The increasing challenge of soil salinization is a significant constraint on agricultural productivity globally. About 7% of the world’s land area is salt-affected to some degree, and this trend is on the rise (Wicke et al., 2011). Glycophytes, which are the primary source of food, home remedies, and drugs, are sensitive to salt and suffer yield reductions even in mildly saline conditions. Halophytes, on the other hand, have been identified as a viable alternative in recent decades. These plants are adapted to grow in saline environments, such as coastal areas and salt marshes, and can tolerate high salt concentrations of 200 mM or more in their tissues, surviving in conditions that most other plants cannot. They make up about 2% of the world’s flora (Flowers and Colmer, 2008; Flowers and Muscolo, 2015). However, it is only recently that their potential has been recognized for crop production, as they do not require as much freshwater for irrigation and can tolerate high levels of salt and other environmental stresses (Ventura and Sagi, 2013). Halophytes have a variety of applications, including food, medicine, and biofuels. Species of the genus Salicornia are particularly noteworthy due to their commercial potential, with current large-scale cultivation in Europe, North America and middle East for human consumption as a vegetable, as well as attempts to produce oilseed and animal feed using seawater irrigation (Chaturvedi et al., 2021).

The increasing interest and widespread embrace of functional foods have paved the way for a fresh market opportunity for halophytes. This stems from the revelation of various bioactive compounds found in halophytes, which not only contribute to their nutritional value but also assist in specific bodily functions. Research indicates that Salicornia spp. possess numerous nutraceutical properties attributed to their phenolic content. They have been used traditionally in the treatment of conditions such as hepatitis, constipation, nephropathy, diarrhea, and have demonstrated antihyperglycemic and antihyperlipidemic activities. Some of the phenolic compounds (quercetin and isoquercetin) in Salicornia spp. are recognized as generally safe by the United States Food and Drug Administration (USFDA) and can be administered in specific concentrations. The concentrations of phenolic compounds in Salicornia spp. can vary due to factors like biomass maturity, pre-treatment methods, and extraction techniques. In terms of antiviral properties, phenolic compounds found in Salicornia spp. have demonstrated inhibitory effects against viruses such as H1N1, HBV, HCV, and HIV-1. These effects are attributed to the compounds’ ability to interact with viral and host proteins and enzymes, preventing viral replication and infectivity (Fredsgaard et al., 2023). Salicornia species are rich in minerals (Barreira et al., 2017), flavonoids and polyphenols (Essaidi et al., 2013; Silva et al., 2021; Limongelli et al., 2022). Polyphenols have been found to be strong antioxidants that can neutralize free radicals by donating an electron or hydrogen atom (Tsao, 2010). Flavonoids possess antioxidant activity and function as a free radical scavenger and a chelator of transition metal, such as iron, a key metal in the generation of ROS through the Fenton reaction (Murota and Terao, 2003) and the quenching of singlet-oxygen atom (Mukai et al., 2005). The chemical research on various Salicornia spp., such as S. europaea, S. arthrocnemum, S. bigelovii, S. perennis, and S. disarticulata, has demonstrated their significant anti-oxidative properties. This finding supports their traditional use, as they all contain phenolic compounds (Rhee et al., 2009). Hence, functional feeds – such as diets rich in Salicornia – may play a crucial role in enhancing the overall health and performance of aquatic organisms in aquaculture, particularly in species like shrimp. By incorporating functional ingredients into their diets, it is possible to boost their immune system and improve antioxidant functions. This could help the shrimp to better combat diseases and oxidative stress, ultimately leading to improved growth, survival rates, and overall welfare. This is particularly vital in aquaculture, where maintaining the health and vitality of the stock is essential for successful and sustainable production.

Salicornia ramosissima is considered an alternative to salt and its fresh and tender green tips (leaves) are used as food ingredients for human diet, mainly in salads, canned or gourmet gastronomy (Ferreira et al., 2018). Recent research has shown that Salicornia spp. can grow in constructed wetlands and can effectively take up nutrients, such as nitrogen and phosphorus, from wastewater into its tissues (Ventura and Sagi, 2013)

Salicornia, particularly S. bigelovii and S. ramosissima, has emerged as a promising feed ingredient in aquaculture, supported by some highlighting its nutritional benefits. Ríos-Durán et al. (2013) demonstrated that S. bigelovii seed meal can be used as a source of protein in fish feed, while the health-modulating potential of Salicornia spp was demonstrated in fish (Marçal et al., 2023; Barreto et al., 2024) and farmed sheep (Taghipour et al., 2021; Nasrabadi et al., 2022), suggesting broader applicability across various species. Together, these findings emphasize the potential of Salicornia as a valuable feed source in the aquaculture industry. However, as the plant matures, it undergoes lignification and accumulates high levels of salt, rendering it less palatable and suitable for consumption. This study aims to investigate the underutilized, non-edible parts of S. ramosissima.

The whiteleg shrimp is the most farmed animal species globally in terms of the number of individuals, with an estimated 300-620 billion produced annually (Waldhorn and Autric, 2022). This industry generates over 33 billion USD per year (FAO, 2022). To sustain its rapid growth, the demand for aquafeeds, which typically comprise over 50% of production costs, has increased significantly. These feeds often rely on conventional ingredients like fish meal, soybean meal, and wheat meal, raising concerns about their sustainability (FAO, 2022).

Hence, the success of shrimp farming, and aquaculture in general, depends on the emergence of novel ingredients that can make formulated diets more economical and reduce the industry’s ecological footprint.

In summary, this study aimed to explore the viability of integrating S. ramosissima biomass into inert diets for juvenile whiteleg shrimp. Additionally, it sought to examine the potential advantages of this inclusion as a functional feed, potentially leading to positive impacts on health and welfare. The assessment encompassed parameters such as growth performance, survival rate, overall health, and oxidative status of the shrimp.

Salicornia ramosissima, a halophyte rich in bioactive compounds, has garnered significant attention as a potential source of natural antioxidants and other health-promoting substances. Its high content of polyphenols, including flavonoids, tannins, and phenolic acids, has been linked to various beneficial effects, such as anti-inflammatory and antioxidant properties (Najar et al., 2023). To investigate the potential of S. ramosissima as a functional feed ingredient for aquaculture, we evaluated the effects of incorporating its non-food biomass into the diet of whiteleg shrimp. This study aimed to assess the impact of S. ramosissima on growth performance, survival, immune function, and oxidative stress response. To our knowledge, this is the first study to explore the use of S. ramosissima as a growth modulator or nutraceutical ingredient in shrimp feeds.

Upon scrutinizing the chemical profile analyses, it’s noted that there were some modifications in the chemical profile on the muscle of the shrimp with the inclusion of salicornia in aquafeeds, but these results are complex and it is difficult to find a pattern that relates the inclusion of salicornia with the changes in chemical profile that we found. When we look for organic acids, succinic acid in shrimp muscle increase with salicornia inclusion. It is important to highlight this organic acid as it has been shown to have an important role in the intestinal microbiome of shrimp, modulating it and improving its digestive and immunological capabilities (Duan et al., 2018, 2020). This could indicate some health-promoting effect on shrimp. About amino acids, the groups fed with aquafeeds including salicornia, there was an increase in the total values of amino acids, as well as the essential amino acids leucine, lysine, threonine, valine and phenylalanine, which are fundamental for the normal growth and metabolism of fish (Wilson, 1986). This is beneficial for the overall health and growth of the shrimp, as amino acids are essential for protein synthesis and indicate some health-promoting effect. Intriguingly, the SL5 group exhibited a striking reduction in amino acid relative abundance, chiefly attributed to a notable decrease in glycine levels. Glycine is a key component of many biological processes. The reduction prompts questions about the overall nutritional quality of this specific diet, SL5 (Wang et al., 2013). Therefore, it is very important to look at these results very carefully.

Fatty acids manifested significant differences between the control and the SL10 group, with lower levels. However, DHA and EPA levels were maintained close to control values, and at 55 days there was an increase in DHA and EPA values in the groups fed with diets with 5% salicornia inclusion compared to the control. These two unsaturated fatty acids are the most relevant Omega-3 fatty acids, due to their recognized relevance in human nutrition (Narayan et al., 2006). The presence of essential amino acids and the omega-3 fatty acids DHA and EPA contribute to the nutritional value of shrimp as food for humans.

While S. ramosissima inclusion did not significantly affect shrimp weight, SGR, or survival after 31 and 55 days, feeding efficiency was negatively impacted. Shrimp fed S. ramosissima diets had significantly higher FCR and feed intake, particularly noticeable after 31 days in the SL10 group (leaves and seeds). This contrasts with previous work on juvenile European sea bass conducted in the framework of the same project by Barreto et al. (2024), where S. ramosissima incorporation up to 10% did not negatively impact growth or feed utilization. This species-specific difference emphasizes the importance of considering species-specific digestive capabilities and nutrient requirements when evaluating novel feed ingredients. A non-significant trend towards lower dry matter content was also observed in the SL10 group, possibly linked to increased moisture content and higher ash levels, which could have contributed to the altered feed efficiency. These findings highlight the need for further investigation into the mechanisms underlying the species-specific response to S. ramosissima and optimization of its inclusion levels in shrimp feeds to maximize benefits while mitigating any negative effects on feed efficiency.

In the present study, feeds with S. ramosissima inclusion showed the ability to modulate the shrimp hepatopancreas antioxidant enzymes, specifically SOD and CAT enzyme activities. These enzymes cooperate in transforming toxic species in harmless water products, since SOD converts superoxide radicals into hydrogen peroxide and molecular oxygen, while CAT converts hydrogen peroxide into water (Weydert and Cullen, 2010). The most pronounced effects were visible after 31 days of feeding with SS (5 and 10% inclusion levels) and with SL (10% of inclusion). Moreover, S. ramosissima stems showed the potential to diminish the oxidative damage (measured as LPO), regardless of the dose incorporated. Interestingly, a down-regulation of the glutathione S-transferase gene (gst) was observed in shrimp fed diets rich in S. ramosissima regardless the biomass source or inclusion level after 31 days of feeding. Zhou et al. (2009) in a study with whiteleg shrimp, pointed out that GSTs are important components of various detoxification, antioxidant defence and stress-tolerance pathways. Therefore, the inclusion of S. ramosissima in aquafeeds for whiteleg shrimp juveniles may be a natural way of enhancing the animal’s health status. In fact, the gene expression of antioxidant enzymes (gst and gpx) indicates an overall profile of down-regulation. There seems to be an agreement between the general antioxidant profile at 31 days of feeding, measured both in activity and gene expression, which also aligns with the lipid peroxidation data (with emphasis on the reduction in SS). Overall, the data suggest that S. ramosissima dietary inclusion acts as an enhancer of the oxidant status, particularly when stems are incorporated and after 31 days of feeding. However, this effect appears to diminish after 55 days.

These results appear to confirm the contribution of non-enzymatic antioxidants by S. ramosissima, thereby sparing endogenous resources and creating a more favourable oxidant status. While research on Salicornia spp. in aquaculture feeds is relatively scarse, with a few papers reporting that it can partially substitute fish protein in diets for O. niloticus fingerlings at levels up to 50% of the dietary protein (Ríos-Durán et al., 2013). And recent studies by Barreto et al. (2024) and Marçal et al. (2023) have revealed promising potential benefits. Barreto et al. (2024) observed significant modulatory effects on fish muscle biochemical profiles, including a decrease in lactic acid and an increase in succinic acid levels, suggesting potential health-promoting effects. Marçal et al. (2023) identified a tissue-specific antioxidant improvement and a genotoxic trigger that ultimately led to increased DNA integrity. These findings highlight the promising potential of S. ramosissima as a functional ingredient in aquaculture feeds, offering a novel, low-value raw material that can be valorized to improve fish health and sustainability. Furthermore, studies on farmed sheep have demonstrated the potential of Salicornia spp. to enhance antioxidant responses (Taghipour et al., 2021; Nasrabadi et al., 2022). Suggesting a broader potential for Salicornia as a functional feed ingredient across various aquaculture species. In this study, higher FCR values were obtained with diets where S. ramosissima biomasses were included, which would increase the final production costs. However, potential antioxidant beneficial effects were observed when using the stems, which is considered a by-product of S. ramosissima cultivation. Therefore, by using a product that would otherwise be disposed instead of wheat meal, the price of the aquafeeds could be lower.

Moreover, the inclusion of S. ramosissima in shrimp diets was able to slightly modulate some of the plasma immune parameters and gene expression analysed on the hepatopancreas. No differences were observed in cell counts during the feeding trial, and a general decrease regardless of dietary treatments was observed following the bacterial bath challenge. Regarding the immune parameters analysed in haemolymph plasma, differences between dietary treatments were only observed after 31 days during the feeding trial, suggesting that the diet effect seems to disappear with a longer feeding period regime.

Decreases in plasma nitric oxide levels and lysozyme activity were observed in shrimp fed SS5, SL5 and SL10 after 31 days of feeding. However, whiteleg shrimp fed S. ramosissima stems (SS10) showed a higher lysozyme activity at that same time, suggesting a slightly increase of the immune response boosted by stems. Lysozyme is an enzyme with bacteriolytic properties, which can be found in both prokaryotes and eukaryotes. Its main function is to break down the β-1, 4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, leading to bacterial lysis (Jollès and Jollès, 1984). It has been shown that lysozyme exhibits antimicrobial activity against both Gram-negative and Gram-positive bacteria, including Vibrio species that are harmful to shrimp (Hikima et al., 2003; Supungul et al., 2010). Interestingly, it was observed that shrimp fed S. ramosissima stems at 5% and 10% inclusion levels (SS5 and SS10, respectively) had higher levels of lysozyme activity after the bacterial challenge as well as respiratory burst (RB) particularly at 10% inclusion level, and both after 48 h following bacterial bath challenge, suggesting that shrimp fed diets with S. ramosissima stems might be more prone to deal with the pathogen.

A mild modulation of gene expression during the feeding trial was observed, and particularly interesting to highlight is the up-regulation of spermine synthase after 55 days of feeding in all experimental diets with S. ramosissima regardless of the biomass type or the inclusion level. Spermine synthase is an enzyme that catalyses the conversion of spermidine to spermine, which are polyamines involved in various cellular processes such as cell growth, proliferation, and differentiation (Pegg and Michael, 2010). Interestingly, Machado et al. (2023) observed a modulation of the methionine-related pathways with the up-regulation of hepatopancreas mRNA expression of SAM-synthase (SAM-synth) and also the spermine synthase (SMS) in shrimp fed a diet rich in AQUAVI^® Met-Met pointing to stimulation of polyamine biosynthesis route and key for cell proliferation.

The expression of the rab (Rab GTPase) gene responsible for the formation, transport, and fusion of intracellular organelles crucial for immune response was higher in the hepatopancreas of challenged shrimp fed S. ramosissima stems (SS) at 5% and 10% inclusion levels already at 4 h after infection. The Rab protein is an important regulatory component involved in cell biosynthesis, endocytosis, and vesicle transport (Zong et al., 2008). Recent research has also demonstrated its essential role in the development and maturation of phagocytes and innate pathways that aid in the removal of foreign pathogens (Wu and Zhang, 2007). The upregulation of Rab GTPase gene expression in response to the experimental treatment suggests increased activity of the protein. This could indicate enhanced membrane trafficking and vesicle transport, which may be important for the immune response to the challenge. For example, increased endocytosis could facilitate the uptake of pathogens or immune-stimulating molecules, while increased exocytosis could promote the secretion of immune effectors. These findings support the potential health-promoting benefits of Salicornia as mentioned earlier.

To gain a comprehensive understanding of the dietary impact on hepatopancreas gene expression, a multivariate analysis combining data from various genes was conducted. The results revealed that shrimp fed SL10 were markedly distinct from those fed the remaining experimental diets after 55 days of feeding. However, it was impossible to draw any firm conclusions from the bacterial challenge gene expression results, as no significant differences were observed among shrimp fed on different diets. Nevertheless, a clear effect of the bacterial bath challenge was evident, as demonstrated by a sharp distinction between shrimp sampled at 0 hours (55 days of feeding) and those collected after 4 hours of the bacterial bath. Furthermore, a distinct separation was noted between 4 hours and the cluster of 24- and 48-hours post-infection.

In summary, this study demonstrates that integrating S. ramosissima biomass into the diets of juvenile whiteleg shrimp does not compromise their final weight, survival, or overall health. This breakthrough offers a substantial opportunity to reinforce halophyte production and advance sustainable aquafeed practices, potentially supplanting commonplace ingredients like wheat meal, a valuable resource also utilized in human consumption. Moreover, the study’s promising outcome underscores the potential antioxidant advantages stemming from the utilization of S. ramosissima stems, conventionally viewed as cultivation by-products often relegated to waste. This innovative strategy not only taps into an overlooked resource but also presents a cost-effective alternative to traditional aquafeed components like wheat meal. Despite potential challenges related to feed efficiency, the inclusion of S. ramosissima biomass shows promise as a cost-effective and sustainable alternative to traditional feed ingredients. Although further research is needed to optimize the use of S. ramosissima biomass in shrimp feed. The cultivation of S. ramosissima is in line with the current global emphasis on resource optimization, waste reduction, and the exploration of alternative energy sources, positioning it as a viable element of a sustainable circular economy.