In the past few decades, aquaculture production has continued to grow globally, making an important contribution to ensuring world food security (FAO, 2022). The aquaculture industry's rapid growth has led to a significant need for fishmeal (FM), which serves as a pivotal protein source in aquafeeds. However, the epidemic of corona virus disease 2019 and the occurrence of El Nino and other unpredictable climatic events in recent years have led to an overall downward trend in global fishmeal production (Jannathulla et al., 2019). The huge supply-demand conflict has caused the price of FM to continue to rise from $452 per tonne (2000) to $1810 per tonne (2020). Rising FM prices in turn further stimulate predation of marine resources by fishers, which will jeopardize the sustainability of marine populations and further increase the cost of aquafeeds directly affecting the economic efficiency of the aquaculture industry, ultimately causing major economic and ecological problems (Shannon and Waller, 2021). Therefore, the development of new protein sources that can replace FM has become an urgent need for the global aquaculture industry.

Pacific white shrimp (Litopenaeus vannamei) are widely farmed worldwide because of their rapid growth rate, disease resistance, delicious flavor and strong economic efficiency (Xie et al., 2007; Xie et al., 2014). Compared to livestock animals, aquatic animals usually have a higher protein content in their bodies and therefore also require more dietary protein from food to provide the essential amino acids for body protein synthesis. Like other aquatic animals, the shrimp requires an elevated level of dietary protein for its growth. FM is used extensively in the feed of shrimp because of its high protein content, its good palatability and its more complete nutritional composition, but it also poses some economic and environmental problems (Cai et al., 2023; Yang et al., 2021b). Thus, the search for equivalent alternatives to FM also contributes to the sustainability of shrimp farming. In addition, shrimp can withstand extremely high stocking densities, and has been previously reported to successfully raise 50,000 shrimps per acre in concrete ponds. The key to intensive farming is to stock animals at high densities, and this feature of the shrimp is in line with the current concept of transforming the aquaculture industry from "crude" to "intensive" (Rodríguez-Olague et al., 2021). However, being able to farm at high densities does not mean that shrimp are suitable for living at high stocking densities, instead changes in stocking density can impact on growth, immunity and metabolism of aquatic animals in many ways (AftabUddin et al., 2020; Apún-Molina et al., 2017; Façanha et al., 2016; Tang et al., 2018). Researchers have found that one possible reason could be that excessive stocking densities can stress the aquatic animal and force it to use the part of its energy that would otherwise be used for growth to respond to environmental stress. The material and energy required for the growth and development of shrimps is mainly derived from dietary protein in the feed. Several studies have shown that inadequate dietary protein intake significantly reduces growth performance and immunity in shrimp, which is more pronounced during intensive aquaculture (Lalramchhani et al., 2020; Shyne Anand et al., 2021). But then again, high protein levels mean high cost. Therefore, in recent years, many feed manufacturers have opted to use low-quality, cheap protein sources such as feather meal to replace FM. However, low-quality protein fails to meet the nutritional requirements of shrimp, resulting in a large amount of unavailable protein being wasted in the environment. This, in turn, contributes to severe environmental pollution, which hinders the healthy and sustainable development of aquaculture (Burford and Williams, 2001; Cai et al., 2023). This is why finding sustainable, high-quality protein to replace the low-quality in feed is so important in intensive shrimp farming.

Currently, animal and plant protein sources represented by feather meal, blood meal, soybean meal and cottonseed meal are often used in shrimp feeds as an alternative to FM (Goda et al., 2007; Pahlow et al., 2015). However, the unbalanced amino acid composition, subpar palatability, and numerous anti-nutritional factors present in plant protein sources (Enyidi et al., 2014; Li et al., 2009), coupled with the inconsistent quality and certain safety hazards associated with animal protein sources (Bureau et al., 2000), have hindered their widespread adoption as viable substitutes for FM. In contrast, microalgae appear to be a more promising source of protein for aquafeeds (Sarker et al., 2018). Microalgae exhibit a favorable amino acid profile, boasting high protein and oil content, while also being wealthy in antioxidants and anti-immune stress compounds, such as beta-1,3-glucan (Shao et al., 2014; Shi et al., 2017b). Moreover, microalgae can swiftly colonize diverse environments, spanning from freshwater to brackish and saltwater, utilizing both photosynthesis and heterotrophic growth, thus exemplifying their exceptional sustainability (Dineshbabu et al., 2019; Vuppaladadiyam et al., 2018). Chlorella sorokiniana (CHL) stands as one of the pioneering microalgae to be commercialized, attributed to its exceptional characteristics, including a high protein content, elevated unsaturated fat content, rich nutritional profile, rapid growth rate, remarkable reproductive efficiency, and the presence of numerous trace elements, minerals, and Chlorella growth factor (CGF) (Carneiro et al., 2020; Zhou et al., 2021).

It's worth mentioning that, unlike plant and animal proteins, CHL has consistently flourished in water environments. Therefore, the process of ingestion of CHL diets by aquatic animals also mimics their natural habits, and helps preserve their inherent flavor (Uchechukwu, 2017). Currently, the substitution of CHL for FM has been documented in various species, including Danio rerio (Carneiro et al., 2020), Macrobrachium rosenbergii, Carassius auratus (Shi et al., 2017a), Clarias gariepinus (Raji et al., 2019a), and Micropterus salmoides (Xi et al., 2022). These substitutions have been demonstrated to have benefits, such as enhanced growth and sexual maturity, increased immune response, elevated digestion, and improved body composition and intestinal health, among other positive effects. Moreover, studies have also discovered that the addition of 9.72% CHL to shrimp diets remarkably enhanced its growth rate, digestion and improved its body composition (Pakravan et al., 2018), although the digestibility of CHL protein is lower than that of FM in Pacific white shrimp (Nederlof, et al., 2023). Nevertheless, there is a shortage of research examining the efficacy of CHL as a substitute for FM in shrimp under different stocking densities.

In this study, a two-way ANOVA was used to investigate the utilization capacity of CHL by shrimp at different stocking densities in terms of growth performance, immunity, intestinal health, immunometabolic pathway as well as the interaction of microorganisms and immune factors to afford a theoretical guidance for its application as a new protein source under intensive aquaculture industry.

Protein is the most critical and costly nutrient in shrimp feed, and the content and quality of dietary protein is usually the main basis for judging the merits of the feed (Wang et al., 2017). In shrimp feed, this part of the role is usually played by FM. However, due to the many limitations of FM, research on the replacement of fishmeal by CHL is gaining attention. It is widely recognized that replacing FM in diets with appropriate amounts of CHL can better achieve the goal of healthy and rapid growth of farmed animals (Carneiro et al., 2020; Raji et al., 2019b). With the rapid development of intensification, the impact of stocking density on aquatic animals had become more and more concerned, because it affects the growth and health of aquatic animals in all aspects and directly affects the economic efficiency (Alanärä and Brännäs, 1996; Conte, 2004; Holm et al., 1990). This study investigated the value of CHL in intensive shrimp farming by analyzing the impact of CHL on growth, immunity, intestinal digestibility, microbiota structure and immunometabolic pathway in shrimp at different stocking densities.

As a superior novel protein source, the effect of CHL in place of FM on the growth performance of shrimp was evident. Compared with the FM group, the SR, WGR, SGR and PRE in the CHL group displayed an upward trend. This growth-improving effect possibly ascribe to the various nutrients contained within CHL, such as CGF, vitamins, minerals, free amino acids, and some immune-related glycoproteins and phytohormones (An et al., 2016). Such beneficial effects in the CHL group were also reflected in feed utilization and intestinal digestive enzyme activity. Animal feed utilization is usually expressed in terms of FCR and FI. At the same time, digestive enzymes play a crucial role in the digestive system of animals that directly reflect the acceptability and adaptability of the body to diet type and dietary nutrient concentrations. In M. salmoides, replacing 31.7% to 32.6% of fishmeal with 15.03% to 15.43% of Chlorella resulted in better growth performance (Xi et al., 2022). In the research by Liu et al. (2022) it was found that CHL replacement of FM in the diet markedly increased the intestinal digestive capacity and feed utilization of Oncorhynchus mykiss, and to some extent promoted the growth of O. mykiss. For crustaceans, in the research of Safari et al. (2022a) CHL substitution for FM improved several growth indicators including SGR, FCR, SR, PER and Protein productive value (PPV) in Pontastacus leptodactylus and when CHL substitution reached between 78.13% and 85.49%, crayfish obtained the maximum SGR, WGR and WD as well as the minimum FCR. In a study of M. rosenbergii, it was found that the addition of 4%, 6% and 8% CHL to aquafeeds markedly improved shrimp growth rate and digestibility (Maliwat et al., 2021). The findings in this paper align with those of previous research, indicating that replacing FM with some CHL in aquafeeds can improve digestibility and feed utilization, thereby reducing farming costs and promote growth in aquatic animals. Specifically, the activity of trypsin, lipase and amylase as well as SR, WGR, SGR and PRE were markedly higher in the CHL group than in the FM group at high stocking densities, while the opposite was true for FCR and FI. This indicates that the increase in stocking density is beneficial to the utilization of CHL by shrimp, which also implies that CHL has significant potential as a novel protein source in the intensive farming of shrimp.

The SR of shrimp in pathogen-challenge experiments is often considered a direct indication of the strength of resistance to disease. In the V. parahaemolyticus challenge experiment, the SR of the shrimp in the CHL group was higher than that in the FM group at the same stocking density, and there was a highly notable difference at high stocking densities. This situation has been similarly reported in Oreochromis niloticus (Abdel-Tawwab et al., 2022a), C. gariepinus (Raji et al., 2018a), and P. leptodactylus (Safari et al., 2022b). The reasons for this may be due to the fact that CHL contains a variety of active substances compared to FM, which have excellent antibacterial properties to prevent the invasion of pathogenic bacteria and improve the immunity of shrimp (Guedes et al., 2015; Ibrahim et al., 2015; Mahmoud et al., 2020). In the studies of Grammes et al. (2013) on Salmo salar and Pakravan et al. (2018) on shrimp, it was found that the addition of CHL to the diet significantly improved the survival of aquatic animals in harsh environments. These are also consistent with the results of the pathogenic bacteria assay in this experiment, suggesting that dietary CHL may be important in improving the resistance of aquatic animals in the face of environmental stress. This result was also validated in non-specific immune indicators. The hepatopancreas is the most important immune organ of crustacean, and the immune effect of the hepatopancreas is achieved by regulating the secretion of various immune-related enzymes such as ACP, AKP, AST, AST, CAT, PO, SOD, and LYS, so the activity of these immune-related enzymes is also an important indicator of the immunity of shrimp. In Raji et al.'s (2018b) study, it was discovered that dietary CHL substitution for FM markedly improved the antioxidant capacity of C. gariepinus by increasing the activity of antioxidant enzymes including CAT, PO, and SOD. In studies conducted by Abdel-Tawwab et al. (2022b) and Safari et al. (2022c), it was observed that dietary CHL markedly improve the antioxidant enzyme activity in O. niloticus fingerlings and Astacus leptodactylus, while also enhancing their immune enzyme activities, such as lysozyme (LYS). It was hypothesized that the observed enhancement in immune enzyme activity might be attributed to the activation of the organism's immune system by the presence of high levels of lipopolysaccharides and carotenoids in CHL. In this experiment, the immune-related indexes including AST, ALT, CAT, SOD, PO and LYS in the CHL group were higher than those in the FM group, and most of them were markedly different at high stocking density. This result is similar to the results of previous studies and is also consistent with the results of toxicity experiments, indicating that partial FM replacement with CHL at a same dietary protein level can improve the immunity of shrimp.

The reciprocity between the intestinal microbiota and host's immune and metabolic responses is critical to the health of aquatic animals (Chen et al., 2023; He et al., 2017; Wu et al., 2021). It has been found that the occurrence of disease and reduced growth performance in aquatic animals may be directly related to the disruption of the structure of the intestinal microbiota (Holt et al., 2020). It has been shown that environmental stress and feed composition can markedly affect the structure of the intestinal microbial community of shrimp. For example, Suo et al. (2017) found that ammonia and sulfide stress disrupted the intestinal microbiota structure of shrimp, causing a reduced content of beneficial bacteria and an elevated content of pathogenic bacteria. A study by Zhou et al. (2020) found that dietary inulin could optimize the intestinal microbiota structure of shrimp under low salinity stocking conditions and promote the colonization of beneficial bacteria. Nevertheless, studies exploring the effects of CHL substitution for FM on shrimp and even aquatic animals at different stocking densities by intestinal microbiota sequencing technology have not been reported. Based on this status, the present study compares the gut microbiota structure of shrimp that consumed two protein sources.

Alpha diversity analysis showed that the alpha diversity index in the CHL group was generally higher than that in the FM group at the same stocking density, and notable differences were mainly found at high stocking densities. This implies that dietary CHL can promote the diversity and richness of the shrimp microbiota. Proteobacteria are usually the dominant phylum in the intestinal microbiota of animals, but because they contain many pathogenic bacteria including Aeromonas, Edwardsiella, Pseudomonas and Vibrio, this makes them a common cause of disease in animals (Lopetuso et al., 2016). Planctomycetes and Actinobacteria are common phyla in the intestine of aquatic animals. The former bacteria are involved in the hydrolysis of heteropolysaccharides in addition to the effective breakdown of proteins, providing energy and substances for shrimp growth (Wiegand et al., 2018). Meanwhile, the latter bacteria produce bioactive substances that benefit the organism and contribute to the preservation of gut homeostasis (Binda et al., 2018; Van Keulen and Dyson, 2014). An interesting finding showed that a reduction in the ratio of the Firmicutes to the Bacteroidetes was directly related to the weight loss of the animals (Ley et al., 2006). In our current investigation, alterations in the microbiota of shrimp within the CHL group closely resembled those within the FM group as stocking density increased. Both exhibited a noticeable decline in abundances of beneficial bacteria and a concurrent rise in abundances of causative bacteria. However, at the same stocking density, the abundance of Planctomycetes, Actinobacteria and the ratio of Firmicutes to Bacteroidetes were higher in the CHL group than in the FM group, whereas the abundance of Proteobacteria was lower than in the FM group. This further demonstrates that dietary CHL is not only harmless to shrimp intestinal microbiota diversity, but also promotes the propagation of beneficial bacteria. At the genus level, this effect becomes more pronounced. Ruegeria and Motilimonas are considered beneficial bacteria in the usual sense and Ruegeria is considered to have a potential character in defending aquatic animals from pathogenic Vibrio (Miura et al., 2019). The genus Vibrio is the predominant bacterial genus found in shrimp intestines, and previous studies have reported that excessive amounts of Vibrio can easily alter the health of shrimp and cause disease (Wang et al., 2020). Spongiimonas, a member of the phylum Bacillus, contain various pathogenic factors as conditional pathogens and are usually considered to be harmful to organisms (Hou et al., 2020). In our current investigation, the levels of Motilimonas in the CHL group was markedly greater than that in the FM group under low stocking density conditions while the levels of Spongiimonas in the CHL group was markedly below that in the FM group under high stocking density conditions. Further, when stocking densities were the same, Ruegeria abundance was markedly higher in the CHL group than in the FM group while Vibrio abundance was markedly below in the FM group. This implies that the alterations in intestinal microbiota structure in the CHL group were associated with increased disease resistance compared to the FM group. These discoveries are also compatible with the outcomes observed in experiments involving challenges with pathogenic bacteria and microbiota functional analysis, suggesting that dietary CHL can improve disease resistance in shrimp by improving the composition of its intestinal microbiota, especially at high stocking densities. This was further verified by the results of the transcriptome analysis.

After years of development, transcriptome sequencing technology has become an important tool to explore mechanisms related to growth, metabolism and immunity of organisms (Yang et al., 2021a; Ye et al., 2023). Transcriptome analysis revealed that both dietary FM and dietary CHL were enriched in similar functions and pathways in response to alterations in stocking density in both differential GO analysis and differential KEGG analysis, but interestingly, the FM group was strongly influenced by stocking density while the CHL group was little influenced. This outcome aligns with other findings from this study, suggesting that dietary CHL can largely reduce the effect of stocking density on shrimp. The composition and enrichment of DEGs in the FM vs CHL groups at the same stocking density were further analyzed to investigate how dietary CHL changed the impact of stocking density on shrimp. Considering that higher stocking densities brought higher environmental stress, and that the DEGs enriched to GO terms as well as KEGG pathways in the FM vs CHL group at high stocking densities covered the low to medium stocking densities, the results of the FM vs CHL group at high stocking densities are more representative. In particular, the CHL group upregulated 2/3 of the DEGs and most of the upregulated DEGs were related to metabolism, catalysis and immunity, suggesting that dietary CHL is a response to environmental stress by enhancing the metabolic and immune activities of shrimp. On the other hand, KEGG analysis based on DEGs in the FM vs CHL group at high stocking densities showed that dietary CHL mitigates the impact of environmental stress on shrimp mainly by regulating immune and metabolic related pathways, including Toll and immune deficiency (Imd) pathways, Toll-like signaling pathway, janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, glycolysis–gluconeogenesis and HIF-1 signaling pathway. Also, this study revealed that key genes (such as glut1, gapdh, cact, socs2, and hex-t2) regulating these pathways were adaptively expressed with different types of dietary protein sources.

The inborn immune response serves as the organism's initial defense mechanism against foreign pathogen invasions. It has multiple functions, including the activation of complement and coagulation pathways, pro-inflammatory signaling cascades, regulatory effects, phagocytosis, and apoptosis. This system is widely present in most invertebrates and mammals (Cai et al., 2022; Janeway and Medzhitov, 2003). Toll and Imd pathways are essential pathways in the invertebrate immune response and are vital for inducing the expression of immune-related genes during invasion in the face of pathogens (Tanji and Ip, 2005). The cact gene is an important component of the Toll and Imd pathways, in which it plays a negative regulatory role (Cai et al., 2022). Specifically, Toll and Imd signaling pathways require NF-kappa B transcription factors for activation, whereas the protein encoded by the cact gene binds to and inhibits NF-kappa B transcription factors, thereby depriving Toll and Imd signaling pathways of activation and ultimately causing silencing of the immune response (Hayden et al., 2006; Hayden and Ghosh, 2004). The egfr gene is an important gene in Toll-like signaling pathway, and silencing of this gene leads to enhanced intracellular viral replication and impaired expression of antiviral genes (Yamashita et al., 2012). The JAK-STAT signaling pathway, together with the Toll-like receptor pathway and the Imd pathway, are three major immune pathways in the innate humoral immune response of shrimp, and are important for activating its immune response to defend against pathogenic invasion. Similar to cact genes, socs2 genes also negatively regulate the JAK-STAT signaling pathway and regulate the termination of JAK-STAT signaling process. As for metabolism, gapdh and hex-t2 genes are metabolic genes and also key genes in glycolysis–gluconeogenesis. The hex-t2 gene is a key gene encoding a rate-limiting enzyme in glycolysis–gluconeogenesis, and the gapdh gene encodes a protein concerned in the sixth step of the glycolytic reaction while its family genes are also implicated in glycerol- 1,3P and glyceraldehyde-3P reciprocal conversion. High expression of gapdh and hex-t2 genes can facilitate the breakdown of glucose for energy and carbon molecules by catalyzing the onset of glycolytic reactions and ultimately increase the growth rate of the organism. In research by Xiao et al. (2022) it was found that the expression of gapdh and hex-t2 genes was markedly higher in fast-growing Silurus lanzhouensis than in slow-growing S. lanzhouensis, and it was hypothesized that this might be due to the enrichment of metabolic genes such as hex-t2 in the carbohydrate metabolic pathway that affects muscle growth and ultimately leads to the difference in growth rate. Similar to the gapdh and hex-t2 genes, the ald1 gene translates a protein that is a metabolic enzyme prevalent in the cytoplasm and catalyzes the fourth step of the glycolytic reaction (Pirovich et al., 2021). The protein produced from the pck2 gene serves as the restricting factor in the tricarboxylic acid cycle (Yang et al., 2009). Research has indicated that nutritional cues can influence endocrine and metabolic functions in O. mykiss by modulating the expression of the pck2 gene (Marandel et al., 2019). Furthermore, the innate immune system requires a rapid supply of energy and sufficient bactericidal substances to defend against pathogenic invasion (Sonoda et al., 2007; West et al., 2011). And according to Imtiyaz and Simon (2010) and Sun et al. (2020a) the rapid onset of glycolytic reactions is exactly the source of energy and substances needed for the immune response. As an important immune-related pathway, the HIF-1 signaling pathway can also influence metabolic functions by regulating the expression of a series of glycolysis-related enzymes including glut1, which can have profound effects on both immune and metabolic responses (Nowicki and Gottlieb, 2015). In the study of Sun et al. (2020b) on Apostichopus japonicus, it was found that the HIF-1 signaling pathway could promote the inflammatory response and combine with glycolytic reactions to produce energy and bactericidal substances, thus improving the immunity of A. japonicus. Similar to previous studies, in our research, shrimp immunity and growth rate were markedly improved in the CHL group compared to the FM group. This suggests that dietary CHL may activate or suppress immune and metabolic pathways such as Toll and Imd signaling pathway and glycolysis–gluconeogenesis through upregulation of glut1, gapdh, egfr, ald1, pck2 and hex-t2 genes and downregulation of cact and socs2 genes expression, that ultimately have beneficial effects on shrimp.

It is well established that the structure of the intestinal microbiota is intricately connected to host health, and that the immune and metabolic systems of an organism may have a mutually beneficial relationship with the intestinal microbiota to regulate the homeostasis of the organism and adapt to environmental changes (Ouyang et al., 2023; Wang et al., 2019; Zheng et al., 2020). Meanwhile, the regulation of immune and metabolic systems is mainly accomplished by regulating the expression of related genes, so it is important to learn the relationship between DEGs and intestinal microbiota to investigate the mechanism of dietary CHL on shrimp under different stocking densities. In the present study, dietary CHL resulted in a positive correlation between the abundance of beneficial bacteria such as Motilimonas and immune and metabolic genes such as glu1 in the intestine of shrimp compared to dietary FM, and this correlation was markedly enhanced with increasing stocking density. This result demonstrates that there may be a mechanism of action between immune genes and intestinal microbiota in the presence of dietary CHL to jointly regulate the growth and immunity of shrimp, thus promoting the nutrition and metabolism of shrimp, but further research is required to determine whether the host shrimp actively controls the microbiota by altering gene expression or passively responds to the changing microbiota. However, there is no doubt that dietary CHL has a significant positive modulatory effect on immunometabolic response and intestinal microbiota of shrimp in high stocking densities.

Finally, it is important to note that the stocking density, protein source, and their interaction had notable impacts on shrimp in various aspects in the present study, such as growth performance, disease resistance, immune indicators, digestive enzymes, microbiota structure, and immunometabolic factor. This result implies that no matter how much one emphasizes the importance of protein sources or stocking densities individually in the shrimp farming process, the importance of their interactions cannot be ignored.

In this study, the effect of using C. sorokiniana in place of some fishmeal in the diet was evaluated on the farming of Pacific white shrimp (L. vannamei) at different stocking densities. The results showed that dietary CHL was effective in improving growth performance, immunity, intestinal health and disease resistance of shrimp, and this improvement effect is more obvious at high stocking density. In addition, at high stocking densities, dietary CHL may also result in good synergistic relationships between immune and metabolic genes and intestinal beneficial bacteria to jointly promote shrimp growth and maintain shrimp health. This finding demonstrates that CHL has great potential as a protein source in intensive farming of shrimp.