Introduction: In order to ensure lipid quality of cultured fish and an environmentally sustainable production, new alternative sources of EPA and DHA are needed to replace traditional lipid sources, such as fish oil.Methods: Different combinations of alternative marine lipid sources rich in n-3 LC-PUFA available in the market were herein evaluated to replace traditional fish oil (sardine oil) in diets for European sea bass (Dicentrarchus labrax). A commercial-type diet (CTRL), containing 1.6% of EPA + DHA, 5% sardine oil and 8% rapeseed oil was used as a negative control. Another diet (SARDINE) formulated with 8.5% sardine oil, 4.5% rapeseed oil and 2.5% EPA + DHA was used as the positive control. Three experimental diets were formulated to completely replace sardine oil with alternative sources, targeting approximately the same EPA + DHA level as the positive control: the SALMON diet contained 9.9% salmon by-product oil mixed with 3.1% of an algal oil rich in EPA and DHA, while the ALGARAPE and the ALGASOY diets included 4.4% of the algal oil and 8.6% of either rapeseed or soybean oil, respectively. A sixth diet (ALGABLEND) was formulated to partially replace sardine oil with salmon by-product oil and rapeseed oil, balanced with 2% of algae biomass. The experimental diets were hand-fed to 118 g fish for 54 days. Results: All diets were well-accepted by fish and no significant differences were found in feed efficiency, growth performance, somatic indexes or whole body composition among treatments. At the end of the trial, regardless the dietary EPA + DHA level, all fillets contained more than 250 mg of EPA + DHA per 100 g fresh weight, meeting EFSA recommendations for cardiovascular risk prevention for European adults (> 250 mg day ^-1). Discussion: Overall, this study demonstrated that combining expensive sources of n-3 LC PUFA (Veramaris® or Algaessence Feed™ with low-priced sustainable oils (salmon by-products oil or vegetable oils) allows fortifying European sea bass flesh with EPA and DHA, without major textural changes. This approach is a successful strategy for mitigating the negative effects associated with the high inclusion of vegetable oils. However, the retention of n-3 LC-PUFA in muscle was not significantly increased, suggesting that there is a maximum dietary threshold beyond which β-oxidation might be promoted, and hence there is no advantage in increasing the dietary level of these fatty acids in European sea bass diets.

Vibriosis, an often-fatal disease induced by pathogenic members of the Vibrionaceae family, causes severe economic losses in aquacultures. To mitigate/avoid vibriosis outbursts, it is vital to detect and quantify these pathogens as early as possible. However, standard microbiological methods are time-consuming and often underestimate cell counts, which calls for the development of valid alternatives. In this study, real-time polymerase chain reaction (qPCR) was employed to detect the pathogenic species Vibrio alginolyticus, Listonella anguillara, and Vibrio harveyi using a new primer pair targeting the groEL gene. In addition, the DNA extraction efficiency of three methods, two commercial kits and the boiling method, was compared. The most efficient method was the DNeasy Blood and Tissue kit, with a detection limit ranging between 154 and 600 CFU mL⁻¹ in the case of V. alginolyticus and L. anguillara, and 48 CFU mL⁻¹ for V. harveyi. Thus, this study presents the development and evaluation of a method for the early quantification of all three species in saline suspensions. However, the results obtained by spiking a microalgae sample with V. harveyi emphasize the importance of adjusting the DNA control’s standard curve to the relevant extraction matrices, as it affects the DNA extraction efficiency and may hamper an accurate quantification with qPCR.

The increase of antimicrobial resistant strains is leading to an emerging threat to public health. Pathogenic Vibrio are responsible for human and animal illness. The Enterobacteriaceae family includes microorganisms that affect humans, causing several infections. One of the main causes of human infection is related to the ingestion of undercooked seafood. Due to their filter-feeding habit, marine invertebrates, such as clams, are known to be a natural reservoir of specific microbial communities. In the present study, Vibrionaceae and coliforms microorganisms were isolated from clams. A microbial susceptibility test was performed using the disk diffusion method. From 43 presumptive Vibrio spp. and 17 coliforms, three Vibrio spp. with MICs to colistin >512 mg L⁻¹ were found. From the 23 antimicrobial resistance genes investigated, only the three isolates that showed phenotypic resistance to colistin contained the mcr-1 gene. Genotypic analysis for virulence genes in EB07V indicated chiA gene presence. The results from the plasmid cure and transformation showed that the resistance is chromosomally mediated. Biochemical analysis and MLSA, on the basis of four protein-coding gene sequences (recA, rpoB, groEL and dnaJ), grouped the isolates into the genus Vibrio but distinguished them as different from any known Vibrio spp.

Understanding the ecological dynamics of zooplankton communities is crucial to precisely assessing the health of marine ecosystems and their management. Metabarcoding has contributed to a better understanding of biodiversity in marine environments. However, this methodology still requires protocol optimisation. Here, we used a complementary approach combining molecular and morphological identification methods to identify the zooplankton community inhabiting the Berlengas Archipelago, Portugal. The presence of non-indigenous species was also assessed. The results showed that the metabarcoding approach outperformed the classical morphological identification method, detecting more species with higher resolution. Nevertheless, the classical method was able to identify species that were not detected by the molecular approaches, probably due to a lack of reference data in the databases. The comparison between different molecular approaches showed that COI and bulk DNA gave better results than 18S rRNA and eDNA by detecting higher species diversity. However, complementarities were observed between them. Molecular tools also proved effective in identifying several potential non-indigenous species, identifying, for the first time, several potentially unreported NIS inhabiting the Portuguese marine ecosystems. Overall, our results confirmed the importance of combining both classical and molecular methods to obtain a more refined assessment of the zooplanktonic communities in marine environments.

Managing fishery resources is crucial to ensure the marine environment continues to provide diverse goods and services. To overcome difficulties of classical methods used for fish stock management, molecular tools have shown potential to address this issue assessing both targeted and non-targeted species. This study aims to evaluate the spatiotemporal diversity of fish using 12S rRNA gene eDNA metabarcoding sequencing in the Berlengas archipelago and compare two seawater eDNA sampling sources: samples collected by fishermen during their activities and those collected by our research team. The results indicated that autumn presented the highest diversity and that the area around Berlenga Island was the richest area, increasing biodiversity across the region. Fisher-collected samples were generally less diverse than those by the research team but detected species typical of deeper and open-ocean habitats, validating this sampling method. Our study also highlighted eDNA’s role in monitoring fish species by detecting unexpected species for the region, such as Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua), while cautioning against false positives like orange clownfish (Amphiprion percula) and blue tilapia (Oreochromis aureus). Future optimisation of our eDNA sampling methodology could better refine marine ecosystem dynamics around the UNESCO Biosphere Reserve of the Berlengas Archipelago, Portugal.