Dietary phosphorus restriction induced phospholipid deficiency, endoplasmic reticulum stress, inflammatory response and gut microbiota disorders in Lateolabrax maculatus

Zixiang WuJiarong GuoKang-le LuKai SongLing WangRuijuan MaChunxiao ZhangXueshan Li^*

• Jimei University, Xiamen, China

Being indispensable for animal physiology, phosphorus not only ensures proper bone development and maintenance but also drives fundamental biological processes including phospholipid production, genetic material synthesis, cellular communication, and systemic metabolism regulation (1)(2)(3)(4). Teleosts primarily rely on dietary phosphorus due to limited physiological capacity to absorb dissolved phosphorus from water (5). The content of phosphorus in commercial feed is important for both the environment and economy (6). Phosphorus homeostasis in aquaculture systems presents critical environmental and physiological trade-offs.Excessive dietary phosphorus inputs contribute to aquatic eutrophication through effluent discharge (7), driving the aquaculture industry toward precision nutrition strategies. While the formulation of low-phosphorus (LP) feeds has gained prominence for ecological sustainability, insufficient phosphorus provision induces multisystemic dysfunction in aquatic species. Chronic phosphorus deficiency manifests as growth retardation, and metabolic dysregulation characterized by adipose tissue accumulation and skeletal mineralization defects (8,9). As a critical regulatory factor in lipid metabolism, phosphorus modulates adipose deposition in fish (10,11).Within intensive aquaculture systems, excessive abdominal fat accumulation reduces dress-out percentage and compromises feed conversion efficiency, thereby elevating production costs (12). Investigating the molecular interplay between phosphorus deficiency and lipid metabolism not only advances ecological conservation objectives but also establishes a theoretical foundation for precision nutritional management in aquaculture practices. Therefore, the role of phosphorus in various metabolic processes of organisms is worth investigating, which can contribute to the development of low-phosphorus feeds and few phosphorus emissions.The endoplasmic reticulum (ER) serves as a central hub for multiple cellular processes, orchestrating calcium ion homeostasis, lipid metabolic regulation, and the synthesis, post-translational modification, and intracellular trafficking of proteins (13)(14)(15). In teleost species, phosphorus assumes critical importance in phospholipid biosynthesis, with the ER serving as the principal site for phospholipid anabolism. This organelle facilitates the enzymatic conversion of inorganic phosphorus into structural and functional phospholipid molecules through coordinated biochemical pathways (16). Phospholipids are essential components of the membranes of the ER and must be present in regular levels to maintain correct ER function (17).Abnormalities in the composition or fluidity of the ER membrane may compromise ER function and induce ER stress (18). ER stress can cause a range of negative effects such as insulin resistance, disruption of lipid metabolism as well as inflammatory responses (19)(20)(21). Furthermore, it is found in mammals that the unfolded protein response (UPR) triggered by ER stress activates caspase-2-mediated NLRP3 inflammasome assembly (22), establishing a molecular bridge between ER stress and systemic inflammation. However, the association involving phosphorus and ER stress should be investigated further.The vertebrate gastrointestinal tract constitutes a complex microbial ecosystem harboring dynamic symbiotic communities that exert essential influences on host nutritional assimilation and physiological homeostasis (23,24). This intricate microbiota-host interface coordinates metabolic cross-talk through enzymatic diversification, micronutrient biosynthesis, and immunoregulatory signaling cascades.Many metabolic illnesses have been linked to alterations in the gut micro-ecosystem (25,26). Dietary variables, such as macronutrients and micronutrients, can alter the composition and functionality of the gut microbiota (27). Phosphorus is necessary for both the host animal to maintain the normal metabolism and for the microbiota colonizing the animal's GIT (28). The deficiency of phosphorus can reduce the ratio of probiotic/pathogenic bacteria (29). Emerging evidence underscores the critical role of probiotic microbiota in enhancing nutrient assimilation, preserving intestinal barrier integrity, and modulating immune responses (30,31). Conversely, ecological perturbations within the intestinal microbiota significantly increase disease susceptibility, manifesting as metabolic dysregulation, chronic stress responses, and growth impairment (32,33). Despite these advances, the mechanistic interplay between dietary phosphorus availability and microbial community dynamics remains poorly characterized, particularly in aquatic vertebrates.The immune system employs both innate and adaptive mechanisms to maintain physiological homeostasis against pathogenic challenges (34,35). Notably, nutritional imbalances can dysregulate NF-κB and JAK-STAT signaling pathways, compromising phagocytic activity of macrophages and neutrophil recruitment efficiency (36,37). Emerging evidence from recent studies highlights the critical involvement of phospholipid metabolic homeostasis in modulating immune system functionality, underscoring its pivotal regulatory role in immunophysiological processes (38). However, the association involving phosphorus and ER stress should be investigated further, particularly regarding how phosphorus deficiency-induced phospholipid depletion modulates these inflammatory cascades in aquatic species.The spotted seabass (Lateolabrax maculatus), a carnivorous teleost species widely farmed in China, exhibits distinct nutritional requirements for sustainable aquaculture.Our laboratory established 0.72% available phosphorus as the optimal dietary level for freshwater-reared specimens through rigorous dose-response trials (39). Strategic reduction of dietary phosphorus content presents a viable approach to mitigate aquaculture-derived phosphorus emissions, yet requires precise calibration to avoid compromising fish health and growth performance. However, the effects of low phosphorus diet on growth, metabolism and gut microbiota of spotted seabass remain to be studied. The present study is conducted to investigate the effects of low phosphorus on spotted seabass from the perspective of phospholipid content and function, ER stress, lipid metabolism and gut microbiota. This study also serves as a theoretical reference point for the application of low-phosphorus feeds and strategies to reduce phosphorus emissions. The study protocol received ethical approval (Permit number: 2011-58) from Jimei University's Animal Ethics Review Board, with all procedures conducted in strict accordance with established animal welfare standards. Diets were formulated to contain either 0.37% available phosphorus (low-phosphorus, LP) or 0.75% available phosphorus (normal-phosphorus, NP). The specific composition and formulation details of these diets are documented in Table S1. The feed preparation was informed by prior research conducted within our laboratory (40).Utilizing a recirculating aquaculture system (RAS) at Jimei University, the feeding trial was conducted in six 200-L fiberglass tanks. Healthy spotted seabass (Lateolabrax maculatus) with an initial mean body weight of 3.53 ± 0.34 g were procured from a commercial hatchery in Zhangzhou, Fujian Province, China. Following a 7-day acclimation period, 180 fish of uniform size were randomly allocated to the tanks (30 fish per tank). Fish were fed their respective experimental diets (LP or NP) to apparent satiation twice daily (08:00 and 17:00) for 10 weeks. The RAS maintained optimal water quality through continuous aeration and a 40% daily water exchange regimen. Key parameters were monitored and stabilized as follows: temperature 26-27°C, dissolved oxygen >6.5 mg/L, pH 6.9-7.2, and total ammonia nitrogen <0.2 mg/L. Tank assignments followed a completely randomized design to eliminate spatial bias. After 24 hours of food deprivation, fish weight was measured to calculate growth metrics. Prior to tissue collection, fish were sedated with MS-222 anesthetic (60 mg/L solution, Sigma). From each tank, we obtained blood samples from 12 individuals through tail vein puncture using 27-gauge needles. These samples clotted overnight at 4°C before centrifugation (1,283 × g, 10 minutes) to isolate serum for -80°C storage. Three biological replicates were set up for each treatment in this study, and the samples from four fish were mixed together as one replicate to reduce variability between individual samples. Four jejunal samples were pooled per tank for microbiome analysis, with each specimen immediately flame-sterilized using an alcohol burner post-collection to mitigate cross-contamination risks. Liver, intestinal, and abdominal fat tissues were flash-frozen in liquid nitrogen and archived at -80°C for subsequent analyses. Serum biochemical parameters, including alkaline phosphatase (ALP) activity, triacylglycerol (TG), phosphorus (P), and calcium (Ca) concentrations, were analyzed using commercial diagnostic kits (Jiancheng Bioengineering Institute, Nanjing, China). Quantification of phosphoglyceride (PG) and sphingomyelin (SM) levels in serum, hepatic, and adipose tissues was performed via enzyme-linked immunosorbent assay (ELISA) kits (Hengyuan Biotechnology, Shanghai, China). Liver enzymatic activities of choline phosphotransferase 1 (CHPT1) and ethanolamine phosphotransferase 1 (EPT1) were assessed using specific ELISA kits (Meimian Biological Technology, Yancheng, China). Liver, jejunum, and abdominal fat tissue samples underwent triplicate RNA extraction using the established protocol from our established methodology (41). Subsequently, reverse transcription quantitative PCR (RT-qPCR) was performed on a QuantStudio 6Pro system (Applied Biosystems) under optimized conditions: 95°C/10min initial denaturation, 40 cycles of 95°C/15s, and 60°C/1min. Primer design specifically targeted conserved regions within the spotted seabass transcriptome, with all oligonucleotides (Supplementary Table S2) exhibiting 90-110% amplification efficiency validated through standard curves (R²>0.99). Gene expression normalization employed the ΔΔCt algorithm, utilizing β-actin (CV<5% across biological replicates) as the endogenous control. Liver sections (5 μm) were cryosectioned and stained with H&E (5% acetic acid differentiation) and ORO (0.3% in isopropanol) following established protocols (42,43). Bright-field imaging used a Leica DM5500B microscope (40×/NA 0.75) with Köhler illumination. For TEM, glutaraldehyde-fixed tissues were osmicated, dehydrated, and embedded in EPON 812 resin. Ultrathin sections (70 nm) stained with uranyl acetate/lead citrate were analyzed on a JEOL JEM-1400 TEM at 80 kV (44). Jejunal microbial DNA was extracted using HiPure Soil DNA Kits (Magen Biotechnology) with bead-beating lysis. DNA quality (A260/A280=1.82±0.05) was verified by NanoDrop 2000. The 16S rRNA V3-V4 regions were amplified with 338F/806R primers, purified using AxyPrep kits, and quantified via Qubit assays. Statistical analyses were stratified by data type. For microbial community data, β-diversity dissimilarity matrices were subjected to permutational multivariate analysis (PERMANOVA) with 999 permutations using the Adonis function in the vegan package (v2.5.3). Differential taxa identification was performed through Welch's two-sample t-tests on operational taxonomic units (OTUs) exhibiting >0.1% relative abundance. PICRUSt2-derived functional profiles and α-diversity indices (Shannon, Simpson) were compared between the two groups using Welch's unequal variances t-test in R v4.1.2. Statistical analyses of non-microbiome data were performed using independent two-tailed t-tests in SPSS 25.0 (IBM, USA), with results presented as mean ± SEM. Significance thresholds were set at ns P≥0.05, * P<0.05, ** P<0.01, and *** P<0.001. In addition, the comparison of specific values in this paper follows the order of LP vs. NP, if there is no special explanation. Fish fed the LP diet had a final body weight of 38.50 ± 0.66 g, representing a significant reduction compared to fish fed the NP diet (69.78 ± 2.29 g) (P < 0.001; Fig. 1A). This growth retardation was further corroborated by weight gain (WG) metrics, where fish fed the LP diet (969.92 ± 17.91%) showed significantly lower values than fish fed the NP diet (1727.49 ± 21.42%) (P < 0.001; Fig. 1B). However, fish fed the LP diet exhibited a significant increase in abdominal fat percentage (8.81 ± 0.10%) versus fish fed the NP diet (7.87 ± 0.05%) (P < 0.001; Fig. 1C). Serum phosphorus concentrations in fish fed the LP diet (6.68 ± 0.29 mmol/L) exhibited a significant decrease compared to fish fed the NP diet (10.85 ± 0.73 mmol/L) (P < 0.01; Fig. 2A), while serum calcium levels remained stable between the two groups (P = 0.366; Fig. 2B). Concurrently, alkaline phosphatase activity in fish fed the LP diet (17.28 ± 1.09 mmol/L) showed a significant increase relative to fish fed the NP diet (12.79 ± 0.64 mmol/L) (P < 0.05; Fig. 2C). Molecular analysis of intestinal transporters demonstrated phosphorus-specific regulation, with fish fed the LP diet displaying remarkable increases in napi-iib, pit1, and pit2 genes expression compared to fish fed the NP diet (P < 0.05; Fig. 2D). In contrast, napi-iia gene expression showed no significant difference (P = 0.304) between the two groups.Systemic phospholipid quantification identified consistent depletion patterns in fish fed the LP diet across all examined tissues. Phosphoglyceride (PG) and sphingomyelin (SM) contents in fish fed the LP diet were decreased in serum (PG: 71.94 ± 2.60 ng/L vs. 93.94 ± 2.60 ng/L, SM: 16.98 ± 0.78 ng/L vs. 24.00 ± 1.30 ng/L), liver (PG: 30.64 ± 0.47 ng/L vs. 37.63 ± 1.17 ng/L, SM: 41.38 ± 0.38 ng/L vs. 44.80 ± 1.14 ng/L), and abdominal fat tissues (PG: 274.12 ± 7.79 ng/L vs. 319.74 ± 12.12 ng/L, SM: 58.12 ± 2.48 ng/L vs. 72.69 ± 3.18 ng/L) compared to fish fed the NP diet, with all intergroup differences reaching statistical significance (P < 0.05; Fig. 2E-J). In abdominal fat and liver tissues, fish fed the LP diet exhibited a remarkable upregulation of ER stress related genes (grp78, perk, atf6, xbp1s) expression compared to fish fed the NP diet (P < 0.05; Fig. 3A,E). Notably, ire1 gene expression remained stable in both abdominal fat (P = 0.469) and liver (P = 0.774) tissues between the two groups. Specifically, fish fed the LP diet showed a significant elevation with triacylglycerol (TG) content in serum (7.39 ± 0.20 mmol/L vs. 6.12 ± 0.08 mmol/L) and liver (0.80 ± 0.07 mmol/gpot vs. 0.55 ± 0.01 mmol/gpot) compared to fish fed the NP diet (P < 0.05; Fig. 3C,G). Notably, liver total cholesterol (TC) content in fish fed the LP diet (0.056 ± 0.007 mmol/gpot) was significantly higher than fish fed the NP diet (0.019 ± 0.003 mmol/gpot) (P < 0.001; Fig. 3H), while serum TC concentrations displayed no significant difference between the two groups (P = 0.970; Fig. 3D). Intriguingly, coordinated lipid metabolic changes were observed across tissues. In both liver and abdominal fat tissues, fish fed the LP diet exhibited a remarkable downregulation of lipolysis genes (pgc-1, atgl, cpt-1) expresssion, whereas lipogenesis (fas, acc1, acc2) and regulatory factors genes (srebp-1, pparγ) expresssion were remarkably upregulated relative to fish fed the NP diet (P < 0.05; Fig. 3B,F). Nevertheless, chrebp-1 gene expression remained stable in both tissues (P = 0.598). Hepatic CHPT1 activity in fish fed the LP diet (61.06 ± 1.392 ng/L) was significantly increased relative to fish fed the NP diet (52.53 ± 0.869 ng/L) (P < 0.001; Fig. 3I), contrasting sharply with unaltered EPT1 activity (P = 0.170; Fig. 3J).Fish fed the LP diet exhibited a remarkable upregulation of il-1β and tnf-α genes expression compared to fish fed the NP diet (P < 0.01). In contrast, il-6 gene expression in abdominal fat tissue showed no remarkable difference between fish fed the LP and NP diets (P = 0.433; Fig. 3K). The ORO sections revealed that the spotted seabass fed the LP diet had a greater fat accumulation in liver compared to fish fed the NP diet (P < 0.05; Fig. 4A vs. B,I). In H&E sections, the vacuolization in liver of spotted seabass fed the LP diet was more serious than fish fed the NP diet (P < 0.05; Fig. 4C vs. D,J). In addition, under the ultrastructure, the hepatocyte endoplasmic reticulum structure of spotted seabass fed the LP diet was severely damaged, the endoplasmic reticulum was loosely stacked, and the mitochondria-associated membranes (MAMs) was disorganized (Fig. 4E vs. F). Meanwhile, the abdominal fat tissue of spotted seabass fed the LP diet showed adipocyte hypertrophy under H&E staining (Fig. 4G vs. H). Venn diagram quantification identified 56 shared operational taxonomic units (OTUs) between the two groups, with fish fed the LP diet harboring 13 unique OTUs compared to 29 in fish fed the NP diet (Fig. 5A). Alpha diversity metrics demonstrated significantly reduced community heterogeneity in fish fed the LP diet, exhibiting markedly lower Simpson index and markedly decreased Pielou evenness relative to fish fed the NP diet (P < 0.05; Fig. 5B-C). Multivariate analysis confirmed distinct clustering patterns, with principal coordinates analysis (PCoA) based on Bray-Curtis distances revealing remarkable separation between gut microbiota profiles of fish fed the LP and NP diets (P < 0.05; Fig. 5D-E).At the phylum level, Proteobacteria and Firmicutes dominated intestinal communities across both dietary regimens. Fish fed the LP diet exhibited a markedly higher relative abundance of Proteobacteria (93.12% vs 66.47%) and markedly lower Firmicutes representation (6.56% vs 33.31%) compared to fish fed the NP diet (P < 0.05; Fig. 6A-B).At the genus level, fish fed the LP diet were dominated by the bacterial taxa Plesiomonas (81.13%), Acinetobacter (11.45%), and Bacillus (4.96%) (Fig. 6C). In fish fed the NP diet, Plesiomonas (48.08%), Lactococcus (27.82%), Acinetobacter (17.02%) and Bacillus (5.16%) were dominant bacterial taxa. Potential pathogenic bacteria, e.g. Plesiomonas, was significantly more abundant (P < 0.05), while the abundance of potential probiotic, e.g. Lactococcus, was significantly lower in fish fed the LP diet compared to those fed the NP diet (P < 0.05; Fig. 6D).LEfSe analysis revealed that fish fed the LP diet had significantly higher levels of translational machinery, and immune regulation (Fig. 7C,D). Based on our laboratory's prior research establishing 0.72% available phosphorus (NP)as the appropriate dietary phosphorus level for Lateolabrax maculatus, whereas 0.37% available phosphorus (LP) demonstrated a significant deficiency relative to the optimal value for investigating phosphorus deprivation effects, two experimental diets were formulated accordingly (39). This experimental design specifically replicated these established available phosphorus concentrations (0.75% NP vs. 0.37% LP) to systematically examine phosphorus deficiency manifestations. Consistent with established nutritional physiology paradigms (45,46), fish fed the LP diet exhibited significantly poorer growth performance and elevated abdominal fat deposition compared to fish fed the NP diet. Serum phosphorus levels were markedly reduced in fish fed the LP diet, while alkaline phosphatase (ALP) activity showed a significant elevation, a biochemical pattern aligning with observations in phosphorus-deficient teleosts (47)(48)(49). This profile reflects enhanced osteoblastic activity under phosphorus restriction, as previously documented in mammalian models (50). Molecular analysis revealed a significant upregulation of intestinal sodium-phosphate cotransporter genes phospholipid synthesis, while adipose tissue serves as the primary site for lipid storage and mobilization (55)(56)(57). Phospholipid-bound phosphorus plays critical roles in biological membrane architecture, functional maintenance, and metabolic regulation (58,59). These amphipathic molecules, classified into phosphoglycerides (PG) and sphingomyelins (SM) based on backbone structures (60), are principally synthesized in the endoplasmic reticulum (ER) where they maintain ER structural integrity (61). Their compositional variations directly modulate membrane fluidity, protein-lipid interactions, and vesicular trafficking (62), with emerging evidence linking phospholipid metabolism to ER stress responses (63,64). Experimental data revealed systemic phospholipid depletion in fish fed the LP diet compared to fish fed the NP diet, with PG and SM levels significantly reduced in serum, liver, and abdominal fat tissue. Concurrently, ER stress related genes (grp78, perk, atf6, xbp1s) expresssion showed a significant upregulation in fish fed the LP diet versus fish fed the NP diet, suggesting phospholipid insufficiency-induced ER membrane destabilization.Interestingly, CHPT1 activity, catalyzing the terminal Kennedy pathway step crucial for phospholipid homeostasis (65), was significantly elevated in fish fed the LP diet.This elevation coincided with ER stress activation, mirroring mammalian models where xbp1-mediated pathways regulate chpt1 expression under ER stress (66). These observations suggest a potential compensatory mechanism wherein phospholipid biosynthesis is upregulated to mitigate LP diet-induced ER stress.Evidence in the literature showed that ER stress could induce disturbed lipid metabolism, which resulted in abnormal fat deposition in the organism (67).Furthermore, ER stress could promote the entry of srebp1c into the nucleus and activate the expression of lipid synthesis-related genes (fas and acc) expression (68).Xbp1 promoted the expression of lipid synthesis transcription factors pparγ (69), and atf6 activation could also promote TG synthesis by increasing fas and acc2 activity (70). In the current study, fish fed the LP diet exhibited higher serum TG level, increased expression of lipogenesis-related genes (fas, acc1, acc2), and key transcription factors of lipid metabolism (srebp-1 and pparγ), along with lower expression of lipolysis-related genes (pgc-1, atgl, and cpt-1) compared to fish fed the NP diet. As a result, the alterations in lipid metabolism observed in fish fed the LP diet are likely a consequence of ER stress.Moreover, the expression of inflammatory factors in the abdominal fat tissues of spotted seabass fed the LP diet was upregulated compared to those fed the NP diet.ER stress was closely related to the inflammatory response (71). PERK triggers the translocation of NF-κB into the nucleus, leading to the transcription of various inflammatory factors, such as il-1β and tnf-α (72). In this experiment, the observed decreased growth performance in spotted seabass fed the LP diet was probably due to ER stress resulting from impaired phospholipid synthesis, which subsequently triggered elevated inflammatory responses.Dietary phosphorus availability exerts profound influence on gut microbial ecosystems, as nutritional substrates directly shape microbial community structure (73). Phosphorus's essential role in microbial proliferation was firstly demonstrated in rumen microbiota studies (74,75), with subsequent research confirming its regulatory effects on fish intestinal microbiomes (76). Under the current experimental conditions, fish fed the LP diet exhibited reduced gut microbial diversity and ecological destabilization compared to fish fed the NP diet.Significant decreases in operational taxonomic unit richness and alpha diversity indices were observed in fish fed the LP diet compared to those fed the NP diet through microbial community analysis. Such microbial simplification has been epidemiologically linked to metabolic dysregulation and increased pathogenic colonization risks across vertebrate taxa (77,78). Multivariate analysis through principal coordinates (PCoA) confirmed distinct clustering patterns between the two groups, indicating phosphorus-dependent microbiome restructuring.At the phylum level, Proteobacteria and Firmicutes dominated intestinal communities in both groups, aligning with teleost gut microbiota baselines (79,80). However, genus-level shifts emerged under phosphorus restriction: fish fed the LP diet showed significant reduction in Lactococcus abundance and marked elevation of Plesiomonas compared to fish fed the NP diet. LEfSe biomarker analysis corroborated these compositional changes.The microbial profile alterations carry functional implications. Plesiomonas, identified as a potential opportunistic pathogen in aquatic species (81), may compromise intestinal barrier integrity and large-scale death of aquatic animals. As a Gram-negative bacterium, its surface contains lipopolysaccharide (LPS), which has been extensively documented to induce intestinal immune dysregulation (82,83).Conversely, Lactococcus lactis demonstrates probiotic properties through growth promotion and pathogen inhibition (84)(85)(86), with proven capacity to modulate intestinal immunity. This dual shift, pathogenic proliferation combined with probiotic depletion, likely disrupt the intestinal mucosal immunity of fish fed the LP diet and contribute to the growth retardation observed in fish fed the LP diet.The gut microbiota functions as a symbiotic metabolic interface, critically modulating host nutrient processing and homeostasis (87). Functional metagenomic prediction revealed significant depletion of lipid and phospholipid metabolic pathways in fish fed the LP diet compared to fish fed the NP diet, aligning with observed systemic lipid dysregulation. Concurrent reductions occurred in carbohydrate metabolism, amino acid cycling, energy transduction, and vitamin processing pathways -all essential for organismal growth and development.This microbial metabolic impairment corresponds with physiological observations, as optimal microbiota composition enhances host nutrient assimilation and metabolic efficiency (88,89). Notably, the reduced abundance of Lactococcus lactis in fish fed the LP diet versus fish fed the NP diet can compromise nutrient bioavailability, given this species' documented capacity to upregulate intestinal growth factors and nutrient absorption mechanisms (84,85). These collective microbial shifts likely contribute to the metabolic inefficiency and growth retardation observed under phosphorus restriction.Although this study identified phospholipid synthesis limitations caused by low phosphorus levels, it still lacks in-depth exploration of specific aspects such as the exact affected types of phospholipids and the precise mechanisms triggering endoplasmic reticulum (ER) stress. Future experiments will further investigate the specific phospholipid changes induced by low phosphorus and the regulated mechanisms in ER. In this study (Fig. 8), LP led to the decreased content of phospholipid in spotted seabass, which in turn induced ER stress, disturbed lipid metabolism and inflammatory response. Additionally, the LP diet resulted in reduced microbial diversity and modifications in the gut microbiota composition, thereby compromising intestinal immune competence. These negative changes likely contributed to the poorer growth and higher abdominal fat percentage observed in spotted seabass fed the LP diet. Gene nomenclature and inflammatory factor details are provided in Supplementary Table S2.Data represent mean ± SEM (n=9/group) with asterisks indicating significance (*P<0.05, **P<0.01, ***P<0.001) determined by two-tailed t-tests. Fig. 8. LP led to decreased content of phospholipid, ER stress, inflammatory responses and disruption of lipid metabolism as well as gut microbiota. These negative effects contributed to poorer growth and higher percentage of abdominal fat in spotted seabass fed the LP diet.