**1. Introduction**

In intensive farming facilities, fish are reared at high densities, which may increase stress and susceptibility to diseases, resulting in lower production yields. Consequently, there is an increasing pressure for disease management strategies, beyond the use of antibiotics or vaccination. In this sense, health promoting feeds designed not only to fulfil the nutrient requirements but also to strengthen the immune system are viewed as a way to reduce aquaculture dependency on chemotherapeutics and to mitigate its negative environmental effects [1,2]. Novel applications based on algal products are a fast emerging and a developing area, expected to reach 56.5 billion US\$ by 2027 with a compound annual growth rate of 6% in the period from 2019 to 2027 [3]. The ability to grow in different environments and conditions as well as to produce large numbers of secondary metabolites makes microalgae a suitable raw material for different applications. These organisms are regarded as sustainable alternative sources of bioactive compounds, mostly sought out for the development of functional feeds, foods and health products [4–6].

*Chlorella vulgaris* is a green microalga with a wide distribution in freshwater, marine and terrestrial environments that is capable of rapid growth under autotrophic, mixotrophic and heterotrophic conditions [7]. These characteristics made *C. vulgaris* a successful candidate for large-scale cultivation and commercial production [8]. As with other microalgae species, *C. vulgaris* produces a different array of health-promoting biomolecules [9,10]. Notably, natural pigments such as lutein and astaxanthin extracted from *Chlorella* sp. show immunostimulatory and antioxidant protective effects [4,11,12]. Furthermore, these microalgae are characterised by a very high crude protein content (>50%) and a balanced amino acid (AA) profile, synthesising all essential AA in a considerable amount [4]. Already, *C. vulgaris* biomass has been successfully used in aquafeeds as a source of protein, improving growth performance, oxidative status and immune response in several fish species [13–17]. For instance, dietary supplementation of *Chlorella sp.* at 0.4 to 1.2%, stimulated the innate immunity of gibel carp (*Carassius auratus gibelio*), namely by increasing IgM, IgD, Interleukin-22 and chemokine levels [18]. Also, Zahran and Risha [16] reported that feed supplementation with powdered *C. vulgaris* protected Nile tilapia against arsenic-induced immunosuppression and oxidative stress.

Nonetheless, as with other algal biomasses, at high fishmeal replacement levels, studies start to report impaired growth performances [19,20]. Microalgae generally show thick cell walls that hinder the access of fish gut enzymes to intracellular nutrients. Hence, algae nutritional value increases if access is provided to macro and micronutrients [21–23]. Hydrolyses improve digestibility through the application of chemical or enzymatic methods to disrupt the cell wall and hydrolyse intact proteins [24]. The enzymatic method is sometimes advantageous because of milder processing conditions and peptide bond specificity, giving rise to digestible peptides believed to be more effective than the whole protein or the free AA [24,25]. Peptide bioactivity is influenced by molecular weight and peptide chain size [26]. In fact, low molecular weight peptides (<3 kDa) are described as having immune-stimulating or anti-inflammatory properties [26–28].

Several studies, have evaluated marine protein hydrolysates (MPH) as a dietary ingredient and their effects on growth performance, immune response and disease resistance in fish [26]. Results are promising, as the dietary inclusion of MPH has been shown to induce growth, antioxidant activity and fish immunity [28–32] as well as improve fish immune response and disease resistance to specific bacterial infections [27,33–35]. Moreover, regarding microalgae, different *C. vulgaris* protein hydrolysates and extracts have already been studied concerning its different bioactivities, namely, anticancer and antibacterial effects [36], as well as antioxidant and immune modulatory properties [37]. Results mentioned above suggest that *C. vulgaris* has the potential to act as a dietary supplement with nutraceutical properties and to stimulate the immune system. Therefore, the present study aimed to evaluate the effects of short-term dietary supplementation, with a 2% *C. vulgaris* biomass and a 0.1% supplementation with *C. vulgaris* soluble peptide-enriched extract, on the immune and the oxidative stress defences (health status; experiment one) and on the

inflammatory response after an inflammatory insult (experiment two) of gilthead seabream (*Sparus aurata*).
