Oxford Nanopore Technology (ONT) allows for the rapid profiling of aquaculture microbiomes. However, not all the experimental and downstream methodological possibilities have been benchmarked. Here, we aimed to offer novel insights into the use of different library preparation methods (standard-RAP and native barcoding-LIG), primers (V3–V4, V1–V3, and V1–V9), and basecalling models (fast-FAST, high-HAC, and super-accuracy-SUP) implemented in ONT to elucidate the microbiota associated with the aquatic environment and farmed fish, including faeces, skin, and intestinal mucus. Microbial DNA from water and faeces samples could be amplified regardless of the library–primer strategy, but only with LIG and V1–V3/V1–V9 primers in the case of skin and intestine mucus. Low taxonomic assignment levels were favoured by the use of full-length V1–V9 primers, though in silico hybridisation revealed a lower number of potential matching sequences in the SILVA database, especially evident with the increase in Actinobacteriota in real datasets. SUP execution allowed for a higher median Phred quality (24) than FAST (11) and HAC (17), but its execution time (6–8 h) was higher in comparison to the other models (0.6–7 h). Altogether, we optimised the use of ONT for water- and fish-related microbial analyses, validating, for the first time, the use of the LIG strategy. We consider that LIG–V1–V9-HAC is the optimal time/cost-effective option to amplify the microbial DNA from environmental samples. However, the use of V1–V3 could help to maximise the dataset microbiome diversity, representing an alternative when long amplicon sequences become compromised by microbial DNA quality and/or high host DNA loads interfere with the PCR amplification/sequencing procedures, especially in the case of gut mucus.

The significant microbiota variability represents a key feature that makes the full comprehension of the functional interaction between microbiota and the host an ongoing challenge. To overcome this limitation, in this study, fish intestinal microbiota was analyzed through a meta-analysis, identifying the core microbiota and constructing stochastic Bayesian network (BN) models with SAMBA. We combined three experiments performed with gilthead sea bream juveniles of the same hatchery batch, reared at the same season/location, and fed with diets enriched on processed animal proteins (PAP) and other alternative ingredients (NOPAP-PP, NOPAP-SCP). Microbiota data analysis disclosed a high individual taxonomic variability, a high functional homogeneity within trials and highlighted the importance of the core microbiota, clustering PAP and NOPAP fish microbiota composition. For both NOPAP and PAP BNs, >99% of the microbiota population were modelled, with a significant proportion of bacteria (55–69%) directly connected with the diet variable. Functional enrichment identified 11 relevant pathways expressed by different taxa across the different BNs, confirming the high metabolic plasticity and taxonomic heterogeneity. Altogether, these results reinforce the comprehension of the functional bacteria–host interactions and in the near future, allow the use of microbiota as a species-specific growth and welfare benchmark of livestock animals, and farmed fish in particular.

A confinement stress test with 75% tank space reduction and behavioural monitoring through tri-axial accelerometers externally attached to the operculum was designed. This procedure was validated by demonstrating the less pronounced stress response in gilthead sea bream than in European sea bass (950–1200 g). Our study aimed to assess habituation to high stocking densities with such procedure in gilthead sea bream. Animals (420–450 g) were reared (June–August) in a flow-through system at two stocking densities (CTRL: 10–15 kg/m³; HD: 18–24 kg/m³), with natural photoperiod and temperature (21–29 °C), and oxygen levels at 5.2–4.2 (CTRL) and 4.2–3.2 ppm (HD). At the end, blood and muscle were sampled for haematology and transcriptomic analyses, and external tissue damage was assessed by image-based scoring. Four days later, fish underwent a 45 min confinement stress test over two consecutive days. HD fish showed reduced feed intake, growth rates and haematopoietic activity. Muscle transcriptome changes indicated a shift from systemic to local growth regulation and a primed muscle regeneration over protein accretion in HD animals with slight external injuries. After stress testing, HD fish exhibited a decreased recovery time in activity and respiration rates, which was shorter after a second stressor exposure, confirming habituation to high densities.

Intestinal microbiota populations are constantly shaped by both intrinsic and extrinsic factors, including diet, environment, and host genetics. As a result, understanding how to assess, monitor, and exploit microbiome–host interplay remains an active area of investigation, especially in aquaculture. In this study, we analyzed the taxonomic structure and functional potential of the intestinal microbiota of European sea bass and rainbow trout, incorporating gilthead sea bream as a final reference. The results showed that the identified core microbiota (40 taxa for sea bass and 20 for trout) held a central role in community organization, despite taxonomic variability, and exhibited a predominant number of positive connections (>60% for both species) with the rest of the microbial community in a Bayesian network. From a functional perspective, core-associated bacterial clusters (75% for sea bass and 81% for sea bream) accounted for the majority of predicted metabolic pathways (core contribution: >75% in sea bass and >87% in trout), particularly those involved in carbohydrate, amino acid, and vitamin metabolism. Comparative analysis across ecological phenotypes highlighted distinct microbial biomarkers, with genera such as Vibrio, Pseudoalteromonas, and Paracoccus enriched in saltwater species (Dicentrarchus labrax and Sparus aurata) and Mycoplasma and Clostridium in freshwater (Oncorhynchus mykiss). Overall, this study underscores the value of integrating taxonomic, functional, and network-based approaches as practical tools to monitor intestinal health status, assess welfare, and guide the development of more sustainable production strategies in aquaculture.

The microalgae Nannochloropsis oceanica cultivated from brewery waste was considered a sustainable feed ingredient for gilthead sea bream ( Sparus aurata ), evaluating physiological, molecular and microbial responses. Over 96 days, fish were fed four isoproteic and isolipidic diets: a commercial‐based diet (D1), and three fishmeal‐free diets with increasing Nannochloropsis inclusion levels (0%, D2; 2%, D3; 14%, D4). The D4 diet was additionally supplemented with docosahexaenoic acid (DHA)‐rich algae oil instead of fish oil (FO). Growth performance remained almost equal in fish fed all the fishmeal‐free diets with only a slight and final improvement (<4%) in the control D1 fish, although an enhanced expression with a discriminant capacity was reported for hepatic growth‐related markers ( igf1 and ghr1 ) in D4 fish. Gene expression profiling also revealed a down‐regulation of fatty acid synthesis in combination with the up‐regulation of tissue lipid‐uptake genes, mostly linked to the dietary DHA enrichment. Antioxidant capacity was increased in both D3–D4 fish, suggesting a progressive activation of enzymatic and non‐enzymatic defences with the dietary microalgae diets (D3–D4). Moreover, intestinal transcriptomic profiling revealed a tightly regulated immune milieu, with coordinated modulation of pro‐ and anti‐inflammatory pathways indicative of an immune surveillance state induced by dietary microalgae. Water microbiota also shifted with diet composition, indicating a link between feeding strategy and environmental microbial communities. Taking all together, Nannochloropsis inclusion, particularly when combined with DHA‐rich microalgae oil, enhances fish health, immune and antioxidant status and beneficial gut microbiota markers. Altogether makes the circularly‐produced Nannochloropsis oceanica a valuable resource for the sustainable development of aquaculture feeds.

In fish gut microbiome studies, there are no standardized protocols regarding sampling region or post-feeding time, nor clear consensus on whether analyses should target resident (autochthonous) or transient (allochthonous) bacteria. This study examined the dynamics and interactions of both microbial communities in the anterior and posterior intestine of farmed gilthead sea bream and evaluated the resident microbiome at 24 and 48 h post-feeding. Microbial DNA was sequenced using the Oxford Nanopore Technology platform. Data were analyzed through statistical and discriminant approaches, as well as a Bayesian network framework to assess bacterial interactions. Transient communities showed higher richness and diversity, regardless of intestinal section, suggesting a more specialized and stable microbial environment in the mucus layer. The two communities differed markedly in structure and composition. Variations associated with intestinal region were less pronounced, particularly for autochthonous bacteria, and post-feeding fluctuations in the resident microbiome were minimal. Functionally, results indicated relevant synergies between communities. Protein metabolism pathways were enriched in autochthonous bacteria, whereas allochthonous microorganisms contributed mainly to bile acid and carbohydrate metabolism. Overall, resident and transient bacteria constitute distinct communities in the gut of gilthead sea bream, with numerous genera present in both but most being differentially represented and interconnected.