Sea anemones are valuable for therapeutic research as a diversified source of bioactive molecules, due to their diverse bioactive molecules linked to predation and defence mechanisms involving toxins and antimicrobial peptides. Acid extracts from Actinia equina tentacles and body were examined for antibacterial activity against Gram-positive, Gram-negative bacteria, and fungi. The peptide fractions showed interesting minimum inhibitory concentration (MIC) values (up to 0.125 µg/mL) against the tested pathogens. Further investigation and characterization of tentacle acid extracts with significant antimicrobial activity led to the purification of peptides through reverse phase chromatography on solid phase and HPLC. Broad-spectrum antimicrobial peptide activity was found in 40% acetonitrile fractions. The resulting peptides had a molecular mass of 2612.91 and 3934.827 Da and MIC ranging from 0.06 to 0.20 mg/mL. Sequencing revealed similarities to AMPs found in amphibians, fish, and Cnidaria, with anti-Gram+, Gram-, antifungal, candidacidal, anti-methicillin-resistant Staphylococcus aureus, carbapenemase-producing, vancomycin-resistant bacteria, and multi-drug resistant activity. Peptides 6.2 and 7.3, named Equinin A and B, respectively, were synthesized and evaluated in vitro towards the above-mentioned bacterial pathogens. Equinin B exerted interesting antibacterial activity (MIC and bactericidal concentrations of 1 mg/mL and 0.25 mg/mL, respectively) and gene organization supporting its potential in applied research.

Stress coping style (SCS) is crucial for animal welfare in the context of breeding. The link between behavioural traits and physiological responses to external stimuli is increasingly recognized and could orient the selection of appropriate SCSs for welfare-oriented breeding. This study aimed to evaluate how SCS influences the physiological responses of Sparus aurata, a widely used species in aquaculture, following Vibrio anguillarum stimulation. To this end, the serum protein electrophoretic profile, analysed by capillary electrophoresis, was used as an innovative parameter to assess physiological variations. S. aurata individuals were categorized into three SCS groups—bold, shy, and intermediate —based on a risk-taking test. Serum was collected at day 0 (pre-inactivated V. anguillarum inoculation) and at 30 and 60 days post-inoculation. Analyses revealed an increase in the β₂-globulin fraction, putatively associated with molecules involved in the physiological response following inactivated pathogen inoculation, accompanied by a decrease in the γ fraction over time. This trend was particularly pronounced in bold fish, while shy ones showed a similar but less marked pattern. Overall, the results suggest that proactive individuals exhibit a more marked physiological modulation to inactivated pathogen stimulation than reactive ones, highlighting modulation of serum protein electrophoresis as a sensitive bioindicator of physiological response in S. aurata.

Equinin B ( GQCQRKCLGHCSKKCPKHPQCRKRCIRRCFGYCL), a marine peptide from Actinia equina exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria. To identify a smaller active region, the peptide was cleaved into three fragments: GQCQRKCLGHCS (EB-1), KKCPKHPQCRK (EB-2) and RCIRRCFGYCL (EB-3). Only the 11-residue C-terminal fragment showed selective activity against Gram-positive bacteria, including Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus hirae, while remaining inactive against Escherichia coli. Peptide modifications, achieved by replacing cysteine residues with arginine, generally did not enhance activity, but in the C-terminal fragment they reduced hemolytic activity and increased bacterial specificity. Membrane depolarization assays confirmed that the unmodified fragment strongly disrupts bacterial membranes, whereas the modified variant showed minimal depolarization, highlighting its markedly reduced membrane-disruptive potential. In silico modelling revealed that the unmodified fragment (EB-3) can adopt multiple membrane-disruption modes, from transient shallow pores to carpet-like mechanisms, while the cysteine-to-arginine variant interacts mainly via partial insertion anchored by arginine residues. Phenylalanine appears to interact with the membrane, and reducing hydrophobicity by its removal abolished antibacterial activity. These findings highlight the 11-residue C-terminal fragment as a tunable, membrane-targeting motif with mechanistic novelty, offering a blueprint for developing safer, selective antimicrobial peptides with reduced cytotoxicity.

Equinin B (GQCQRKCLGHCSKKCPKHPQCRKRCIRRCFGYCL), a marine peptide from Actinia equina exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria. To identify a smaller active region and explore tunable properties, three peptide fragments were synthesized: GQCQRKCLGHCS (EB1), KKCPKHPQCRK (EB2), and RCIRRCFGYCL (EB3), yielding peptides with key AMP-like properties, including the most positively charged and most hydrophobic regions. Only the 11-residue C-terminal fragment showed selective activity against Gram-positive bacteria, including Staphylococcus epidermidis, Bacillus subtilis, and Enterococcus hirae, while remaining inactive against Escherichia coli. Peptide modifications, achieved by replacing cysteine residues with arginine, generally did not enhance activity, but in the C-terminal fragment EB3 they reduced hemolytic activity and increased bacterial specificity. Membrane depolarization assays confirmed that the unmodified fragment EB3 strongly compromises bacterial membranes, whereas the modified variant showed minimal depolarization, highlighting its markedly reduced membrane-perturbing potential. In silico modelling revealed that the EB3 can adopt multiple membrane-disruption modes, from transient shallow pores to carpet-like mechanisms, while the cysteine-to-arginine variant interacts mainly via partial insertion anchored by arginine residues. Phenylalanine appears to interact with the membrane, and reducing hydrophobicity by its removal abolished antibacterial activity. These findings highlight the 11-residue C-terminal fragment as a tunable, membrane-targeting motif with mechanistic novelty, offering a blueprint for developing safer, selective antimicrobial peptides with reduced cytotoxicity.

Annelids are widely recognized as a major taxonomic and functional component of marine benthic systems. With more than 20,000 species formally described and a comparable number likely awaiting desc...