capacity, and Immune Responses of Juvenile Taimen Hucho Taimen

The effects of dietary glutathione on growth performance, skin antioxidant capacity, and immune responses and of juvenile taimen (Hucho taimen) were investigated. The experimental fish had a mean initial weight of 5.36 ± 0.13 g and were fed for eight weeks on diets containing graduated amounts of glutathione (0, 100, 200, 400, and 800 mg kg−1). After the 7 d lipopolysaccharide challenge, the immune parameters of the skin mucus, and antioxidant ability were determined. The treatment groups (400 – 800 mg kg−1) showed a higher survival rate and weight gain rate (P < 0.05). There were higher levels of skin mucus protein, lysozyme activity, and alkaline phosphate activity associated with dietary glutathione supplementation (P < 0.05). Dietary glutathione supplementation improved the minimal inhibitory concentration and antimicrobial activity of the skin mucus (P < 0.05). Fish in the treatment groups showed higher superoxide dismutase, glutathione peroxidase, and catalase activity (P < 0.05), whereas the malondialdehyde content was lower (P < 0.05) than those in the control group. Supplementary glutathione decreased the expression levels of TNF-α, IL1β, IL6, IL8 in the skin and ensured the relatively high expression levels of IκBα after lipopolysaccharide challenge. In conclusion, dietary glutathione (400 – 800 mg kg−1) improved the growth performance, decreased lipopolysaccharide-induced skin inflammatory response, indicating that glutathione has the potential anti-inflammatory effects for preventing inflammation diseases in juvenile taimen.

Anti-breast cancer synthetic peptides derived from the Anabas testudineus skin mucus fractions

Previous study has shown the antimicrobial activities of mucus protein extracted from Anabastestudineus. In this study, we are interested in characterizing the anticancer activity of the A.testudineus antimicrobial peptides (AMPs). The mucus was extracted, fractioned, and subjected to antibacterial activity testing to confirm the fish's AMPs production. The cytotoxic activity of each fraction was also identified. Fraction 2 (F2), which shows toxicity against MCF7 and MDA-MB-231 were sent for peptide sequencing to identify the bioactive peptide. The two peptides were then synthetically produced and subjected to cytotoxic assay to prove their efficacy against cancer cell lines. The IC50 for AtMP1 against MCF7 and MDA-MB-231 were 8.25 ± 0.14 μg/ml and 9.35 ± 0.25 μg/ml respectively, while for AtMP2 it is 5.89 ± 0.14 μg/ml and 6.97 ± 0.24 μg/ml respectively. AtMP1 and AtMP2 treatment for 48 h induced breast cancer cell cycle arrest and apoptosis by upregulating the p53, which lead to upregulate pro-apoptotic BAX gene and downregulate the anti-apoptotic BCL-2 gene, consequently, trigger the activation of the caspase-3. This interaction was supported by docking analysis (QuickDBD, HPEPDOCK, and ZDOCK) and immunoprecipitation. This study provided new prospects in the development of highly effective and selective cancer therapeutics based on antimicrobial peptides.

Assessment of Responses of Common Carp, Cyprinus carpio, Exposed to Sub-Lethal Concentrations of Lead, by Molecular and Mucosal Biomarkers

Heavy metals are one of the most persistent pollutants in waters, and molecular biomarkers as bioindicators could uncover early warning signals of stress suffered by organisms exposed to these pollutants. This research was conducted to explore the impact of lead contaminant on expression levels of hsp70 gene as well as protein pattern and innate immune indicators of skin mucus in Common carp as a biological model. The fish were exposed to different concentrations of Pb(NO3)2 as a source of lead (0, 7.875, 15.75, and 31.5 mg/L of lead) for 14 days. After the completion of the experiment, the liver and gill tissue plus mucus samples were collected from the fish. Next, the effects of exposure to sublethal doses of lead were investigated on expression levels of hsp70 gene, protein pattern, and innate immune indicators of skin mucus. The results indicated that the expression level of hsp70 gene significantly increased (at 7.5% & 30% LC50) in the liver tissue compared to the control group (P<0.05). The protein pattern of treatment samples was different from that of the control group. Also, in the mucus innate immune parameters, the levels of alkaline phosphatase increased significantly at high concentrations of lead contamination in comparison with the control group (P<0.05). The protein levels first revealed an ascending trend (P<0.05), and then a descending trend (at 30% LC50), though it was not a significant downward trend compared with the control group (P>0.05). The results suggested that these physiological indicators in Cyprinus carpio could be used as molecular and mucosal biomarkers for ecotoxicological studies.