The efficacy of intraperitoneal injection of an oil-based bivalent autogenous vaccine and the commercial vaccine AlphaJect 3000 (Pharmaq AS) to prevent atypical furunculosis and vibriosis in turbot was analyzed. The effect of both vaccines on health parameters and survival of fish after challenge with V. anguillarum and A. salmonicida subsp. achromogenes was tested. The autogenous vaccine conferred high levels of protection and long-lasting immunity against both pathogens with a single dose. However, severe side effects were observed in turbot injected with this autovaccine and minor negative effects with the AlphaJect 3000 vaccine and the adjuvant Montanide or Eolane. All vaccinated fish showed remarkable antibody agglutination titers, higher than those of control fish, which were maintained 160 d after vaccination. In conclusion, the autogenous bivalent vaccine induces long-lasting protection against atypical furunculosis and vibriosis in turbot, after administration of a single dose, at the cost of high side effects in fish. Therefore, the development of new vaccines should focus on autovaccines and the use of liquid paraffin adjuvants that increase protection with reduced or no side effects.

Betanodavirus infection poses a significant threat to marine fish species in the Mediterranean, affecting both aquaculture and wild populations. Despite increasing evidence of viral circulation in farmed and wild fish, data on natural outbreaks in wild groupers remain limited. This study investigated mortality episodes in wild dusky groupers (Epinephelus marginatus) within two marine protected areas (MPAs): Portofino MPA (Liguria, Italy) and Larvotto MPA (Principality of Monaco) during 2018–2019. Pathological examinations and virological diagnostics confirmed that the causative agents were betanodavirus strains belonging to the RGNNV genotype. Phylogenetic analyses revealed high genetic similarity among viral strains detected at geographically distant sites and across host species, suggesting potential regional connectivity mediated by mobile vectors or environmental transport. Seawater temperature analysis indicated that extreme and prolonged high-water temperatures were prodromal and coincided with observed outbreaks, supporting a role for thermal stress in triggering outbreak onsets. These findings highlight the susceptibility of wild dusky grouper populations to betanodavirus and underscore the interplay between host behavior, environmental conditions, and pathogen dynamics. The study emphasizes the importance of integrated health surveillance strategies within and around MPAs to monitor fish health and environmental parameters, thereby conserving wild fish populations and biodiversity.

Among all the mucosal barriers, the skin and its surrounding mucus are possibly the main defensive tools used by fish against the environment. Due to its less-invasive extraction, the study of its production and functions has gained high interest in the last years. However, there are still many gaps in research, such as the possible alteration of mucus composition or the skin integrity resulting from the sampling process. In the current study, skin mucus extraction impacts were determined by comparing the effects of one-single extraction (SEG; single extraction group) and three successive extractions (REG; repetitive extractions group, separated by 4 days) on mucus properties and on skin epithelial integrity. In terms of analytical evaluation, plasma biomarkers and plasma antibacterial capacity were also determined. With regard skin histology and skin barrier properties, both SEG and REG did not evidence differences with respect to intact skin (ØEG). Interestingly, the repetitive mucus extraction protocol seemed to activate skin mucus turnover, significantly increasing the number of low-size mucus cells (cell area < 100 µm²) and reducing the number of high-size mucus cells (cell area > 150 µm²). Repetitive extraction of skin mucus diminished the amounts of soluble protein and glucose in mucus with regard to one-single extraction and increased cortisol exudation. These metabolites remained unaltered in plasma, indicating the different response among both sampling targets. Despite mucus biomarkers modification, the antibacterial capacity against the pathogenic bacterial (P. anguilliseptica and V. anguillarum) was maintained in both plasma and mucus irrespective of the number of mucus extractions. Overall, the mucus sampling protocol scarcely affected skin integrity and mucus antibacterial properties and only modified metabolites exudation, evidencing a feasible and minimally invasive method for studying fish health and welfare as an alternative or as a complement to plasma. The knolwdege provided here highlighted that this methodology is putatively transferable to farm culture conditions and showed that it is very useful for the study of threatened species aimed at preserving fish welfare.