Alberto Ruiz

In many meta-analyses and literature reviews on fish microbiota, the provenance of the animals (farmed vs. wild) is often overlooked. Given the well-established role of diet as a key factor in shaping gut microbiota, this study investigates the impact of dietary nature by comparing the microbiota of gilthead seabream (Sparus aurata) fed a commercial diet versus a wild-type diet, all reared within a recirculating aquaculture system. Over a 60-day period, we tracked changes in gut bacterial diversity, structure, and composition following a shift from a commercial feed to a diet exclusively based on pink shrimp (Parapenaeus longirostris). Gut bacterial communities were assessed using 16S rRNA gene sequencing (Illumina MiSeq platform) with primers targeting the V3-V4 hypervariable regions. Twenty days after the dietary change, microbial diversity (Shannon index) increased in fish fed the shrimp diet compared to those fed the commercial diet, while Dominance index values decreased. Additionally, inter-individual (beta-) diversity based on Bray-Curtis distances also differed between dietary treatments. These results support further that microbiota comparisons between farmed/captive and wild fish are challenging due to the unpredictable feeding regimes and dietary variations in wild fish. However, the diet impact on microbiota diminished over time, with the differences in intra- and inter-individual diversity being reduced after 40 days, which suggests an adaptation of microbial communities to dietary changes. At this point, gut microbial communities also showed a similar taxonomical composition. Moreover, a core microbiota consisting of species belonging to the genera Ralstonia, Paraburkholderia, Fulvimonas, Pseudomonas, and Cutibacterium was maintained in all sampling times under both dietary treatments. Overall, this study serves as a conceptual approach that shows a long-term adaptation of the gut microbiota after a radical dietary change, probably driven by host-inherent factors. Furthermore, these results may be a valuable insight for feed manufacturers aiming to develop sustainable and cost-effective ingredients since they suggest that some alternative feeds and ingredients do not have adverse long-term effects on fish gut microbiota.

The combined production of three varieties of lettuce (romaine, iceberg, and red leaf) with flathead grey mullet (Mugil cephalus) was tested in triplicate in three independent coupled aquaponic units with no thermal control. For this purpose, a total of 114 fish (2.5 kg/m³) were stocked in each fish tank (2 m³), and 92 lettuces were planted in the hydroponic unit (6 m²). As no thermal control was included in the design of the aquaponic system, water temperatures declined from maximum values of 20.4 °C to minimum values of 5.0 °C, which directly affected fish growth. However, the conditions imposed by the aquaponic system were suitable for promoting lettuce’s growth and external appearance, as no pests or leaf discoloration were noticed. Lettuce survival was similar among the three tested varieties (98.5 ± 1.7%). The yields for the romaine and iceberg varieties were 384 ± 100 g/lettuce and 316 ± 70 g/lettuce, respectively, and that for the red leaf variety was lower, at 176 ± 75 g/lettuce. Yield values ranged between 3.6 and 4.4 kg/m² depending on the replicate considered (4.0 ± 0.4 kg/m²). According to present results, each aquaponic unit required ca. 2.6–2.7 L of water per unit of lettuce produced.

In the present study, we propose the use of swabs in non-lethal sampling procedures to collect the mucosa-adhered gut microbiota from the posterior intestine of fish, and therefore, we compare the bacterial communities collected by conventional scraping and by swabbing methods. For this purpose, samples of the posterior intestine of rainbow trout (Oncorhynchus mykiss) were collected first using the swabbing approach, and after fish euthanasia, by mucosa scraping. Finally, bacterial communities were compared by 16S rRNA gene Illumina sequencing. Results from the current study revealed that similar values of bacterial richness and diversity were found for both sampling procedures. Similarly, there were no differences between procedures when using qualitative metrics (Jaccard and unweighted UniFrac) for estimating inter-individual diversity, but the quantitative metrics (Bray-Curtis and weighted UniFrac) showed a higher dispersion when samples were obtained by swabbing compared to scraping. In terms of bacterial composition, there were differences in abundance for the phyla Firmicutes and Proteobacteria. The cause of these differential abundances may be the inability of the swab to access to certain areas, such as the basal region of the intestinal villi. Moreover, swabbing allowed a higher representation of low abundant taxa, which may also have an important role in host microbiome regardless of their low abundance. Overall, our results demonstrate that the sampling method is a factor to be considered in experimental design when studying gut bacterial communities to avoid potential biases in the interpretation or comparison of results from different studies. In addition, the advantages and disadvantages of each procedure (swabbing vs scraping) are discussed in detail, concluding that swabbing can be implemented as a reliable and non-lethal procedure for posterior gut microbiota studies, which is of particular interest for animal welfare and the 3Rs principle, and may offer a wide range of novel applications.

Inter-species microbial transplantations offer the possibility of transferring species-specific microbes and their associated functionality. As a conceptual approach, an intestinal microbiota transplant (IMT) between two marine carnivorous fish species that thrive in different environmental conditions was conducted: from donor Atlantic salmon (Salmo salar) to recipient gilthead seabream (Sparus aurata), after obliterating its basal microbiota with an antibiotic treatment. To confirm that the gut microbiota was able to recover after antibiotics without the influence of the diet, a group of gilthead seabream not submitted to the IMT was kept fasted as an internal control. To assess the effect of the diet after the IMT, two groups of gilthead seabream were respectively fed with their typical diet and with Atlantic salmon diet. At 36 days post-IMT, the gut of the individuals fed with their typical diet was dominated by the feed-associated bacteria, while those fed with the salmon diet had developed a unique microbiota from the convergence of the diet, donor, and recipient microbiota. These results suggested that an intestinal microbiota transplantation may be effective if the basal microbiota from the gut is first cleared and a targeted dietary modification is provided to maintain and enrich the novel bacteria species over time.

This work studied the potential of a combination of pungent spices (capsicum, black pepper, ginger, and cinnamaldehyde) to be used as a supplement in diets of gilthead seabream (Sparus aurata; 44.1 ± 4.2 g). During 90 days, fish were fed three experimental diets with low inclusion of fish oil and containing poultry fat as the main source of lipids, supplemented with graded levels of the tested supplement: 0 (control), 0.1 (SPICY_0.1%), and 0.15% (SPICY_0.15%). As a result, the pungent spices enhanced the growth performance, the activity of the bile-salt-activated lipase in the intestine, and decreased fat deposit levels within enterocytes. The SPICY_0.1% diet reduced the feed conversion ratio and the perivisceral fat index and lipid deposits in the liver. Moreover, the ratio of docosahexaenoic acid/eicosapentaenoic acid in fillet increased in fish fed the SPICY_0.1% diet, while the hepatic levels of docosahexaenoic acid and total n-3 polyunsaturated fatty acids increased in fish fed the SPICY_0.15% diet. Furthermore, there was an effect on the expression of some biomarkers related to lipid metabolism in 2-h postprandial fish (fasn, elovl6, scd1b, cyp7a1, lpl, and pparβ), and in 48 h fasted-fish fed with the SPICY_0.1% diet, a regulation of the intestinal immune response was indicated. However, no significant differences were found in lipid apparent digestibility and proximate macronutrient composition. The spices did not affect biomarkers of hepatic or oxidative stress. No differences in microbial diversity were found, except for an increase in Simpson’s Index in the posterior intestine of fish fed the SPICY_0.1% diet, reflected in the increased relative abundance of the phylum Chloroflexi and lower relative abundances of the genera Campylobacter, Corynebacterium, and Peptoniphilus. In conclusion, the supplementation of gilthead seabream diets with pungent spices at an inclusion of 0.1% was beneficial to enhance growth performance and feed utilization; reduce fat accumulation in the visceral cavity, liver, and intestine; and improve the fish health status and condition. Results suggest that the tested supplement can be used as part of a nutritional strategy to promote a more judicious use of fish oil in fish diets due to its decreasing availability and rising costs.

Given their role in lipid digestion, feed supplementation with bile salts could be an economic and sustainable solution to alterations in adiposity and intestinal inflammation generated by some strategies currently used in aquaculture. An important part of the metabolism of bile salts takes place in the intestine, where the microbiota transforms them into more toxic forms. Consequently, we aimed to evaluate the gut immune response and microbial populations in gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of bile salts with proven background as a regulator of lipid metabolism and fat content. After the 90-day feeding trial, a differential modulation of the microbiota between the anterior and posterior intestine was observed. While in the anterior intestine the relative abundance of Desulfobacterota doubled, in the posterior intestine, the levels of Firmicutes increased and Proteobacteria, Actinobacteriota, and Campylobacterota were reduced when supplementing the diet with bile salts. Even so, only in the anterior intestine, there was a decrease in estimated richness (Chao1 and ACE indices) in presence of dietary bile salts. No significant differences were displayed in alpha (Shannon and Simpson indices) nor beta-diversity, showing that bile sales did not have a great impact on the intestinal microbiota. Regarding the gene expression profile in 2 h postprandial-fish, several changes were observed in the analyzed biomarkers of epithelial integrity, nutrient transport, mucus production, interleukins, cell markers, immunoglobulin production and pathogen recognition receptors. These results may indicate the development of an intestinal immune-protective status to tackle future threats. This work also suggests that this immune response is not only regulated by the presence of the dietary bile salts in the intestine, but also by the microbial populations that are in turn modulated by bile salts. After a fasting period of 2 days, the overall gene expression profile was stabilized with respect to fish fed the unsupplemented diet, indicating that the effect of bile salts was transient after short periods of fasting. On the balance, bile salts can be used as a dietary supplement to enhanceS. auratafarming and production without compromising their intestinal health.

The effects of porcine plasma protein hydrolysate (PPH) on growth, feed efficiency, and immune responses was evaluated in Sparus aurata. Fish were fed two isoproteic (48% protein), isolipidic (17% fat), and isoenergetic diets (21.7 MJ/kg) diets, one of them containing 5% PPH at the expense of fishmeal. Both diets were tested for 92 days. A significant increase in growth was observed in fish fed the PPH diet in comparison to the control group (182.2 ± 4.4 vs. 173.8 ± 4.1 g), as well as an increase in feed intake without worsening FCR values. An ex vivo assay, with splenocytes incubated with lipopolysaccharide, was conducted to evaluate the cellular immune competence of fish. Genes involved in humoral immunity (lys, IgM), pro- (tnf-α, il-1β), and anti-inflammatory (tgf-β1, il10) cytokines were upregulated in the PPH group in comparison to the control group. The inclusion of PPH in diets enhanced the antibacterial capacity of skin mucus, as the co-culture of selected bacteria (E. coli, V. anguillarum, and P. anguilliseptica) with skin mucus indicated. The present results showed that the PPH in low fishmeal diets (2%) promoted growth and feed efficiency, as well as enhancing the immune response, which indicates that this is a safe and functional ingredient for aquafeeds.