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- Antibacterial activity of PDDA-stabilized GO-AgNP nanocompositesPublication . Sadoq, Badr-Edine; Elamine, Youssef; Leal, Inês; Bouajaj, Adel; Britel, Mohammed Reda; Maurady, Amal; Power, Deborah MaryGraphene oxide (GO)-silver nanoparticle (AgNP) nanocomposites are widely studied for their antimicrobial synergy. In this study, the antibacterial activity of GO– AgNP nanocomposites prepared with poly(diallyldimethylammonium chloride) (PDDA) as a stabilizing linker was investigated. The composites were characterized by UV–Vis spectroscopy, FTIR, zeta potential analysis, XRD, TGA, and MPAES. GO–PDDA–AgNPs exhibited a strong surface plasmon resonance band at ~ 420 nm, new C–H stretching bands at 2865–3012 cm⁻¹ from PDDA, and a high positive zeta potential (+ 57.5 mV) compared to bare GO (–40 mV), confirming successful functionalization and improved colloidal stability. Antibacterial activity was evaluated against E. coli and S. aureus using disk diffusion assays, growth curves, and MIC determination. GO and AgNPs alone showed no inhibition at concentrations up to 20 µg/mL, whereas PDDA alone produced inhibition zones of 6–11.5 mm against S. aureus (2.5–100 µg/mL) and induced selective bacterial aggregation. The minimum inhibitory concentration (MIC) of PDDA was between 6.25 and 12.5 µg/mL for S. aureus and between 25 and 50 µg/mL for E. coli, indicating greater potency toward Gram-positive bacteria. The GO–PDDA–AgNP composite inhibited S. aureus with zones of 8.5–10 mm at 10–20 µg/mL, while no significant inhibition was observed for E. coli at the tested concentrations. Molecular docking simulations examining interactions between PDDA and quorum-sensing regulatory proteins AgrA in S. aureus and LsrR in E. coli predicted higher binding affinity to AgrA (–5.11 kcal/mol) than LsrR (–3.76 kcal/mol). While in vitro assays using a Chromobacterium violaceum CV026 biosensor showed no inhibition of AHL-mediated signalling, it should be noted that this model differs mechanistically from the Gram-positive agr system, leaving the predicted AgrA interaction as a potential target for future investigation. Ultimately, this study demonstrated that PDDA is the primary antibacterial component of the GO–PDDA–AgNP composite, exhibiting potent activity against Gram-positive bacteria through a mechanism involving selective bacterial aggregation.
- Assessing fish welfare in small-scale commercial fixed-net fisheries off the southern portuguese coastPublication . Samel, Vighnesh Nilesh; Costa, Rita; Marçalo, Ana; Frade, Magda; Bentes, Luis; Saraiva, João; Gonçalves, Jorge Manuel Santos; Guerreiro, Pedro Miguel; Claudio D'IglioDespite a growing interest in animal welfare in production systems, research on fish welfare remains limited, particularly in commercial fisheries. Fish caught in fixed-net fisheries experience multiple stressors from the time of capture to mortality on deck considered detrimental to their welfare. We examined the impact of bottom-set gill nets and on-board handling on catch welfare using behavioural and physiological indicators. Vitality assessments were performed on four commercially important fish species on-board fishing vessels through a devised vitality scale that included behaviours, morphological condition and reflexes as indicators of welfare. Physiological stress parameters (Cortisol, Glucose, Lactate and Osmolality) were evaluated in blood collected on deck and analysed in relation to the vitality scores. The vitality at arrival on deck as well as the rate of decrease in vitality differed significantly amongst the tested species. Furthermore, Generalised Linear Models predicted that several biological, operational, and environmental variables significantly affect the extent of time the fish shows activity, and hence, on the welfare. Elevated average cortisol levels were found at all the vitality stages highlighting the stress experienced by fish due to the fishing process. The findings of this study enable us to recommend welfare-friendly methods in set-net fisheries to promote better fishing standards.
- Assessment of cell disruption methods in an integrated multi-product biorefinery for Nannochloropsis Oceanica: from process design to economic analysis.Publication . Cunha, Pedro; Carvalho, Bernardo; Kholany, Mariam; Pereira, Hugo; Varela, JoãoMicroalgae are bioresources with significant potential within a sustainable, circular, bio-economy. However, high production costs have limited the widespread use of algae biomass. This study aimed to develop a multi-product biorefinery for Nannochloropsis oceanica that generates multiple revenue streams from the biomass, thereby enhancing the economic viability of algal production. The effectiveness of cell wall disruption using high-pressure homogenization and enzymatic hydrolysis was evaluated. Enzymatic hydrolysis solubilized nearly half (48.2 ± 1.5%) of the dry cell weight, compared to only 27.3 ± 3.2% with high-pressure homogenization, resulting in more concentrated water-soluble fractions and significantly higher protein extraction yields. Lipid extracts obtained after enzymatic hydrolysis had higher lipid (72.0 ± 5.3% w/w) and eicosapentaenoic acid (28.1 ± 6.9% w/w) contents than those from high-pressure homogenization (38.8 ± 6.1% w/w lipids; 9.1 ± 0.6% w/w eicosapentaenoic acid), despite similar lipid extraction yields (around 30%). Increasing the ethanol volumetric ratio from 58% to 75% v/v significantly improved lipid extraction yields (57.4 ± 3.1%) in the enzymatic hydrolysis-based biorefinery, with even higher yields observed upon scaling up (70.1%). All fractions, including lipid extracts, exhibited a balanced essential amino acid profile that exceeded the WHO/FAO/UNU-recommended values. A preliminary economic analysis indicated that lipid production was more cost-effective when cells were permeabilized by enzymatic hydrolysis than by high-pressure homogenization.
- Automated prediction of spawning nights using machine learning analysis of flatfish behaviourPublication . Qadir, Abdul; Duncan, Neil; González-López, Wendy Ángela; Serratosa, Francesc; Fatsini Fernández, ElviraSenegalese sole (Solea senegalensis) broodstock exhibit distinct behaviours (Rest the Head, Guardian, Follow, and Locomotor activities) that are important for breeding success. Understanding and monitoring these behaviours are essential to understand successful breeding of Senegalese sole. However, manually analysing these behaviours represents a significant challenge for human observers and is a labour-intensive process. Moreover, due to reproductive dysfunctions in Senegalese sole, aquaculture operations currently depend on wild origin breeders for successful spawning a reliance that is unsustainable in the long term. Therefore, to address these limitations, this study introduces a custom-designed framework based on computer vision and machine learning techniques. The model integrates object detection and tracking mechanisms to recognize and monitor reproductive behaviours of Senegalese sole within aquaculture environments. By combining advanced tracking algorithms, our model effectively extracts and analyses behavioural patterns from video datasets. The automated model behavioural analyses compared with manual analyses demonstrated strong performance, with accuracy, precision, and specificity exceeding 87 %, and a Pearson correlation of R = 0.99 between manual observation data and automated data. The model analysed videos to accurately identify behaviours with minimal human intervention, thereby saving a substantial number of hours and opened up the possibility to analyse behaviours over longer periods, generating more data. This is the first study to automatically analyse reproductive behaviours across full-night video recordings in Senegalese sole, providing new insights into how behavioural patterns relate to spawning. These behavioural changes in relation to spawning enable the model to effectively predict spawning and non-spawning nights with accuracies ranging from 70 % to 100 %. Such predictive capability can reduce dependence on wild origin breeders, support timely gamete collection, improve reproductive planning, and serve as a potential tool for hatchery automation.
- Chemical and bioactivity profiling of the invasive macroalga rugulopteryx okamurae collected in southern Portugal supporting biotechnological valorisation approachesPublication . D’Unienville, Amandine; Lasnel, Lucas; Macquigneau, Wadi; Trentin, Riccardo; Pais, Adriana C. S.; Rodrigues, Maria João; Santos, Sónia A. O.; Custódio, LuísaThe invasive brown macroalga Rugulopteryx okamurae has rapidly expanded across the Mediterranean–Atlantic region, generating severe ecological impacts. Nevertheless, the considerable amount of biomass available creates opportunities for valorisation within circular bioeconomy frameworks. This study provides an integrated characterization of the chemical profile and bioactivities of freshly collected floating biomass of R. okamurae from southern Portugal. Proximate composition was determined, and lipophilic (hexane) and hydrophilic (water) extracts were analyzed by GC–MS and spectrophotometric methods. Antioxidant activity was assessed using complementary radical-scavenging, reducing power, and metal-chelation assays, and enzyme inhibition was evaluated against targets associated with neurodegenerative, metabolic, and dermatological disorders. The lipophilic fraction was dominated by long-chain alkanes (≈101 mg/g extract) and sterols, particularly fucosterol (≈43 mg/g extract), but exhibited low radical-scavenging capacity (no EC50 reached in DPPH or ABTS assays), and no relevant enzyme inhibition. In contrast, the water extract contained measurable phlorotannins (6.61 mg PGE/g extract) and showed moderate antioxidant (ABTS: EC50 = 5.17 mg/mL; FRAP: EC50 = 0.78 mg/mL) and enzyme inhibition activities (BChE: IC50 = 5.17 mg/mL; tyrosinase: IC50 = 0.78 mg/mL). Compared with previous studies on R. okamurae, this work applies a systematic fractionation of biomass from southern Portugal into polar and non-polar fractions and, for the first time, correlates the resulting detailed chemical profiles with multiple bioactivities. This approach revealed a clear functional differentiation between fractions, with bioactivity being mainly associated with polar metabolites. Overall, these findings highlight the value of structured extraction strategies for biomass valorisation and support the sustainable management of R. okamurae.
- Clean production of microalgae high-value lipid fraction: influence of different pretreatments on chemical and cytotoxic profiles of Chlorella vulgaris supercritical extracts and life cycle assessmentPublication . Vladic, Jelena; Radman, Sanja; Zizak, Zeljko; Besu, Irina; Jerkovic, Igor; Galileu Speranza, Lais; Hala, Ahmad Furqan; Kovacevic, Strahinja; Gouveia, Luisa; Pereira, HugoMicroalgae have emerged as a promising natural resource rich in bioactive compounds. Health-beneficial properties of microalgae, coupled with advantageous characteristics such as high biomass productivity, adaptability, robustness, and carbon dioxide mitigation, position them as a viable solution for global sustainable food production. This study explored clean and environmentally friendly processes to enhance the recovery of lipid bioactive fractions. Microwave (MW), enzymatic (ENZ), and ultrasound (US) pretreatments were applied to improve environmentally friendly extraction of lipid-based components using supercritical CO2. The effects of these pretreatments on extraction yield, chemical profiles, and cytotoxic properties of Chlorella vulgaris (Cv) and smooth C. vulgaris (sCv) extracts were investigated. Additionally, a Life Cycle Assessment (LCA) was conducted to evaluate environmental impacts. MW pretreatment achieved the highest yield increases, from 2.58 times (Cv) to 3.15 times (sCv). UHPLC-ESI-HRMS analysis revealed shifts in the distribution of pigments and derivatives caused by pretreatments, with ENZ extracts showing the most pronounced changes: pigments increased from 9.24% (control Cv) to 40.92% (Cv) and from 12.52% (control sCv) to 71.12% (sCv). Cv extracts exhibited greater activity against MDA-MB-453 cells, while sCv extracts from US pretreatment demonstrated the strongest effect on HeLa cells. The LCA indicated reduced environmental impacts of the pretreatment-enhanced processes up to 65% compared to the control. A scenario analysis was presented to show further possible impact reduction by recirculating the CO2 solvent and substituting the energy source. These findings provide valuable insights into sustainable and scalable green processes for recovering microalgal bioactive components.
- Comparative proteomic analysis of trioza erytreae nymphs developed on Citrus ×limon and Citrus ×sinensis host plantsPublication . Magalhães, Tomás Thormann Abranches de; Anjos Guerreiro, Liliana Isabel Tomé; Power, Deborah Mary; Pereira, José Alberto Cardoso; Duarte, Amilcar; Tomás Marques, NatáliaTrioza erytreae is a vector of Huanglongbing (HLB), a highly damaging citrus disease. Lemon plants (Citrus ×limon) are the preferred host for T. erytreae, although the underlying mechanisms behind this remain to be fully elucidated. A comparative proteomic analysis of T. erytreae nymphs in their fourth and fifth instars that were fed either lemon or sweet orange (SwO) was carried out to investigate the interaction with its hosts. A 24-hour sucrose feeding assay was conducted to understand proteomic responses to a nutrient-poor diet. Proteomic profiling using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS) identified a total of 1,477 psyllid proteins with high confidence. Oviposition and nymphal development were also evaluated across citrus hosts, revealing higher numbers of nymphs developing on lemon than on SwO. Feeding on SwO enriched pathways related to “transmission across chemical synapses” and “metabolism of proteins”. Responses observed under a 24-hour sucrose-only diet enriched the biological processes “response to external stimulus”, “response to stress” and “cytoskeleton organization”. In contrast, these enrichments were absent on lemon host, suggesting that lemon provides a more favourable environment for psyllid development. In addition, nymphs developed on lemon exhibited enhanced energy metabolism and an increase in translation initiation factors. Overall, the results demonstrate that development strongly depends on host plant species, with SwO impairing optimal growth and lemon promoting successful nymphal development.
- The copepod/artemia trade-off in the culture of long snouted seahorse hippocampus guttulatusPublication . Palma, Jorge; Hachero-Cruzado, Ismael; Correia, Miguel J.; Andrade, José Pedro AndradeThis study evaluated the effects of copepod use and copepod conditioning strategies on the growth and survival of long-snouted seahorse (Hippocampus guttulatus) juveniles from 1 to 60 days post-parturition (DPP). Four dietary treatments were tested: Artemia enriched for 24 h with Isochrysis galbana (control), daily collected copepods, copepods unfed for 48 h, and copepods enriched for 24 h with I. galbana. Juveniles fed copepod-based diets exhibited significantly higher growth and survival (p < 0.05) than those fed enriched Artemia. Mean standard length increased from 1.3 ± 0.1 cm at release to 5.9 ± 0.2, 7.5 ± 1.4, 7.1 ± 1.2, and 7.3 ± 1.1 cm at 60 DPP for the enriched Artemia, daily collected copepods, unfed copepods, and enriched copepods treatments, respectively. Wet weight increased from 0.002 ± 0.001 g to 0.44 ± 0.07, 0.81 ± 0.40, 0.68 ± 0.30, and 0.76 ± 0.40 g, while final survival reached 20%, 60%, 33.3%, and 56%, respectively. Compared with enriched Artemia, copepod-based diets markedly enhanced juvenile performance, supporting faster growth and promoting favorable behavioral traits that contributed to improved survival. These results demonstrate that copepods constitute a superior live feed for early juvenile H. guttulatus; however, copepod conditioning strategies directly influence their nutritional quality and, consequently, seahorse growth and survival. The use of copepods throughout the first 60 DPP is therefore not only feasible but strongly recommended for optimizing juvenile H. guttulatus rearing performance.
- Developing productivity and susceptibility attributes towards a rapid management-risk assessment of seaweedPublication . Milla-Figueras, David; Torres, P.; Botelho, A. Z.; Larrea, A.; Parente, M. I.; Afonso, P.The challenge of balancing conservation with sustainable use of marine resources faced by fisheries managers is quite often constrained by limited data on key species. This has become particularly true in the case of seaweed, which provide essential habitats for a diverse range of species in marine ecosystems and have long been valued by humans as food or, more recently, for their industrial applications. The expanding seaweed industry relies heavily on harvesting but historical declines in seaweed populations raise concerns over sustainability, leveraged by a general data-poor scenario for these species. To address this, we propose adapting the Productivity and Susceptibility Analysis (PSA) method—traditionally used in fishery assessments—for seaweeds. This novel approach incorporates the unique biology of seaweeds, including growth, reproduction, and susceptibility to environmental changes and harvesting practices, to evaluate their vulnerability. We applied this adapted PSA framework to a case study of seaweed harvesting in the Azores Archipelago, mid-north Atlantic, demonstrating its applicability in future science-based seaweed fisheries management srtategies. Among the 18 species studied, we identified five of high vulnerability (Cystoseira pustulata, Fucus spiralis, Gongolaria abies-marina, Laminaria ochroleuca, and Ericaria selaginoides). Our adapted PSA framework also considers local ecological impacts, stakeholder input, and relevant environmental factors, providing targeted management recommendations. While such a PSA does not replace stock assessments, it serves as a valuable risk assessment tool, identifying species needing greater management attention despite data limitations. Ultimately, this framework supports local, national and international policy and management measures, contributing to the conservation of coastal marine ecosystems.
- Dietary 1.3-1.6 yeast β-glucans enhance immune response and disease resilience in European seabass challenged with Tenacibaculum maritimumPublication . Orelhas Cabano, Miguel; Richard, Nadège; Schulthess, Julie; Pinto, Lourenço Ramos; Cunha, Francisco Pinto; Peixoto, Maria João; Aragão, Cláudia; Costas, BenjaminIntroduction: Nutritional strategies have emerged as promising tools to modulate immune responses in marine fish, particularly at mucosal surfaces involved in host-pathogen interactions. Dietary immunomodulators such as yeast-derived bglucans (BG) have gained attention for their capacity to enhance innate immune defenses, however their role in coordinating these mechanisms during bacterial infections remains incompletely understood. Methods: This study evaluated the effects of two dietary inclusion levels (0.06 and 0.12%) of purified yeast b-1,3/1,6-glucans on the immune responses of European seabass (Dicentrarchus labrax) following a bath challenge with Tenacibaculum maritimum specifically. After four weeks of feeding with experimental diets, fish were exposed to a sub-lethal infection, and immune parameters were accessed in the skin, intestinal mucosa and at systemic level. Results: Growth performance and feed efficiency were not affected by BG supplementation. At 24 h post-infection, fish fed with the 0.12% BG diet maintained circulating leukocyte counts and displayed increased plasma lysozyme activity, indicating enhanced early immune readiness. Moreover skin immune responses showed an early upregulation of pro-inflammatory and antimicrobial related genes, followed by increased expression of regulatory cytokines at 7 days post-infection, suggesting an efficient transition from immune activation to resolution. In contrast, intestinal immune responses remained largely unchanged, indicating a compartmentalized modulation focused on the primary site of infection. Conclusion: Overall, dietary BG supplementation exerted a dose dependent immunostimulatory effect in European seabass, with 0.12% BG eliciting the most significant chances in disease resilience