1. Introduction

The use of sustainably produced protein and lipid sources for the replacement of fish meal (FM) and fish oil (FO) in aquafeeds are in need to support future growth of the aquaculture sector. Alternative ingredients derived from low-trophic-level organisms such as bacteria, yeast, algae, and insects are currently receiving increasing attention. Microalgae biomasses can be environmentally friendly and are rich in essential fatty acids, amino acids, pigments, and vitamins [1, 2], and their use in aquafeeds can result in enhanced fish quality [3]. For instance, the diatom Phaeodactylum tricornutum contains high levels of eicosapentaenoic acid (EPA) [4], as well as phenolic compounds and carotenoids, which have been suggested to enhance fillet quality in salmon [5]. Use of P. tricornutum in diets for Senegalese sole larvae led to enhanced growth performance [6] and higher dietary nutrient digestibility in rainbow trout [7] and it has also been suggested as an effective replacement for dietary FO in gilthead sea bream larval diets [8, 9].

Microalgae species have been used as FO replacers in fish feeds to enhance the content of n−3 long chain polyunsaturated fatty acids (PUFAs) in white muscle [10]. The traustochytrid Schizochytrium sp., mainly referred to as microalgae, contain high levels of n−3 long chain PUFAs, especially docosahexaenoic acid (DHA) [11]. Dietary Schizochytrium sp. lipids are highly digestible [12, 13] and their use has led to enhanced PUFA concentration in the fillet and improved growth performance in S. aurata [14, 15], while leading to health benefits in other farmed fish species, such as rainbow trout and zebrafish [16, 17]. The combination of Schizochytrium sp. with other microalgae in the diets has shown positive effects on growth and fatty acid accumulation in Pagrus major [18] and Sparus aurata [19]. Katsoulis-Dimitriou et al. [20] showed that microalgae mixtures in sea bream diets can be utilized by the fish gut microbiota metabolizing fucose, potentially improving host metabolism.

Insects are among the most promising nutrient sources that may play a significant protein contributor to aquaculture feeds in the future. Several insects contain a high amount of functional ingredients such as chitin, proteins, fatty acids, and micronutrients [21, 22]. Insect meals (IMs) have been shown to be efficient for FM replacement without adverse effects on fish health [23–25] and have also been suggested to have immunomodulatory, antimicrobial, and antioxidant properties [26–29]. The ability of insects to bioconvert agricultural waste and nonfood ingredients into valuable biomass [26, 30, 31] may contribute to the compliance of food production with circular economy guidelines [32, 33]. Among the species that are considered as potential FM replacement candidates, Hermetia illucens is a highly promising insect with high content of proteins and fats, and a good amino acid profile, although methionine and lysine amounts present are lower than in the FM based protein sources [34–37]. Several studies have been conducted evaluating how H. illucens in the diet affects fish nutrition and health with controversial results [38, 39]. In S. aurata diet, H. illucens meal studies showed to have no effect on fish growth [25, 40, 41], enhance growth [42] or suppress it, depending on FM replacement and feed inclusion level [43–45].

Tunicates are low-trophic-level filter-feeding sea invertebrates that feed on algae-rich water and have the potential for utilization in fish feeds [46]. Their bodies contain, in dry matter, high levels of proteins (e.g., collagen), cellulose, and n−3 fatty acids as well as minerals [47] and bioactive compounds [48]. Such organism is Ciona intestinalis, which is rich in n−3 and n−6 PUFAs and has been suggested as an alternative ingredient for FO replacement [49]. Samuelsen et al. [50] reported that C. intestinalis meal, at 172 g/kg inclusion in feed, which replaced 64% of FM in the diet, resulted in pellet quality features without compromising the fat absorption capacity and fat leakage of the pellets following extrusion.

Availability and cost of raw materials are going to play a key role in FM replacement in industrial aqua-feed production in the future. Among the insects that could be used in fish feeds, Musca domestica appears to be commercially available, with the remaining insect species expected to increase according to demand [51]. The microalgae industry faces challenges in producing large quantities, while microalgal biomasses are not cost-effective, especially when further processed [52]. The cost of raw materials is expected to decrease due to future increased demand of novel ingredients, while the feed formulation has to be tailored made species specific. Another step to increase efficiency of novel ingredients, and therefore, the feed could be genetic selection of fish [53], which could also reduce feed costs.

Diet-associated changes have been demonstrated to ambiguously affect several aspects of fish development and health. In addition to their impact on fish growth [45, 50, 54], novel ingredients in aquafeeds have been reported to modulate both physiological status [55–57] and gut microbial communities [40, 58, 59]. Changes in cellular signaling and energy homeostasis, as well as the recruitment of molecular chaperones, occur during an altered nutrient status to facilitate adaptation to the dietary regime [60–64]. Furthermore, diet-induced stress may also be aggravated due to modifications in gut microbial communities, which contribute to changes in both microbial nutrient production and malabsorption [64, 65]. However, an adequate diet in essential nutrients favors the enrichment of beneficial host bacteria, thus, enhancing the diversity and multifaceted functionality of the gut microbiome [40, 66, 67]. IM in gilthead sea bream diet has controversial effects on fish growth [68] and defatted meal instead of full fat H. illucens meal is proven to have advantages in fish nutrition [44, 69, 70]. On the other hand, tunicate meal (TM) is a novel, alternative, and sustainable ingredient that has the potential to be suitable for FM replacement in the feed of S. aurata. The combination of TM with other known ingredients, such as insect and algae meals, may be the key to the total FM and FO replacement in fish feeds. Such a combination of low-trophic ingredients could be the future of the aquaculture feed industry, due to the ability of these organisms to be sustainably grown on agricultural wastes and be environmentally friendly. In this context, this study aimed to evaluate the global effect of FM and FO replacement with innovative ingredients, such as IM, TM, or their combination with algae meals, respectively, on growth performance, nutrient utilization, physiological status, gut microbiota, liver, and gut histology of gilthead sea bream (S. aurata).

2. Materials and Methods

3. Results

3.4. Heat Shock Response, p38 MAPK, Akt, and AMPK Activation in the Liver

There was a significant effect of the diet on the response of Hsp70 and Hsp90. Both levels decreased in all dietary treatments compared to control (Figure 1a). Specifically, the most significant decrease (p < 0.05) for both examined heat shock proteins (Hsps) was observed in the TM treatment, while the least significant decrease (p < 0.05) was observed in the IM dietary treatment. All dietary treatments exhibited statistically significant differences (p < 0.05) between all dietary regimes.

Regarding the p38 MAPK pathway, a contrary pattern was observed for the phospho p38 MAPK and p38 MAPK under the effect of the experimental dietary treatments (Figure 1b). Phospho p38 MAPK levels significantly increased (p < 0.05) under the effect of 0%FMFO and TM treatments compared to the control, while p38 MAPK levels significantly decreased (p < 0.05) in all dietary treatments. However, p38 MAPK phosphorylation ratio (phospho p38 MAPK/p38 MAPK) exhibited a different pattern. Levels significantly increasing (p < 0.05) in all experimental trials compared to control. Specifically, the most significant increase was observed in TM and the least significant increase in IM dietary group. Regarding p38 MAPK phosphorylation ratio, all dietary groups exhibited statistically significant differences (p < 0.05) between all dietary treatments.

Compared to the abovementioned pathways, a different pattern regarding Akt was apparent in the liver of S. aurata in response to the experimental dietary treatments. Specifically, both phospho Akt and Akt levels significantly decreased (p < 0.05) under the effect of all dietary treatments with the most intense observed in 0%FMFO and TM (Figure 1c). However, Akt phosphorylation ratio (phospho Akt/Akt) exhibited statistically significant increase (p < 0.05) only under the TM treatment, while a significant (p < 0.05) decrease was apparent in the 0%FMFO group compared to the control. IM dietary treatment resulted in no changes compared to the control group.

Regarding AMPK, both phospho and total levels exhibited a pattern of significant decrease (p < 0.05) under the effect of 0%FMFO and TM treatments compared to the control. Similarly, AMPK phosphorylation ratio (phosphor AMPK/AMPK) decreased (p < 0.05) in 0%FMFO and TM dietary treatments, while the IM dietary treatment resulted in a significant increase (p < 0.05) compared to the control (Figure 1d).

3.5. Midgut Bacterial Microbial Analysis

Following the raw data processing, the average number of OTUs detected in the midgut samples of S. aurata fed the control, 0%FMFO, IM, and TM diets was not statistically significant (p > 0.05; Figure 2a). The number of unique OTUs was 45 in control dietary treatment, 45 in 0%FMFO, 38 in IM, and 133 in TM. Several bacterial families were identified in the midgut samples with relative abundance displaying variations among the different dietary treatments (Figure 2b). Chitinophagaceae, Alphaproteobacteria, Haliaceae, Clostridia, Methyloligellaceae, Streptococcaceae, Moraxellaceae, Splingomonadaceae, Flavobacteriaceae, Neisseriaceae, and Corynebacteriaceae were among the dominant bacterial families observed in midgut of fish fed control diet, while Burkholderiales, Micrococcaceae, Comamonadaceae, Propionibacteriaceae, Moraxellaceae, Bacteroidota, and Burkholderiaceae were among dominant families of fish fed 0%FMFO diet. In fish fed IM diet the midgut bacterial dominance consisted of Burkholderiaceae, Planococcaceae, Propionibacteraceae, Comamonadaceae, Flavobacteriaceae, Campylobacteraceae, Streptococcaceae, Moraxellaceae, Staphylococcaceae, and Xanthobacteraceae and in fish fed TM diet consisted of Comamonadaceae, Cellulomonadaceae, Alphaproteobacteria, Methyloligellaceae, Burkholderiaceae, and Vibrionaceae. Bacterial families LWQ8 and Rhodobacteraceae were detected among the dominant bacteria in midgut of fish of all dietary treatments.

The presence of Cellulomonadaceae was not observed in fish fed the 0%FMFO and IM diets, there was absence of Chitinophagaceae in fish fed IM diet, while Rubritaleaceae family was present only in fish of TM dietary treatment. The midgut bacteria richness increased in fish fed with the TM diet. Shannon and coverage indices also increased in fish of the TM dietary treatment, although evenness was reduced, compared to the other dietary treatments, but without statistical significance (p > 0.05).

3.6. Histological Examination

The liver of fish fed the 0%FMFO and IM dietary treatments had large lipid droplets around the pancreatic islets (Figure 3a). Fish fed with the replacement diets as well as the control diet, showed mild lipid droplets accumulation and nuclei displacement detected in some cases compared to a normal liver imaging. Steatosis or hemorrhages were not detected (Figure 3b). Diet did not have a significant effect on anterior and posterior gut histology. All dietary treatments displayed a normal structure with normal distribution of goblet cells with no signs of inflammation (Figure 3c,d).

4. Discussion

In the current experiment, we showed that all dietary FM and FO can be replaced with a mixture of microalgae (S. limacinum and P. tricornutum), insect (H. illucens), and tunicate (C. intestinalis) without impairing growth performance or fish utilization in S. aurata. Nevertheless, fish growth and feed utilization were significantly suppressed when either H. illucens or C. intestinalis meal was added at 211 g/kg, replacing 68% and 46% of the dietary FM, respectively. These results indicate that high level of FM replacement by either IM or TM is not nutritionally adequate, and that the use of microalgal biomass is beneficial for gilthead sea bream performance. Hermetia illucens protein has been proven to be effective at replacing moderate levels of FM protein in the diet of this species. Replacing 30% of FM with a diet of 195 g/kg [25] did not impair gilthead sea bream growth or feed efficiency. Karapanagiotidis et al. [45] tested the use of a defatted instead of a full-fat H. illucens meal together with an inclusion level as high as 174 g/kg and a replacement of 30% of FM protein and found a decreased FI, which in turn impaired growth and feed efficiency, which is similar to the findings by Fabrikov et al. [43] for included 109–180 g/kg. Carvalho et al. [44] also observed reduced FI and growth performance in S. aurata fed diets with 10% H. illucens, replacing 66% of FM, suggesting that S. aurata may have a low ability to digest the IM at increased inclusion levels in the feed. In other studies, total FM replacement using as much as 450 g/kg H. illucens meal did not impair gilthead sea bream growth or feed efficiency [81], while Pulido-Rodriguez et al. [42] reported that even greater growth and feed efficiency were achieved when H. illucens was added at 324 g/kg diet. The high digestibility of H. illucens meal in gilthead sea bream [81, 82] may explain these effects. Chitin found in IM has been suggested as an antinutritional factor affecting the digestibility of nutrients. Though, in the present study the 0%FMFO diet and the IM diet had similar inclusion levels of the same IM H. illucens (20.75% inclusion in 0%FMFO and 21.14% inclusion in IM) their growth performance was different. Fish fed the 0%FMFO diet showed better SGR similar to the control diet than fish fed the IM diet. Therefore, chitin in the dietary treatment group fed 0%FMFO did not slow down or inhibit fish growth performance. Moreover, chitin in IM can have a functional role [29, 83].

Karapanagiotidis et al. [45] suggested that full fat black soldier fly meal at high inclusion levels in S. aurata diets may have negative effects on diet palatability demonstrated by reduced feed consumption and decreased nutrient intake due to incomplete catabolism in fish. Defatting process of H. illucens meal can affect the availability of lipids, becoming bound in the chitin structure [69], especially the MUFA. Pulido-Rodriguez et al. [42] examined the use of partially defatted black soldier fly prepupae meal in S. aurata appetite-related genes and found that the H. illucens meal, even at the inclusion level of 324 g/Kg did not depress the central neuroendocrine mechanism involved in appetite stimulus, resulting in FI, SGR, and FCR similar to the control treatment. According to Randazzo et al. [70], partially defatted black soldier fly prepupae meal increased glycosylated compounds in the midgut and lipid deposition in liver and of S. aurata, with the latter associated with the fatty acid profile of the IM. Furthermore, the H. illucens meal has a balanced EAA profile [34, 68] and contains bio-active compounds, like medium-chain fatty acids and chitin [29, 83–85], which can stimulate the immune system of fish, providing antimicrobial and anti-inflammatory properties [28, 36].

The addition of P. tricornutum in combination to Schizochytrium sp., IM and TM to the diet of gilthead sea bream had a positive effect on the growth performance of the fish. When P. tricornutum meal replaced FM in starter diets for S. aurata at 4% level, fish SGR was unaffected but an evident negative effect on survival was observed [8]. On the other hand, Ribeiro et al. [86] reported that in finishing diets of gilthead sea bream, the inclusion of 2.5% P. tricornutum did not impair growth or feed utilization parameters, improved fish skin pigmentation or acted as a functional ingredient in the diet. Reis et al. [87] examined the use of P. tricornutum meal, either in terms of whole cell or broken cell biomass, at the 1% level in a FM-free diet for juvenile gilthead sea bream and found that growth performance parameters and feed efficiency remained unaffected, while the immune system of fish was stimulated, improving fish health, especially in the case of broken cell biomass. The low level of inclusion of P. tricornutum in the diet of gilthead sea bream at different stages of development seems to be beneficial to the fish. In the present study, the use of 8% Schizochytrium sp. blended with other ingredients in the diet of gilthead sea bream did not affect the growth performance of the fish. Ganuza et al. [14] reported that a lower inclusion level (2.5%) of Schizochytrium sp. in microdiets for S. aurata, as a DHA source, did not affect the larvae performance; although fish growth and survival were reduced when Schizochytrium sp. replaced FO fully in the diet. In contrast, Carvalho et al. [88] reported that the total replacement of FO by commercial products based on Schizochytrium sp. in weaning microdiets for S. aurata improved growth performance and had no effect on survival, but increased the survival rate under stressful conditions, probably due to the increased DHA content of Schizochytrium sp. Similar results on the survival rate were reported by Eryalçin and Yildiz [89], who replaced 10% of FM and all FO with a mixture of microalgae containing P. tricornutum, Schizochytrium sp., Tetraselmis suecuca, Isochrysis sp., and Nannochloropsis oculata meal, in weaning microdiets of gilthead sea bream, allowing growth performance to be unaffected. According to Santigosa et al. [15], the growth performance of seabream with an initial weight of 64.5 g fed 3.5% Schizochytrium sp. oil as a replacement for FO was also unaffected, indicating that the supplementation of feed with microalgae could lead to a diet rich in n−3 and n−6 fatty acids and environmentally friendly. A mixture of Schizochytrium sp. with other microalgae, such as Microchloropsis gaditana, has also been shown to be effective for total FO replacement in diet for juvenile gilthead sea bream without influencing growth and feed utilization, providing valuable EPA and DHA to the fish [19]. Improved growth performance and feed utilization were seen in gilthead sea bream when fed a diet containing a blend of microalgae (3.2% Schizochytrium sp., 5% Chlorella sp., and 0.5% Tetraselmis sp.), 0.4% macroalgae, and 0.02% selenized yeast replacing 37% of FM. Nevertheless, when the dietary levels of microalgae and yeast were doubled, this led to impaired growth [90].

In the present study, in order to formulate the 0%FMFO diet that was devoid of FM and FO, the levels of low protein binder meals, such as horsebean meal and wheat meal were also reduced, as the novel ingredients’ protein level is lower that of FM present in the control diet. The variation in the dietary horsebean meal is not expected to have contributed to the variation in fish performance as it has been shown up to 35% inclusion of horsebeans in the diet did not compromise the growth performance of S. aurata [91] or the digestibility of dietary protein and lipids in D. labrax [92, 93].

There is limited research on the replacement of FM with TM in the diet of gilthead sea bream. In the present study, S. aurata fed a TM diet had increased FCR and decreased SGR. Similar results were also shown in Salmo salar fed TM at 200 g/kg, which was 45% replacement for FM and this increase in FCR occurred due to the high ash content of TM [46]. The present results indicate that TM in the diet of seabream in the present study decreased the fish FI and LER, thus, decreased nutrient and energy intake, resulted in decreased lipid content in fish fillets, while IM increased the lipid content in fish muscle. Increased lipid levels in muscle of fish fed IM diet cannot be related to increased FI, because the FI in this treatment decreased, compared to control, indicating that dietary lipid derived from IM was not catabolized, but deposit in high degree in fish muscle. Kousoulaki et al. [46] also observed increased lipid content in muscle of S. salar fed 20% H. illucens meal diet, as a result of increased amino acid catabolism for energy use. However, Fabrikov et al. [43] reported that 50% replacement of FM with H. illucens meal led to decreased lipid content in S. aurata fillets. The latter may be attributed to differences in feed digestibility and the level of dietary FM replacement. In the present study, the amino acid deposition in the muscle of S. aurata remained unaffected by the inclusion of algae meal in combination to IM and TM in diets. The accumulation of DHA in the fish fillets increased in the fish fed the IM diet and was similar to that in the control diet-fed fish. These results agree with those of Mastoraki et al. [25] and Pulido et al. [94], who reported that the inclusion of H. illucens in S. aurata and D. labrax in the diets did not affect the content of main fatty acids. In addition, Carvalho et al. [44] reported that low-level inclusion of H. illucens did not affect the proximate composition, amino acid, or fatty acid profile of S. aurata. Atalah et al. [8] observed an increase in the lipid content of S. aurata when they were fed weaning diets containing P. tricornutum meal as a replacement for FM. Furthermore, gilthead sea bream fed a mixture of macroalgae, microalgae containing 3.2% Schizochytrium sp. and selenized yeast to replace 33% of the FM had a positive effect on n−3 PUFA accumulation in fish muscle, leading to an increase in the fatty acid content of the fillet [90]. Similar results were also found in gilthead sea bream fillets, either fed 3.5% Schizochytrium sp. oil [15] or a blend containing Schizochytrium sp. and P. tricornutum meals [89]. The successful FO substitution with Schizochytrium sp. meals in salmon feeds demonstrates the feasibility of low trophic level organism to promote the sustainability of the aquaculture sector [95].

Differences in the activity levels of enzymes involved in intermediary metabolism in the liver and foregut of fish fed the experimental diets indicated alterations in the metabolic pathways due to dietary ingredients. Increased L-LDH activity in fish fed TM indicates hepatic and intestinal lactate uptake, which may suggest a glycolytic carbohydrate metabolism dependence [60]. The 0%FMFO diet containing microalgae meal had no effect on hepatic HOAD activity, which is in agreement with the HOAD activity levels in the liver of gilthead sea bream fed nutraceuticals derived from microalgae [96]. On the contrary, HOAD activity levels in the present study increased in the foregut, indicating lipid oxidation. Fish fed the IM diet exhibited increased L-LDH and CS activity levels in the foregut, contrary to decreased CS and L-LDH activities in the foregut of gilthead sea bream fed the Tenebrio molitor diet [97]. According to Antonopoulou et al. [80], the increase in HOAD activity in relation to unaffected levels of L-LDH and CS indicate that liver metabolism mainly utilizes lipid energy reserves.

Hsp70 and Hsp90 levels in the IM, TM, and 0%FMFO diets significantly decreased compared to the control, contrary to the upregulated Hsp70 in Cyprinus carpio fed diets with 75%–100% FM replacement with defatted H. illucens [63]. Similarly, Oncorhynchus mykiss fed a 50% full-fat H. illucens diet exhibited increased Hsp70 levels in the liver, suggesting a possible stress response [97]. The decreased Hsp levels in the present study in the liver of fish fed IM, TM, and 0%FMFO diets could be attributed to the fact that only significant changes in the quantity of amino acids provided by food is necessary for an alteration in Hsp levels [60], whereas amino acid deprivation can lead to Hsp deficiency [98]. However, this needs to be further investigated. Although the involvement of p38 MAPK in the induction of Hsp70 has been proven in fish tissues [99, 100] contradictory results were obtained in the present study, with p38 MAPK being activated in the liver of all experimental diet groups. p38 MAPK phosphorylation likely occurs when dietary lipids cannot meet fish dietary requirements, thus, indicating a stress response [80]. Akt activation, possessing a key role in glucose metabolism [101], increased in the TM compared to the control group, and along with the increased L-LDH activity levels confirmed in TM induced carbohydrate metabolism. Similarly, Liu et al. [102] reported that upregulated hepatic Akt is a potential cause of elevated amino acid metabolism in O. mykiss fed a mixture of IM (Hermetia illucens) and Chlorella sorokiniana. Contrary to Akt phosphorylation, AMPK activation was more apparent in the IM dietary group, as also evidenced by HOAD, probably due to increased lipid exploitation processes [103].

With respect to the gut microbiota, there was no statistical difference of midgut bacterial taxa that detected in fish fed the 0%FMFO, IM, or TM diet, which contained H. illucens, C. intestinalis, P. tricornutum, and S. limacinum meal (separately or mixed), compared to the control diet. In contrast, Cerezuela et al. [9] reported decreased bacterial activity in the intestine of S. aurata fed P. tricornutum meal, suggesting a fragile gut microbial community. However, Piazzon et al. [104] observed a strong interaction between the gut microbiome and S. aurata fed a diet with IM and algae oil, promoting growth and health. Regarding the bacterial richness in S. aurata fed a diet with H. illucens meal, no differences were detected, although changes in the metabolic pathways of the bacterial community may occur [40], even at 50% replacement of FM [43]. According to Katsoulis-Dimitriou et al. [20], the gut microbiota of S. aurata fed diets containing microalgae, were able to adapt to these diets and metabolized fucose, which is a major carbohydrate that can be found in microalgae cells. This ability of gut microbiota to utilize the microalgae fucose has potentially beneficial results to the host, by inducing the short chain fatty acids production [105–107].

The gut microbiota potentially plays an important role in the nutrition of fish by enzyme production [108]. In the present study, the observed dominant bacterial families in fish midgut may have nutritional role, producing enzymes (Bacillaceae [109], Flavobacteriaceae [110, 111], Rhodobacteraceae [112], Cellulomonadaceae [113], and Saccharimonadales in which the LWQ8 family belongs [114]), short-chain fatty acids (Clostridia [115], Halieaceae [116], and Propionibacterium [117]), amino acids (Halieaceae [116], Planococcaceae [118], and Neisseriaceae [119]), vitamins (Propionibacterium [117] and Neisseriaceae [119]), and assist to metabolism (Micrococcaceae [120], Moraxellaceae [121], and Rubritaleaceae [122]) and growth (Micrococcaceae [123], Comamonadaceae [124], and Planococcaceae [125]). Other bacterial families are found to improve the immune system and health status (Streptococcaceae [126], Alphaproteobacteria [127], Burkholderiaceae [120], Propionibacterium [117], and Comamonadaceae [124]), while other are involved in nitrogen cycle (Sphingomonadaceae [128, 129]) and CH₄ oxidation (Methyloligellaceae [130]). Chitinophagaceae bacteria known for their ability to degrade chitin [131] were absent in IM dietary group. This observation may be the result of microbial shifts derived from the rearing substrate, life stage, and processing of the insect, in combination with the microbial adaptation to the host metabolic requirements [34, 132, 133]. Also, Kroeckel et al. [69] did not observe any chitinolytic active bacteria in the midgut of Psetta maxima fed H. illucens meal diet. On the other hand, Flavobacteriaceae, which includes several members known to produce amylase, protease, and chitinase [110, 111], were observed d in fish fed IM. The ability to break down chitin favored its presence in the intestine of fish fed the IM diet, thus, providing valuable nutrients to the fish.

Lipid droplets were found around the pancreatic islets in the 0%FMFO and IM experimental diet groups, while nuclear displacement was observed in the liver of the examined fish. The presence of lipid droplets in pancreatic islets of fish fed IM diet, could correlated to increased lipid content observed in fish muscle of the abovementioned dietary group. Abundant lipid droplets were also found in S. aurata fed a H. illucens diet without altering lipid accumulation [70]. In contrast, the inclusion of H. illucens meal in the S. salar diet did not affect lipid droplets in the liver [38]. Additionally, Karapanagiotidis et al. [19] reported no alterations in the hepatic histology of seabream fed Schizochytrium sp. or Microchloropsis gaditana as a FO replacement. Instead, Di Rosa et al. [41] reported severe alterations in the posterior gut structure of S. aurata fed 50% H. illucens meal, which may be due to increased levels of chitin in the diet.

5. Conclusion

Total replacement of FM and FO with a blend of IM, microalgae, Schizochytrium sp., and TM did not affect gilthead sea bream growth performance compared with the control diet, although replacement of 68% of the FM with IM and replacement of 45% of the FM with TM alone resulted in reduced fish growth performance. The CS activity in the liver increased in the fish of the aforementioned treatments, indicating the use of the Krebs cycle for energy production. In addition, the livers of fish fed the TM diet exhibited an increase in L-LDH and a decrease in Hsps, AMPK, and p38 MAPK activity, implying a reduced stress response. Bacterial taxa present in the midgut of fish from all experimental groups, were involved in fish metabolism and nutrient supply to the host. The differences in fish metabolism and the gut microbiota seemed to be linked to the differences in the experimental diets. According to the histological examination, large lipid droplets appeared around the pancreatic islets of the fish fed the 0%FMFO and IM diets, although mild lipid droplet accumulation was detected in the liver of the fish fed all the experimental diets (replacement diets and the control diet). To conclude, new horizons are opened for the replacement of FM and FO in S. aurata feeds by incorporating into the aquafeed a mixture of innovative ingredients that can be grown sustainably on agricultural sidestream biomasses.

Disclosure

This output reflects the views only of the authors, and the European Union cannot be held responsible for any use which may be made of the information contained therein. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Conceptualization and design: Eleni Mente and Katerina Kousoulaki. Supervision: Efthimia Antonopoulou, Konstantinos Kormas, and Eleni Mente. Writing – original draft preparation: Anna Tampou. Writing – review and editing: Anna Tampou, Katerina Kousoulaki, Antigoni Vasilaki, Nikolaos Vlahos, Eleni Nikouli, Nikolas Panteli, Konstantinos Feidantsis, Kostantinos Kormas, Ioannis T. Karapanagiotidis, Panagiotis Berillis, Ioannis Nengas, Efthimia Antonopoulou, and Eleni Mente. Feeding trial: Anna Tampou and Styliani Andreopoulou. Formal analysis: Anna Tampou, Katerina Kousoulaki, Antigoni Vasilaki, Nikolaos Vlahos, Eleni Nikouli, Nikolas Panteli, Konstantinos Feidantsis, Kostantinos Kormas, Ioannis T. Karapanagiotidis, Panagiotis Berillis, Ioannis Nengas, Efthimia Antonopoulou, and Eleni Mente. Statistical analysis: Anna Tampou, Antigoni Vasilaki, Nikolaos Vlahos, Eleni Nikouli, Nikolas Panteli, Konstantinos Feidantsis, and Panagiotis Berillis.

Funding

This project has received funding from the European Union’s Horizon 2020 Innovation Action, FutureEUAqua, under Grant Agreement No. 817737 and from the European Union’s Horizon Europe Research and Innovation Action AWARE Project, under Grant Agreement No. 101084245.