*2.4. Gut Gene Expression Analysis*

To evaluate the expression of gut health, immunity and oxidative stress related genes (Tables 5 and 6), total RNA was isolated from fish anterior intestine. In experiment one, target genes transcriptomic analysis was not able to ascertain differences attributable to the dietary treatments, which could be related to the high intraspecific variability for some target genes (Table 5). However, cd8α, hsp70 and muc2 genes expression increased from 1 to 2 weeks.

Following the inflammatory insult, changes attributed to dietary treatments were also not found in the majority of analysed genes, except for hsp70, which was down-regulated at 24 h in D2 fed fish (Table 6). Furthermore, tlr1 gene expression was up-regulated and gpx was down-regulated at 24 h in all dietary treatments.


 one).

**Table 5.** Relative gene expression profiling of anterior intestine in gilthead seabream juveniles after 1 and 2 weeks of feeding (experiment



differences in time within the same diet (*<sup>p</sup>* < 0.05). Different capital letters represent significant differences in time regardless of diet (*<sup>p</sup>* < 0.05).

#### **3. Discussion**

A main feature of *C. vulgaris* is its protein content and its balanced AA profile, making it a potential source of bioactive peptides. However, the presence of rigid cell walls limits the fish's ability to access and to utilise the different nutrients inside microalgae cells. In the present study, cell wall disruption was obtained through a combination of chemical and enzymatic processes and the protein fraction was hydrolysed using a serine protease. Protein hydrolysates seem more effective than either intact protein or free AA in different applications for nutrition [25,38]. The current study was devised using two different approaches. First, there was a 2-week feeding trial to evaluate the health status of the fish, aiming to develop future prophylactic strategies (experiment one). After 2 weeks of feeding, fish were subjected to an inflammatory insult to evaluate the inflammatory response (experiment two) and to better discriminate any immunomodulatory effect from the different dietary treatments.

The overall haematological profile from the health status experiment showed some changes, mainly exerted by *C. vulgaris* biomass and peptide-enriched extract supplemented diets (D1 and D2 diets). Fish fed diet D1 showed lower lymphocyte numbers (Table 2). Accordingly, in a previous experiment with poultry, where different preparations of *C. vulgaris* were used, animals fed a supplemented diet with 1% chlorella powder showed decreased lymphocyte numbers [39]. Nonetheless, fish fed D2 diet not only had comparable lymphocyte numbers to CTR, but also showed a higher neutrophil concentration (Table 2). These higher circulating myeloid cell numbers in the D2 group might be of relevance during early responses to infection. Bøgwald et al. [40] have shown that medium-size peptides (500–3000 Da) from cod muscle protein hydrolysate, stimulated in vivo respiratory burst activity in Atlantic salmon (*Salmo salar*) head-kidney leucocytes. In the present study, the peptide-enriched extract protein/peptide profile (Figure S1) is mainly composed of small to medium size particles (<1200 Da) [41]. Size and molecular weight (MW) seem to be particularly important for peptide immunomodulatory activities, with small- to mediumsized particles showing the highest activity [26,28,40,42]. However, an increased leucocyte response in fish fed the D2 diet did not translate into an improved plasma humoral parameters response (NO concentration, antiprotease and peroxidase activities) at 1 or 2 weeks (Figure 1A–C), although those values tended to increase in seabream fed D2 and D3. Accordingly, former studies conducted on Coho salmon (*Oncorhynchus kisutch*) and turbot (*Scophthalmus maximus*) did not show any significant impacts on several innate immune defence mechanisms, when fish were fed MPH supplemented diets [43,44]. Nonetheless, beneficial effects have been reported in different fish species [26]. Khosravi et al. [33] supplemented red seabream (*Pagrus major*) and olive flounder (*Paralichthys olivaceus*) feeds with 2% krill and tilapia protein hydrolysates and supplementation improved lysozyme activity and respiratory burst in both species. Protein hydrolysates were mainly composed of small- (<500 Da) to medium-sized peptides (500–5000 Da). Furthermore, diet D2 shows a higher Hb concentration than D1 and D3 fed fish. The extraction method employed in a *C. vulgaris* biomass to obtain the soluble extract (diet D2) might increase iron availability, since most of the intracellular iron is associated with soluble proteins and iron is an essential element for Hb production [45].

In the present study, when fish were subjected to an inflammatory insult (experiment two), an immune response after the stimulus was observed through the time-dependent response pattern of peripheral leucocytes, plasma and gut immune parameters. Peripheral cell dynamics were significantly changed at 24 h post-stimulus, translating into a sharp increase in circulating neutrophils and a significant decrease in lymphocytes (Table 4), indicating that cells were differentiating and being recruited to the site of inflammation. Also, Hb concentration increased (Table 3) in line with a higher metabolic expenditure due to the inflammatory response, and peroxidase activity showed a clear augmentation following inflammation (Figure 1E). Even though circulating neutrophil numbers tended to increase in D1, 2 and 3 dietary treatments at 48 h following inflammation (Table 4), it was not possible to ascertain a clear Chlorella whole-biomass or extracts effect, a fact that could

be related to high intraspecific variability in response to the stimulus and that reinforces the need for further studies to unravel the potential of these extracts.

Hydrogen peroxide and oxygen radicals are physiologically generated within cellular compartments and their build-up leads to tissue oxidative stress and damage [46]. Free radical effects are controlled endogenously by antioxidant enzymes and non-enzymatic antioxidants and also by exogenous dietary antioxidants that prevent oxidative damage. Chlorella sp. contain several phytochemicals, namely carotenoids, chlorophyll, flavonoids and polyphenols, which exhibit antioxidant activities [47,48]. Earlier studies showed a significant increase in serum SOD activity in gibel carp fed diets containing 0.8–2.0% dry Chlorella powder [20]. Rahimnejad et al. [14] reported increased plasma CAT activity and total antioxidant capacity (TAC) in olive flounder fed diets with 5% and 10% defatted *C. vulgaris* meal. As with other microalgae species, the antioxidant potential of *C. vulgaris* has been mainly assessed on serum and liver, though information is still scarce at the intestinal level. The intestinal epithelium, a highly selective barrier between the animal and the external environment, is constantly exposed to dietary and environmental oxidants. Consequently, it is more prone to oxidative stress and damage, which can impact gut functionality and health [49,50]. The dietary effects of microalgae biomass inclusion have been previously assessed on the intestine of gilthead seabream. Fish were fed diets supplemented with 0.5, 0.75 and 1.5% *Nannochloropsis gaditana* biomass and no signs of nutritional modulation were found for intestinal SOD and CAT transcription [51]. In the present study, D2 fed fish showed higher gut LPO than CTR and D3 at the end of experiment one (Figure 3A), which could be related to the extraction method employed, since most of the pigments present in the *C. vulgaris* biomass are not present in the peptide-enriched extract, diminishing the availability of exogenous dietary antioxidants. As pigments are mostly hydrophobic, they are extracted alongside the lipid fraction present in the insoluble extract (Diet D3). Regarding the activities of key enzymes involved in intestinal redox homeostasis (CAT and SOD), these remained unchanged among experimental groups. Castro et al. [17] replaced 100% FM by *C. vulgaris* biomass in plant protein rich diets for seabass (*Dicentrarchus labrax*) and found no differences in intestinal LPO, tGSH and GSH levels between dietary treatments. However, they reported lower SOD activity and higher GSSG levels in microalgae-enriched diets, suggesting an increased risk for oxidative stress when fish are subjected to pro-oxidative conditions. Such conditions might arise during an inflammatory insult. However, in experiment two of the present study, lipid peroxidation increased at 24 and 48 h (Figure 3D) post-stimulus but to the same extent for all the dietary treatments. It could be hypothesised that fish fed the D2 diet were able to cope with acute inflammation in a similar manner as the other experimental groups, despite their higher intestinal oxidative state. In other studies, *C. vulgaris* powdered biomass has been found to counteract the pro-oxidative effects of arsenic induced toxicity in both the gills and the liver of tilapia [16]. Furthermore, Grammes et al. [51] reported that substituting FM by *C. vulgaris* in aquafeeds containing 20% soybean meal (SBM) is an effective strategy to counteract soybean meal-induced enteropathy (SBMIE) in Atlantic salmon. Likely, this was by maintaining the integrity of the intestinal epithelial barrier and therefore preventing innate immune response activation and ROS generation [52,53].

In the present study, anterior gut transcriptional changes were also evaluated to determine the effect of dietary treatments on the expression patterns of different structural (muc2 and muc13), antioxidant (hsp70; gpx and sod(mn)) and immune related genes (il1β; il34; tlr1; cd8α; igm and hepc). The transcriptomic approach employed was not able to ascertain a clear dietary modulation, at least for the great majority of genes under evaluation in both experiments one and two. However, after the inflammatory insult, the hsp70 gene was down regulated in the D2 fed group after 24 h compared to those fed CTRL (Table 6). Heat shock protein 70 (HSP70) maintains cell integrity and function, and it promotes cell survival under stressful conditions [54]. Leduc et al. [28] reported that genes involved in cellular damage response and repair were also under-expressed in seabass fed a mix of tilapia (TH) and shrimp (SH) protein hydrolysates (5% dry matter diet), mainly composed of low molecular weight peptides. In the same study, fish that were fed the SH alone showed up-regulation of intestinal immune-related genes. Although composed of small-sized peptides, TH did not show the same pattern of stimulation, following what was observed in the current work. According to the authors, the immune-stimulatory effect of the SH was due to low molecular weight peptides, but also to its origin and its degree of hydrolysis [28]. Bioactive peptides are inactive when they are part of the native protein sequence; and, after hydrolysis, bioactivity can be gained depending on specific AA sequences and the size of the newly formed peptides [25]. Nevertheless, in the present study, the observed down-regulation of hsp70 gene expression in the gut of seabream fed D2 suggests a certain degree of anti-stress and/or antioxidant properties from the *C. vulgaris* peptide-enriched extract, in line with that hypothesized above.

In summary, the *C. vulgaris* peptide-enriched extract tested in the present study seems to confer a dual modulatory effect at both peripheral (blood) and local (gut) levels. In particular, it drives the proliferation of circulating neutrophils in resting seabream, which could be of assistance to fight against opportunistic pathogens. Following an inflammatory insult, this peptide-enriched extract may protect the gut against stress, and it should be considered for further studies.

## **4. Materials and Methods**
