Recent studies have demonstrated the impact of diet on microbiota composition, but the essential need for the optimization of production rates and costs forces farms and aquaculture production to carry out continuous dietary tests. In order to understand the effect of total fishmeal replacement by vegetable-based feed in the sea bream (Sparus aurata), the microbial composition of the stomach, foregut, midgut and hindgut was analysed using high-throughput 16S rDNA sequencing, also considering parameters of growth, survival and nutrient utilisation indices.A total of 91,539 16S rRNA filtered-sequences were analysed, with an average number of 3661.56 taxonomically assigned, high-quality sequences per sample. The dominant phyla throughout the whole gastrointestinal tract were Actinobacteria, Protebacteria and Firmicutes. A lower diversity in the stomach in comparison to the other intestinal sections was observed. The microbial composition of the Recirculating Aquaculture System was totally different to that of the sea bream gastrointestinal tract. Total fishmeal replacement had an important impact on microbial profiles but not on diversity. Streptococcus (p-value: 0.043) and Photobacterium (p-value: 0.025) were highly represented in fish fed with fishmeal and vegetable-meal diets, respectively. In the stomach samples with the vegetable diet, reads of chloroplasts and mitochondria from vegetable dietary ingredients were rather abundant. Principal Coordinate Analysis showed a clear differentiation between diets in the microbiota present in the gut, supporting the presence of specific bacterial consortia associated with the diet.Although differences in growth and nutritive parameters were not observed, a negative effect of the vegetable diet on the survival rate was determined. Further studies are required to shed more light on the relationship between the immune system and sea bream gastrointestinal tract microbiota and should consider the modulation of the microbiota to improve the survival rate and nutritive efficacy when using plant-based diets.

BackgroundIn order to ensure sustainability of aquaculture production of carnivourous fish species such as the gilthead seabream (Sparus aurata, L.), the impact of the inclusion of alternative protein sources to fishmeal, including plants, has been assessed. With the aim of evaluating long-term effects of vegetable diets on growth and intestinal status of the on-growing gilthead seabream (initial weight = 129 g), three experimental diets were tested: a strict plant protein-based diet (VM), a fishmeal based diet (FM) and a plant protein-based diet with 15% of marine ingredients (squid and krill meal) alternative to fishmeal (VM+). Intestines were sampled after 154 days. Besides studying growth parameters and survival, the gene expression related to inflammatory response, immune system, epithelia integrity and digestive process was analysed in the foregut and hindgut sections, as well as different histological parameters in the foregut.ResultsThere were no differences in growth performance (p = 0.2703) and feed utilization (p = 0.1536), although a greater fish mortality was recorded in the VM group (p = 0.0141). In addition, this group reported a lower expression in genes related to pro-inflammatory response, as Interleukine-1β (il1β, p = 0.0415), Interleukine-6 (il6, p = 0.0347) and cyclooxigenase-2 (cox2, p = 0.0014), immune-related genes as immunoglobulin M (igm, p = 0.0002) or bacterial defence genes as alkaline phosphatase (alp, p = 0.0069). In contrast, the VM+ group yielded similar survival rate to FM (p = 0.0141) and the gene expression patterns indicated a greater induction of the inflammatory and immune markers (il1β, cox2 and igm). However, major histological changes in gut were not detected.ConclusionsUsing plants as the unique source of protein on a long term basis, replacing fishmeal in aqua feeds for gilthead seabream, may have been the reason of a decrease in the level of different pro-inflammatory mediators (il1 β, il6 and cox2) and immune-related molecules (igm and alp), which reflects a possible lack of local immune response at the intestinal mucosa, explaining the higher mortality observed. Krill and squid meal inclusion in vegetable diets, even at low concentrations, provided an improvement in nutrition and survival parameters compared to strictly plant protein based diets as VM, maybe explained by the maintenance of an effective immune response throughout the assay.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1626-6) contains supplementary material, which is available to authorized users.

The search for new sustainable aquafeeds for the species with greater economic importance, such as the gilthead sea bream in Europe, is one of the main challenges in the aquaculture sector. The present work tested fishmeal replacement by a mixture of plant meals at different levels, as well as the use of marine by-products with attractant properties and high-quality protein in high plant protein diets. In order to do that, effects on growth and biometric parameters, digestibility, amino acid retention, excreted ammonia and proteases and amylase activity were assessed, using six different diets: FM100 (100% of protein provided by fishmeal), FM50 (50% of replacement), FM25 (75% of replacement) and FM0 (100% of replacement), but also FM25+ (75% of replacement and 15% of squid and krill meal inclusion), and FM0+ (100% of replacement and 15% of squid and krill meal inclusion). In group FM0, a clear impact of dietary changes was observed on growth, survival and ammonia excretion. Amino acid retention in group FM0+ was also significantly affected, which can be explained by the limited content of certain amino acids in this diet. On the other hand, no significant differences were observed in most biometric parameters or in enzyme activity. In conclusion, complete fishmeal replacement can be achieved by using a mixture of plant-based sources, but supplementation with complementary marine ingredients can prevent detrimental effects on growth, survival, nutritional parameters and protein metabolism.

Impact of high dietary plant protein with or without marine ingredients in gut mucosa proteome of gilthead seabream (Sparus aurata, L.

A 154‐day trial was performed to assess the use of an alternative protein blend (corn gluten, krill and meat meal) as a substitute for fishmeal in diets for juvenile yellowtail, using four isolipidic (140 g/kg) and isoenergetic diets (24 MJ/kg) with the same digestible protein content (50%). The control diet was FM100, without replacement, and in FM66, FM33 and FM0, fishmeal was replaced at 33 g/kg, 66 g/kg and 100 g/kg, respectively. At the end of the experiment, no differences in growth parameters were observed. Fish fed the FM0 diet exhibited the lowest survival (23%). This high mortality may be due to different factors, such as a dietary amino acid imbalance or some antinutrient factors contained in the alternative ingredients. Feed intake, feed conversion ratio, digestible protein intake and protein efficiency ratio were similar in all diets. However, digestible energy intake and protein efficiency retention were lowest in fish fed the FM0 diet. Apparent digestibility coefficients for protein, energy and amino acids diminished as a substitution for fishmeal increased. Significant differences were observed in the diet whole‐fish body profile amino acid retention (AAR) ratio for the seven essential amino acids. In summary, total fishmeal replacement by the blend assayed was not feasible for yellowtail. The FM66 diet resulted in good growth, high survival and good nutrient efficiency.

In recirculating aquaculture systems, performance of nitrifying biofilters for total ammonia nitrogen (TAN) removal from the culture water and thus minimizing eutrophication depends on numerous elements of design. In this article the combined effect of three of these process parameters (temperature, hydraulic loading and TAN load) is evaluated. Ammonia removal rates (N-TAN divided by biofiltration area and day) were measured for every combination of five different temperatures, three different hydraulic loadings and three different ammonia loads. Every one of the process parameters were influential on nitrification rates and the lowest process parameters values corresponded with significantly lower N-TAN removal rates. A significantly higher mean N-TAN removal rate (0.241 gN-TAN removed m -2 day -1 ) was found for the combination of the highest water temperature (27 ºC), the highest hydraulic loading (11 2 m 3 m -2 h -1 ) and the highest TAN load (9 gTAN m -3 day -1 ), suggesting a positive synergy of the three process parameters on the achievement of greater biofilter performances.

The interaction between diet and intestinal health has been widely discussed, although in vivo approaches have reported limitations. The intestine explant culture system developed provides an advantage since it reduces the number of experimental fish and increases the time of incubation compared to similar methods, becoming a valuable tool in the study of the interactions between pathogenic bacteria, rearing conditions, or dietary components and fish gut immune response. The objective of this study was to determine the influence of the total substitution of fish meal by plants on the immune intestinal status of seabream using an ex vivo bacterial challenge. For this aim, two growth stages of fish were assayed (12 g): phase I (90 days), up to 68 g, and phase II (305 days), up to 250 g. Additionally, in phase II, the effects of long term and short term exposure (15 days) to a plant protein (PP) diet were determined. PP diet altered the mucosal immune homeostasis, the younger fish being more sensitive, and the intestine from fish fed short-term plant diets showed a higher immune response than with long-term feeding. Vibrio alginolyticus (V. alginolyticus) triggered the highest immune and inflammatory response, while COX-2 expression was significantly induced by Photobacterium damselae subsp. Piscicida (P. damselae subsp. Piscicida), showing a positive high correlation between the pro-inflammatory genes encoding interleukin 1β (IL1-β), interleukin 6 (IL-6) and cyclooxygenase 2(COX-2).

BackgroundAlthough high levels of fish meal replacement by alternative protein sources have been achieved without relevant alterations in terms of growth performance, negative effects on immune status were detected. The diet and fish immunity interactions at gut level have been widely discussed, although in vivo approaches have reported several limitations. In this sense, intestine explant culture system can be a valuable complementary tool to study the interactions between pathogenic bacteria and fish gut response, and the possible influence of environmental, breeding, rearing conditions or dietary components on the responsiveness of the innate immune system in fish.The object of this study was to test the impact of total substitution of fish meal by plant protein on the intestinal health of seabream (12g) in two growth stages: phase I (90 days), up to 68 g, and phase II (305 days), up to 250g. In phase II, the effects of the long term and short exposure (15 days) to plant protein diet were determined. In order to determine the effect of plant protein feeding on the innate immune response to bacterial pathogens, and an ex vivo procedure of intestine explants culture was implemented.ResultsFish showed less tolerance to dietary plant protein in phase I than in phase II, while the ex vivo assays indicated that the intestine from fish fed at short-term plant diets showed a higher immune response than at long term feeding.In relation to the immune response to bacterial challenge, a significant expression in pro-inflammatory cytokines IL-1β and IL-6 after 6 hours of exposure to V. algynoliticus, while COX-2 expression was significantly induced by P. damselae subsp. pisicida, showing positive high correlation between them.ConclusionsA differential health status was observed depending of growth stage, being stricter to the plant protein inclusion the younger fish. The new experimental system based on fish intestinal explants culture has been successfully implemented, becoming an effective methodology for ex vivo studies. Under ex vivo conditions, the bacterial challenge induced inflammatory and immune intestinal response, responding stronger those intestine of fish fed during a short-term with a total substitution of fish meal.