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Article

TLR2/TLR5 Signaling and Gut Microbiota Mediate Soybean-Meal-Induced Enteritis and Declined Growth and Antioxidant Capabilities in Large Yellow Croaker (Larimichthys crocea)

by
Lei Zheng
1,
Chao Zeng
2,
Wanqin Zhu
2,
Jiaonan Zhang
3,
Lei Wang
4,
Jianchun Shao
2,4,* and
Wei Zhao
2,4,*
1
Applied Technology Engineering Center of Fujian Provincial Higher Education for Marine Resource Protection and Ecological Governance, Xiamen Ocean Vocational College, Xiamen 361012, China
2
State Key Laboratory of Mariculture Breeding, Key Laboratory of Marine Biotechnology of Fujian Province, College of Marine Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China
3
Fujian Province Key Laboratory of Special Aquatic Formula Feed, Fujian Tianma Science and Technology Group Co., Ltd., Fuzhou 350300, China
4
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
*
Authors to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2024, 12(11), 2016; https://doi.org/10.3390/jmse12112016
Submission received: 18 September 2024 / Revised: 7 November 2024 / Accepted: 7 November 2024 / Published: 8 November 2024
(This article belongs to the Special Issue Nutrition and Physiology of Marine Fish)
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Abstract

:
Soybean meal, renowned for its high yield, cost efficiency, and protein richness, serves as a pivotal plant-based alternative to fish meal. However, high soybean meal inclusion in Larimichthys crocea diets is linked to enteritis and oxidative damage, with unknown mechanisms. Our study aims to elucidate the molecular basis of soybean-meal-induced enteritis and its impact on intestinal microbiota in L. crocea. To this end, four isonitrogenous and isolipidic diets with varying soybean meal levels (0% FM, 15% SBM15, 30% SBM30, and 45% SBM45) were administered to L. crocea for 8 weeks. The results indicated that the SBM30 and SBM45 treatments significantly hindered fish growth, digestive efficiency, and protein utilization. Furthermore, high soybean meal levels (SBM30 and SBM45) activated intestinal Toll-like receptors (TLR2A, TLR2B, TLR5, and TLR22), stimulating C-Rel and mTOR protein expression and elevating ERK phosphorylation. This led to increased pro-inflammatory cytokine production (IL-1β, IL-6, and TNF-α) and decreased anti-inflammatory cytokine expression (IL-4/13A, IL-4/13B, and TGF-β), suggesting a potential signaling pathway for soybean-meal-induced enteritis. Furthermore, enteritis induced by high soybean meal levels led to oxidative damage, evident from increased MDA levels and decreased antioxidant enzyme activities (SOD and CAT). The SBM30 and SBM45 treatments increased Firmicutes and Bacteroidetes abundance in fish gut microbiota, while Proteobacteria abundance decreased. This microbiota shift may enhance soybean meal nutrient utilization, yet high soybean meal concentrations still impair growth. A soybean-meal-rich diet promotes harmful bacteria like Rhodococcus and depletes probiotics like Ralstonia, increasing disease risks. L. crocea has limited tolerance for soybean meal, necessitating advanced processing to mitigate anti-nutritional factors. Ultimately, exploring alternative protein sources beyond soybean meal for fish meal replacement is optimal for L. crocea.

1. Introduction

In 2022, the worldwide production of aquaculture attained a milestone of 130.9 million tons, marking an unprecedented high that constituted 59% of the combined output from both fisheries and aquaculture on a global scale [1]. Notably, aquatic products sourced from aquaculture have gained significant prominence in ensuring global food security and augmenting nutritional supply, serving as a vital source of indispensable nutrients for human consumption, including proteins, omega-3 fatty acids, minerals, and vitamins. The rapid development and future expansion trends in aquaculture are poised to inevitably increase the demand for aquafeed and ingredients, particularly fish meal and fish oil. However, the decline in wild fish catches due to the El Niño phenomenon has further reduced the availability of the fish meal and fish oil required for aquaculture, resulting in a sharp increase in prices. Therefore, identifying suitable protein sources as alternatives to fish meal for the sustainable development of the aquaculture industry has become an urgent issue that needs to be addressed in aquaculture. To date, an array of plant-based protein sources, encompassing soybean meal, peanut meal, rapeseed meal, and cottonseed meal, have been extensively employed as primary substitutes for fish meal in aquafeeds, attributed to their favorable attributes such as substantial yield, cost-effectiveness, high protein concentration, and widespread accessibility [2,3,4]. However, the incorporation of these plant protein sources into aquatic feeds is limited by several challenges, including their inferior palatability, amino acid imbalances, and the presence of anti-nutritional compounds [5,6]. Prior studies have revealed that the excessive utilization of plant protein in feed inhibited fish growth and induced oxidative stress, enteritis, and immunosuppression [7,8,9]. Furthermore, the tolerance level towards plant protein varies among different fish species.
The large yellow croaker (Larimichthys crocea) stands out as the most prolific marine fish species in aquaculture in China, achieving an impressive yield of 257,700 tons in 2022. Its delicious flavor and rich content of protein and unsaturated fatty acids have earned it immense popularity among consumers. A previous investigation revealed that substituting soybean meal for more than 30% of fish meal, while fish meal was included at a 35% level, markedly reduced growth rates, antioxidant capabilities, and digestive efficacy in L. crocea fish weighing 11.78 g initially and also induced enteritis and liver metabolic disturbances [2]. The study conducted by Zhu et al. [10] also documented analogous findings, revealing that the substitution of 60% soybean meal for fish meal, while maintaining a 45% fish meal inclusion level, resulted in growth retardation and the development of enteritis in L. crocea with an initial body weight of 18.20 g. However, none of these studies have evaluated the impacts of soybean meal substitution for fish meal on the intestinal microbiota of L. crocea. Furthermore, the influence of substituting soybean meal for fish meal on enteritis in L. crocea, as well as the intricate signal transduction mechanisms behind it, still remains unclear. Previous studies indicated that variations in dietary protein sources can modify the gut microbiota of fish, and these alterations have, on occasion, been associated with subsequent declines in growth, health, and immunity [5,8,11]. Juvenile L. crocea have stringent nutritional requirements for feed, and there is a generally positive correlation between the size and survival rate of juvenile fish. Therefore, an appropriate feed formulation is beneficial for juvenile L. crocea to achieve optimal growth and health while also satisfying the market’s demand for the sustainability of L. crocea. The primary objective of this research is to determine the optimal level of soybean meal inclusion in the diet of L. crocea, with the evaluation focusing on growth performance, antioxidant capabilities, inflammatory reaction, and intestinal flora composition as key parameters. Additionally, we have conducted a preliminary screening of the recognition receptors associated with soybean-meal-induced enteritis in L. crocea. The results of this study contribute to elucidating the signal transduction mechanisms underlying soybean-meal-induced enteritis, thereby offering valuable insights and a practical reference framework for optimizing the formulation of commercial feeds tailored specifically for this species.

2. Materials and Methods

2.1. Experimental Diets

This study formulated four isonitrogenous and isolipidic diets in accordance with the prescribed recipe (Table 1). The experimental diet formulations were designed based on commercial feeds, tailored to fulfill the nutritional requirements of juvenile L. crocea, encompassing both vitamin and mineral necessities. The FM diet served as the control, utilizing 45% fish meal as its base without any soybean meal addition. Conversely, the SBM15, SBM30, and SBM45 diets incorporated 15%, 30%, and 45% soybean meal, respectively, intended to partially substitute for fish meal. Initially, all dry ingredients underwent grinding and filtration through a 200 μm sieve. Following this, each ingredient was accurately measured as per the recipe and thoroughly combined. Further, fat sources comprising fish oil, soybean oil, and soy lecithin, along with water, were integrated into the premixed blend and thoroughly agitated. The final product, a 2 mm diameter puffed pellets, was dried to a moisture content below 10% in a ventilated oven maintained at 40 °C and subsequently stored at −20 °C for preservation.

2.2. Experimental Fish and Rearing Conditions

Juvenile L. crocea, procured from Fufa Aquatic Products Co., Ltd. (Ningde, China), underwent an eight-week feeding study on aquaculture fish rafts. Prior to experimentation, all fish were accommodated in cages (3 m × 3 m × 4 m) located on the rafts and fed with control group feed for a two-week period to facilitate their adaptation to the experimental conditions. Following this acclimation period, 640 robust and uniformly sized juvenile L. crocea, with an initial weight of 48.5 ± 1.7 g, were randomly distributed into 16 floating seawater cages (1 m × 1 m × 1.5 m), housing 40 fish each. Each experimental feed was then randomly assigned to four of these cages. During the experimental period, the fish were manually fed twice daily, at 6:00 and 18:00, until satiation. The water temperature naturally fluctuated within the range of 27 to 29 °C, and the salinity naturally ranged approximately between 31 and 34‰. Furthermore, the dissolved oxygen content in the water naturally remained above 6.0 mg/L throughout the entire duration of the experiment.

2.3. Sample Collection

Following the completion of the culture experiment, all L. crocea underwent a 24 h fasting period. Next, all fish in each cage were anesthetized with 100 mg/L eugenol for counting purposes, and their individual body weights were recorded to derive growth parameters. Six fish were selected from each cage for the collection of liver and intestinal samples. The liver samples were designated for the analysis of antioxidant enzyme activities, whereas the intestinal samples were intended for the assessment of digestive enzyme activities, gene expression, and protein expression. These samples were promptly snap-frozen in liquid nitrogen and stored at −80 °C for future analyses. Furthermore, intestinal samples from an additional three fish per cage were collected for 16S rDNA high-throughput sequencing. The selection of the previously mentioned samples adheres to the principle of randomness.

2.4. Antioxidant Parameters and Digestive Enzyme Activity

Approximately 0.2 g of liver or gut was measured and homogenized with nine times its weight of pre-chilled normal saline, maintaining a temperature of 4 °C. Subsequently, the mixture was centrifuged at 2000 rpm for 30 min at 4 °C. Once centrifuged, the resulting supernatant was collected for further evaluation of antioxidant parameters and digestive enzyme activities. Utilizing dedicated commercial kits, the activities of superoxide dismutase (SOD) and catalase (CAT) enzymes, in addition to malondialdehyde (MDA) levels in the liver, were accurately determined following the manufacturer’s guidelines. The MDA content was precisely quantified employing the thiobarbituric acid (TBA) method. Specifically, one unit of SOD is characterized as the enzymatic amount that elicits 50% inhibition in color formation during a spectrophotometric assessment at 550 nm. The CAT activity unit refers to the enzymatic quantity capable of catalyzing the breakdown of 1 μmol of H2O2 per minute. Similarly, using commercial detection kits, the enzymatic activities of chymotrypsin, lipase, and α-amylase in the gut were assessed and quantified following the manufacturer’s guidelines. Chymotrypsin activity was specifically measured at 37 °C with a 660 nm absorbance, defining one unit as the enzyme quantity that degrades protein to yield 1 μg of amino acids per milligram of tissue protein per minute. Similarly, α-amylase activity was defined by the enzyme amount capable of hydrolyzing 10 mg of starch per milligram of protein, measured at 37 °C with a 660 nm absorbance. The lipase activity, on the other hand, was determined through the rate of methylresorcinol formation at 580 nm absorbance under 37 °C conditions, directly correlating to the concentration of the catalytic lipase in the supernatant. The reagents employed for measuring antioxidant indices and digestive enzyme activities were sourced from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The protein content in liver and intestinal supernatants was determined using a Bradford protein assay kit (Beyotime Biotechnology, Shanghai, China).

2.5. Quantitative Real-Time PCR (qRT-PCR)

Utilizing a Tissue RNA Extraction Kit (LS1040, Promega Biotech, Shanghai, China), total RNA was isolated from intestinal tissue, adhering strictly to the detailed instructions in the accompanying manual. The subsequent evaluation of the extracted total RNA’s quantity and quality was performed via a Nucleic Acid Protein Detector (NP80, IMPLEN, Munich, Germany) and 1% agarose gel electrophoresis, respectively. Upon confirmation of satisfactory RNA quality, reverse transcription into cDNA was carried out using a Reverse Transcription Kit (LS2050, Promega Biotech, Shanghai, China), following the manufacturer’s guidelines. The qRT-PCR assay was executed in line with the methodology detailed in our prior report by Zhao et al. [12]. The primer sequences employed for the qRT-PCR analysis are specified in Table 2. The expression levels of the target genes were determined using the 2−ΔΔCt method, with normalization to the housekeeping gene β-actin to ensure consistency and comparability across samples.

2.6. Western Blot

To gain insights into the signaling transduction mechanism underlying soybean-meal-induced enteritis in L. crocea, we explored the protein levels of C-Rel, along with the phosphorylation status of extracellular signal-regulated kinase (ERK) and mTOR. Proteins were extracted from intestinal samples employing well-established protocols, followed by quantification and Western Blot analysis, as outlined by Zhu et al. [10]. The primary and secondary antibodies used in this study adhered to the sources and preparation methods detailed in a previous report by Zhu et al. [10]. The intensities of protein bands were quantified using ImageJ software (V 1.8.0) and normalized to the G0 treatment, expressed as relative density.

2.7. Detection of the Gut Microbiota

In this study, bacterial DNA was extracted from gut samples collected in individual cages using a soil DNA extraction kit from Omega Bio-tek (Norcross, GA, USA), adhering strictly to the manufacturer’s prescribed protocol. The quantitative assessment of the extracted DNA’s concentration and purity was conducted using a Nucleic Acid Protein Detector (NP80, IMPLEN, Germany), and the integrity was further validated through 1% agarose gel electrophoresis.
Subsequently, the V3–V4 hypervariable segment of the bacterial 16S rRNA gene underwent amplification via the polymerase chain reaction (PCR), adhering strictly to the amplification protocol outlined by Zhang et al. [13]. The resulting PCR products were then purified in accordance with a previously described protocol outlined by Wang et al. [14]. Following purification, the DNA samples underwent paired-end sequencing on the Illumina MiSeq platform, with technical support provided by Majorbio bio-pharma Technology Co., Ltd. (Shanghai, China). The raw sequencing data obtained were subsequently processed and analyzed in accordance with the established and standardized procedures described by Wang et al. [14]. Using the Kruskal–Wallis H test and one-way analysis of variance (ANOVA), the abundance differences in gut microbiota between different treatments, in terms of phylum and genus classifications, were examined, with a significance threshold of p < 0.05.

2.8. Statistical Analysis

In this study, the weight gain rate (WGR), specific growth ratio (SGR), protein efficiency ratio (PER), feed conversion ratio (FCR), and feed efficiency (FE) were determined following established standard calculation equations, as reported by Zhang et al. [7].
The data are presented in a quantitative manner as the average with a standard deviation (SD) attached (mean ± SD). Before applying a one-way ANOVA analysis, we ensured the data conformed to a normal distribution using the Shapiro–Wilk test and examined their variance homogeneity through Levene’s test. Subsequently, we utilized Duncan’s test to compare the average values among the different treatments, setting a significance level of p < 0.05. SPSS 23.0 statistical software (SPSS, Chicago, IL, USA) was used for data analysis.

3. Results

3.1. Growth Performance

Fish consuming the SBM30 and SBM45 diets displayed a notably diminished final body weight (FBW), WGR, and SGR compared to those fed with the FM diet (p < 0.05). Nevertheless, there was no notable difference in these metrics between the FM and SBM15 treatments (p > 0.05). Furthermore, the PER of fish in the SBM45 treatment was significantly inferior to those in the FM, SBM15, and SBM30 treatments (p < 0.05). Fish consuming the SBM45 diet displayed a notably diminished FE and FCR compared to those fed with the FM and SBM15 diet (p < 0.05) (Table 3).

3.2. Digestive Enzyme Activities and Antioxidant Parameters

The alteration in the activities of digestive enzymes within the gut was observed as a consequence of the experimental diet administered (Table 4). The amylase activity remained comparable among the FM, SBM15, and SBM30 treatments, showing no significant difference (p > 0.05), whereas the SBM45 treatment exhibited a significantly lower amylase activity compared to the SBM15 treatment (p < 0.05). The SBM15 treatment yielded the highest lipase activity, which was markedly superior to the other treatment groups (p < 0.05). In addition, the lipase activity between the FM and SBM30 treatments remained comparable, demonstrating no significant difference (p > 0.05). However, the SBM45 treatment exhibited a lipase activity that was significantly lower than the other treatments (p < 0.05). The FM treatment exhibited the highest chymotrypsin activity, surpassing all other treatments significantly (p < 0.05). Simultaneously, the chymotrypsin activity in the SBM45 treatment was conspicuously inferior to that observed in the SBM15 and SBM30 treatments (p < 0.05).
The hepatic antioxidant parameters exhibited variations in response to the administered experimental diet (Table 5). The MDA content in soybean meal treatments markedly exceeded that of the FM treatment (p < 0.05). Notably, the MDA content in the SBM45 treatment stood out as significantly higher compared to the SBM15 and SBM30 treatments (p < 0.05). In contrast, the CAT activity in soybean meal treatments exhibited a significant decrement compared to the FM treatment (p < 0.05), attaining its nadir in the SBM45 treatment (p < 0.05). Analogously, the SOD activity exhibited significantly lower levels in the SBM30 and SBM45 treatments compared to the FM and SBM15 treatments (p < 0.05).

3.3. Protein and mRNA Expression Levels of Inflammation-Related Factors

The utilization of soybean meal as a dietary component resulted in a significant boost in the mRNA levels of Toll-like receptors (TLRs) (p < 0.05), specifically TLR2A, TLR2B, TLR5B, and TLR22 (Figure 1A). These elevations exhibited a positive correlation with the rising concentration of soybean meal in the diet. Specifically, the TLR2B, TLR5B, and TLR22 expression levels were significantly augmented in the SBM30 and SBM45 treatments compared to the FM treatment (p < 0.05). Analogously, the TLR2A expression level showed a substantial upregulation in the SBM45 treatment, surpassing all other treatment groups (p < 0.05). However, the TLR1 expression level remained statistically unchanged across all treatment groups (p > 0.05).
Similarly, soybean meal treatments led to a marked upregulation in the expression levels of pro-inflammatory cytokines (p < 0.05), particularly Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Tumor Necrosis Factor alpha (TNF-α), demonstrating a positive correlation with the elevated soybean meal content in the diet (Figure 1B). Specifically, the TNF-α expression level was significantly boosted in the soybean meal treatments compared to the FM treatment (p < 0.05). Furthermore, the SBM30 and SBM45 treatments exhibited significantly higher levels of IL-6 than the FM treatment (p < 0.05). Likewise, the IL-1β expression level was notably higher in the SBM45 treatment compared to the FM treatment (p < 0.05).
The expression pattern of anti-inflammatory cytokines exhibited a completely opposite trend compared to that of pro-inflammatory cytokines. The expression levels of anti-inflammatory cytokines, including Interleukin-4/13A (IL-4/13A), Interleukin-4/13B (IL-4/13B), and Transforming Growth Factor-beta (TGF-β), were significantly reduced in the soybean meal treatment (p < 0.05) (Figure 1C). In contrast to the FM treatment, the TGF-β expression levels were notably decreased in the SBM30 and SBM45 treatments (p < 0.05). Among these treatments, the SBM45 treatment demonstrated a marked decrement in IL-4/13B expression, significantly lower than the FM treatment (p < 0.05). Additionally, soybean meal treatments led to a substantial downregulation of IL-4/13A expression, which was significantly lower than the FM treatment (p < 0.05).
Upon comparison with the FM treatment, the protein level of Recombinant Human C-REL (C-Rel) exhibited a notable boost in the SBM45 treatment (p < 0.05), whereas a significant decrease was observed in the SBM15 treatment (p < 0.05) (Figure 2A,B). Similarly, when assessed against the FM treatment, the SBM30 and SBM45 treatments displayed a significant elevation in the phosphorylation level of extracellular-regulated protein kinases (ERKs) (p < 0.05), whereas the SBM15 treatment showed a marked decrease in the same parameter (p < 0.05) (Figure 2A,D). Furthermore, the Mammalian Target of Rapamycin (mTOR) phosphorylation levels in the SBM30 and SBM45 treatments exhibited a marked decrease, significantly lower than those observed in the FM treatment (p < 0.05) (Figure 2A,C). However, in contrast to the FM treatment, the mTOR phosphorylation level in the SBM15 treatment showed a significant elevation (p < 0.05).

3.4. Gut Microbiota

The high-throughput sequencing process yielded raw sequencing data, which underwent rigorous quality filtering, ultimately generating 388,423 reads. Notably, the number of reads varied per sample, ranging from 6312 to 61,581. For this study, we evaluated the completeness of sample detection by assessing the coverage index of randomly selected amplicon sequences detected within the same sample. The results of our investigation indicated that the coverage index surpassed 0.999 for all treatment cohorts, demonstrating that the sequencing depth attained was adequate to encompass virtually all microbial species represented within the samples.
In this study, the ACE, Chao1, and Shannon indices were employed to assess the richness of species within the gut microbiota. The results revealed no discernible differences in the indices across the various treatment groups (p > 0.05) (Table 6). The soybean meal treatments did not significantly alter the α-diversity of the gut microbiota in L. crocea. However, at the phylum and genus levels, the soybean meal treatments did induce changes in the composition of certain bacterial communities within the intestine of L. crocea. Specifically, Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidota were prominently abundant across all four treatments, accounting for 50.58%, 19.73%, 12.66%, and 5.07%, respectively, at the phylum level. Based on these findings, it is reasonable to postulate that these bacterial phyla occupy a dominant position within the gut microbiota of the L. crocea (Figure 3A). A thorough comparative analysis of the microbial community composition at the phylum level unveiled a notable decrease in the abundance of Proteobacteria in the SBM30 and SBM45 treatments. These treatments exhibited statistically significant lower levels of Proteobacteria compared to both the FM and SBM15 treatments (p < 0.05), as depicted in Figure 3B. Conversely, the abundances of Firmicutes and Bacteroidota were observed to be significantly increased in the SBM30 and SBM45 treatments when compared to the FM and SBM15 treatments (p < 0.05) (Figure 3B). On the genus-specific level, Ralstonia and Rhodococcus emerge as the predominant bacterial genera within the gut microbiota of L. crocea, exhibiting relative abundances of 40.63% and 12.76% accordingly (Figure 4A). Further, upon a comparative analysis of the genus-level composition of the microbial community, it became evident that the relative presence of Ralstonia in the SBM30 and SBM45 treatments was conspicuously lower compared to that observed in the FM and SBM15 treatments (p < 0.05). In contrast, Rhodococcus displayed a markedly elevated abundance in the SBM30 and SBM45 treatments, significantly surpassing that in the FM and SBM15 treatments (p < 0.05) (Figure 4B).

4. Discussion

The existence of anti-nutritional factors in soybean meal has been shown to attenuate the appetite of fish and hinder their ability to digest and assimilate nutrients, culminating in a decrement in feed utilization efficiency and subsequent deterioration in the growth performance of fish. Furthermore, the lack of certain crucial essential amino acids emerges as another factor contributing to the reduced growth performance and decreased feed utilization efficiency observed in fish fed diets containing elevated levels of soybean meal [15]. This phenomenon has been corroborated in studies focusing on L. crocea [2], Paralichthys olivaceus [15], and Scophthalmus maximus [16]. The present study yielded analogous findings, demonstrating that the incorporation of soybean meal at concentrations of 30% or higher markedly impedes the growth performance, FE, and PER of L. crocea. Nonetheless, the incorporation of 15% soybean meal in the diet exhibited comparable growth performance, FE, and PER in L. crocea to that of the FM treatment, aligning with previous research findings [2]. Undeniably, an elevated level of soybean meal impairs the FE of L. crocea, exhibiting a profound association with the deterioration in growth performance. The FE is predominantly contingent upon the activity of digestive enzymes present in the gut of fish [17]. The findings of this study revealed that elevated soybean meal levels, specifically 30% and 45%, led to a decrease in the enzymatic activities of α-amylase, lipase, and chymotrypsin, aligning with the observed patterns in the FE. A study conducted by Wang et al. [2] revealed a significant reduction in the enzymatic activity of α-amylase and trypsin in the gut of L. crocea when the dietary soybean meal level exceeded 15.5%. In this study, the initial body weight of the experimental fish was approximately 50 g, contrasting with approximately 10 g in the research conducted by Wang et al. [2]. This finding indicates that, for L. crocea, individuals with a larger body size exhibit a higher tolerance to soybean meal compared to those of a smaller size. The anti-nutritional factors in soybean meal, such as soybean globulin and β-conglycinin, have been found to elicit intestinal inflammation in aquatic animals. This inflammatory response impairs digestive enzyme activity in the gut, thereby compromising the digestion and utilization of feed, ultimately resulting in the inhibition of fish growth [18,19,20]. The results of this study suggest that incorporating soybean meal in excess of 30% in the diet impairs intestinal digestive enzyme activities, leading to a decline in feed efficiency (FE), which ultimately manifests as a notable decrement in the growth performance of L. crocea.
Inflammation holds a crucial position in the immune system of the body, functioning as a defensive shield against the penetration of pathogens, tissue harm, and alien substances. This process entails the synchronized activation of diverse cellular components, particularly white blood cells, macrophages, and lymphocytes, which secrete inflammatory factors to trigger and amplify the inflammatory response. Inflammatory modulators, including pro-inflammatory cytokines like IL-1β, IL-6, and TNF-α, hold a fundamental role in orchestrating the inflammatory cascade. They facilitate the mobilization of additional immune cells to inflamed areas, intensify the immune response, and ultimately neutralize the inflammation’s origin. Conversely, anti-inflammatory cytokines, exemplified by IL-4, IL-10, and TGF-β, work to mitigate the inflammatory response. Released by regulatory T cells and additional immune cells, they safeguard tissues from excessive damage and aid in the resolution of inflammation. These cytokines with anti-inflammatory properties also act to suppress the production of cytokines that promote inflammation and limit the migration of immune cells to areas of inflammation. It is undeniable that the maintenance of a balance between pro-inflammatory and anti-inflammatory cytokines is crucial for preserving immunological homeostasis in fish. Any disturbance in this delicate balance can trigger persistent inflammatory reactions, which subsequently elevate oxidative stress and compromise the integrity of the immune system. This study revealed that soybean meal supplementation notably augmented the mRNA levels of pro-inflammatory cytokines while concurrently inhibiting anti-inflammatory cytokines, particularly at supplementation levels of 30% and 45%. The findings align with previous research conducted on L. crocea [2], S. maximus, and Danio rerio [21]. The inclusion of anti-nutritional agents, notably glycinin and β-conglycinin, in soybean meal can contribute to intestinal mucosal damage [22]. This damage, in turn, stimulates an overproduction of pro-inflammatory cytokines. Concurrently, the expression of anti-inflammatory cytokines is downregulated, hindering their ability to adequately mitigate inflammatory responses in the intestine, thereby intensifying the progression of enteritis. The mTOR signaling pathway assumes a pivotal regulatory function in inflammatory processes [23]. Upon activation, mTOR enhances the transcription of anti-inflammatory cytokines [24]. Prior investigations have revealed that β-conglycin triggers mTOR inactivation, subsequently suppressing the expression level of IL-10 and TGF-β in Ctenopharyngodon idella [25]. This study yielded comparable outcomes, revealing that treatment with high concentrations of soybean meal (30% and 45%) notably reduced the phosphorylation level of mTOR, potentially leading to a further suppression of anti-inflammatory cytokine expression (IL-4 and TGF-β). Notably, the mTOR phosphorylation level exhibited a marked elevation in the low-level soybean meal (15%), potentially suggesting an underlying immune mechanism employed by L. crocea to mitigate intestinal injury and enteritis triggered by soybean meal. Specifically, this lower soybean meal concentration appears to activate the immune response of the fish, effectively countering the release of pro-inflammatory cytokines and striving to maintain a balanced immune homeostasis within the organism. The present study reveals that the dietary inclusion of 15% soybean meal has the potential to induce enteritis in L. crocea, with the severity of the condition exhibiting a direct correlation to the increasing level of soybean meal supplementation.
Toll-like receptors (TLRs) occupy a central position in inflammatory processes, modulating the release of inflammatory factors and shaping the evolution of inflammatory responses via mechanisms encompassing pathogen recognition, immune response activation, and immune response regulation. Consequently, the investigation of Toll-like receptors holds paramount significance in elucidating the underlying mechanisms of soybean-meal-induced enteritis, thereby paving the way for the development of innovative anti-inflammatory therapeutics and treatment strategies. In this study, we delved into the potential influence of soybean meal on TLR expression in the gut of L. crocea. Our findings revealed that the addition of soybean meal increased the mRNA levels of TLR2A, TLR2B, TLR5B, and TLR22 to different extents, hinting that these four TLRs may serve as key mediators in the development of soybean-meal-induced enteritis in L. crocea. Furthermore, prior studies have shown that TLRs have the capacity to initiate the activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, ultimately regulating the expression of downstream cytokines, both pro-inflammatory and anti-inflammatory [26]. The present investigation demonstrated that the inclusion of high percentages of soybean meal, specifically 30% and 45%, led to a significant upregulation of the expression level of C-rel protein, which functions as a downstream effector in the NF-κB signaling cascade. Concurrently, it also intensified the phosphorylation status of the ERK protein, a downstream component of the MAPK signaling cascade. These key discoveries point towards the potential of soybean meal in activating TLRs, subsequently triggering the MAPK/NF-κB signaling axis and ultimately culminating in the augmented expression of pro-inflammatory cytokines. The findings of this study unveil the underlying mechanism behind soybean-meal-induced enteritis in L. crocea, offering valuable insights for the creation of innovative anti-inflammatory medications and therapeutic approaches.
The occurrence of intestinal inflammation has the potential to trigger an excessive generation of reactive oxygen species (ROS), ultimately culminating in oxidative damage (Turan and Mahmood). SOD and CAT play a crucial role in defending the body against oxidative damage: SOD catalyzes the conversion of superoxide anions into hydrogen peroxide and oxygen, while CAT decomposes hydrogen peroxide into water and oxygen [27]. SOD helps mitigate cellular injury by scavenging free radicals and protecting cells from the harmful effects of superoxide anions, while CAT eliminates the toxicity of hydrogen peroxide and acts as a key enzyme in the antioxidant defense system. Together, SOD and CAT form a comprehensive antioxidant defense network that transforms ROS into harmless substances, thus safeguarding the normal physiological functions of organisms and preventing oxidative damage. MDA arises as a consequence of peroxidation, stemming from the assault of free radicals on cellular lipids. The accumulation of MDA serves as a marker for the significant presence of oxidative damage within the organism, thus frequently serving as a crucial indicator in assessing oxidative stress levels. Certainly, previous studies have conclusively shown that soybean meal has the potential to trigger oxidative stress in fish, which in turn leads to a decrease in the activity of antioxidant enzymes and a marked increase in MDA concentrations [2,3]. The current research further validates the conclusion that high concentrations of soybean meal (30% and 45%) suppress the activities of SOD and CAT, concurrent with an elevation in MDA levels. Moreover, our findings reveal that even a 15% inclusion of soybean meal significantly raises MDA levels, indicating a positive correlation between MDA levels and the dietary soybean meal concentration. The key factors that contribute to the diminished activity of antioxidant enzymes in fish are likely the anti-nutritional components found in soybean meal, such as glycinin, β-conglycinin, phytic acid, and tannins. Studies have shown that these components have the potential to compromise the antioxidant defenses of fish, resulting in oxidative harm to their intestinal systems [25,28]. This study reveals a rather low tolerance of the L. crocea to soybean meal, with even a 15% inclusion leading to oxidative stress. Consequently, the enhanced processing of soybean meal to minimize anti-nutritional factors is imperative to potentially elevate its inclusion rate in the diet of fish.
The gut microbiota holds vital importance in numerous aspects of fish physiology, including nutrition absorption, immune system function, intestinal integrity, and overall well-being [29,30]. Consequently, gaining insight into and preserving the equilibrium of this microbial community is essential for fostering fish health and guarding against the onset of illnesses [31]. However, in previous studies, the influence of soybean meal content on the gut microbiota of L. crocea was not explored. In this study, the inclusion of soybean meal did not significantly modify indices such as Shannon, Chao1, and ACE, implying a negligible effect on the diversity and abundance of the gut microbiota. This finding aligns with previous investigations conducted on Micropterus salmoides [32] and S. maximus [33]. In the current investigation, Firmicutes and Proteobacteria were determined to be the predominant phylum-level taxa in cultured L. crocea, affirming findings from prior experimental studies [34,35]. This study revealed an augmentation in the abundance of Firmicutes and Bacteroidetes within the gut microbiota of L. crocea following an elevation in soybean meal concentrations (30% and 45%), accompanied by a notable decrease in the abundance of Proteobacteria. In the study conducted by Zhang et al. [33] on S. maximus, analogous findings were observed, demonstrating an increase in the abundance of Bacteroidetes as the level of extruded full-fat soybeans escalated. Firmicutes and Bacteroidetes are two major phyla in gut microbiota, which are pivotal in sustaining gut well-being, bolstering nutrient assimilation, and modulating the immune system [36]. Among Firmicutes, certain species are adept at degrading complex carbohydrates and cellulose, whereas Bacteroidetes specialize in the degradation of diverse polysaccharides and proteins [37,38]. Hence, with an elevated soybean meal content in the diet, the proliferation of these two microbial communities is likely to enhance the utilization of nutrients in soybean meal by L. crocea. This augmentation could be indicative of an adaptive mechanism adopted by the gut microbiota of L. crocea in response to alterations in their dietary composition. Despite the gut microbiota exhibiting adaptive changes to the soybean meal feed, this study revealed that a high soybean meal level significantly hampered the growth of L. crocea. This indicates that the adaptive changes in the microbiota are not enough to compensate for the decline in growth performance caused by enteritis and oxidative damage. This study revealed that, in addition, a diet rich in soybean meal elevated the presence of Rhodococcus while diminishing the abundance of Ralstonia. Ralstonia, capable of generating butyrate in the gut, possess the ability to regulate the metabolic pathways of short-chain fatty acids, boost energy metabolism, and effectively safeguard against harmful external influences [39]. A significant number of species belonging to the Rhodococcus genus are regarded as potential pathogens that can threaten the health of aquatic animals under certain conditions [40]. After thorough analysis of the current research, we have determined that a diet high in soybean meal enhances the presence of opportunistic pathogens while diminishing the population of probiotics, subsequently elevating the chances of disease infection in L. crocea.

5. Conclusions

Elevated levels of soybean meal (30% and 45%) have adverse effects on the growth, digestion, and feed utilization of L. crocea. Furthermore, even a 15% soybean meal inclusion can provoke enteritis and oxidative stress. The underlying mechanism for soybean-meal-induced enteritis in fish involves the activation of TLRs, subsequently stimulating the MAPK/NF-κB signaling pathway, resulting in the heightened production of pro-inflammatory cytokines. Elevated soybean meal levels induce alterations in the gut microbiota of L. crocea, marked by an increase in opportunistic pathogens and a decrease in probiotics. The gut microbiota of L. crocea undergoes adaptive adjustments in response to high-soybean-meal diets, potentially enhancing the utilization of its nutritional components. However, despite these adaptive changes, the growth of L. crocea is notably hampered by high-soybean-meal diets, presumably due to the unrecoverable setback in growth stemming from severe enteritis and oxidative stress. Fundamentally, the L. crocea demonstrates limited tolerance towards soybean meal, necessitating the implementation of advanced processing techniques to alleviate the presence of anti-nutritional factors, particularly if there is an intention to enhance its inclusion in their dietary formulations. Undeniably, seeking alternative protein sources besides soybean meal to replace fish meal may be a superior option for L. crocea.

Author Contributions

The design of this research was undertaken by W.Z. (Wei Zhao), J.S., L.W., J.Z., and L.Z. W.Z. (Wanqin Zhu) was responsible for the rearing work, while L.Z., W.Z. (Wanqin Zhu) and C.Z. collaborated in collecting experimental samples and measuring key parameters. W.Z. (Wei Zhao) analyzed the data and penned this manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by grants from the National Natural Science Foundation of China (32373148), the Fujian Science and Technology Department (2024J09027, 2022NZ033021), the Applied Technology Engineering Center of Fujian Provincial Higher Education for Marine Resource Protection and Ecological Governance (2024-05), and the Fujian Provincial Youth Teacher Education and Scientific Research Project (Science and Technology) (NO. JAT220625).

Institutional Review Board Statement

The study protocol and all experimental procedures were approved by the Animal Care and Use Committee of Fujian Agriculture and Forestry University (approval code: PZCASFAFU21007).

Informed Consent Statement

Not applicable.

Data Availability Statement

Upon receipt of a reasonable request, the corresponding author is able to provide access to all data presented in this article.

Acknowledgments

The authors express gratitude to all the participants for devoting their time to this study. Additionally, the authors acknowledge with sincere gratitude the contributions of Xuyin Lin, who provided invaluable support during the experimental procedures and in the preparation of this article.

Conflicts of Interest

Author Jiaonan Zhang was employed by the Fujian Tianma Science and Technology Group Co., Ltd.. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Jmse 12 02016 g001
Figure 1. The mRNA expression levels of specific intestinal inflammation-related factors, including Toll-like receptor genes (A), pro-inflammatory cytokine genes (B), and anti-inflammatory cytokine genes (C), in large yellow croaker (Larimichthys crocea) fed with various experimental diets. Values are shown as mean ± SD (n = 4). Different lowercase letters indicate a significant difference (p < 0.05). Indicators without lowercase letters indicate no significant difference (p > 0.05).
Figure 1. The mRNA expression levels of specific intestinal inflammation-related factors, including Toll-like receptor genes (A), pro-inflammatory cytokine genes (B), and anti-inflammatory cytokine genes (C), in large yellow croaker (Larimichthys crocea) fed with various experimental diets. Values are shown as mean ± SD (n = 4). Different lowercase letters indicate a significant difference (p < 0.05). Indicators without lowercase letters indicate no significant difference (p > 0.05).
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Figure 2. Protein level of C-Rel and phosphorylation levels of ERK and mTOR in the gut of large yellow croaker (Larimichthys crocea) fed with experimental diets (A). The protein levels were quantified using ImageJ (B,C). The phosphorylation levels were quantified using ImageJ (D). Values are shown as mean ± SD (n = 4). Different lowercase letters indicate a significant difference (p < 0.05).
Figure 2. Protein level of C-Rel and phosphorylation levels of ERK and mTOR in the gut of large yellow croaker (Larimichthys crocea) fed with experimental diets (A). The protein levels were quantified using ImageJ (B,C). The phosphorylation levels were quantified using ImageJ (D). Values are shown as mean ± SD (n = 4). Different lowercase letters indicate a significant difference (p < 0.05).
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Figure 3. Composition of gut microflora at phylum level (A) and differentially abundant phyla (B) in large yellow croaker (Larimichthys crocea) fed experimental feeds. Values are shown as mean ± SD (n = 3). The red star represents a statistically significant difference (p < 0.05).
Figure 3. Composition of gut microflora at phylum level (A) and differentially abundant phyla (B) in large yellow croaker (Larimichthys crocea) fed experimental feeds. Values are shown as mean ± SD (n = 3). The red star represents a statistically significant difference (p < 0.05).
Jmse 12 02016 g003
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Figure 4. Composition of gut microflora at genus level (A) and differentially abundant genera (B) in large yellow croaker (Larimichthys crocea) fed experimental feeds. Values are shown as mean ± SD (n = 3). The red star represents a statistically significant difference (p < 0.05).
Figure 4. Composition of gut microflora at genus level (A) and differentially abundant genera (B) in large yellow croaker (Larimichthys crocea) fed experimental feeds. Values are shown as mean ± SD (n = 3). The red star represents a statistically significant difference (p < 0.05).
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Table 1. Composition and nutrient levels of the experimental diets (%DM basis).
Table 1. Composition and nutrient levels of the experimental diets (%DM basis).
Ingredient (%)FMSBM15SBM30SBM45
Fish meal45322518
Soybean meal a0153045
Wheat gluten15151515
Wheat flour31.2328.6419.3310.71
Fish oil2.233.094.154.79
Soybean oil1.541.131.231.07
Soy lecithin1.51.51.51.5
Ca(H2PO4)21.51.51.51.5
Vitamin premix b1111
Mineral premix c1111
Methionine00.080.170.25
Lysine00.060.120.18
Proximate analysis
Moisture9.379.659.249.81
Crude protein45.3444.8645.2244.76
Crude lipid10.7510.899.9510.24
Ash9.189.268.658.33
a The soybean meal containing 45% crude protein and 1.4% crude lipid was purchased from Dachang Feed Company, located in Fuzhou, China. b Vitamin premix (kg−1 of diet): vitamin A, 250,000 IU; riboflavin, 750 mg; pyridoxine-HCl, 400 mg; cyanocobalamin, 1 mg; thiamin, 250 mg; menadione, 250 mg; folic acid, 125 mg; biotin, 10 mg; a-tocopherol, 2.5 g; myo-inositol, 8000 mg; calcium pantothenate, 1250 mg; nicotinic acid, 2000 mg; choline chloride, 8000 mg; vitamin D3, 45,000 IU; vitamin C, 7000 mg. c Mineral premix (kg−1 of diet): ZnSO4·7H2O, 0.04 g; CaCO3, 37.9 g; KCl, 5.3 g; KI, 0.04 g; NaCl, 2.6 g; CuSO4·5H2O, 0.02 g; CoSO4·7H2O, 0.02 g; FeSO4·7H2O, 0.9 g; MnSO4·H2O, 0.03 g; MgSO4·7H2O, 3.5 g; Ca (HPO4)2·2H2O, 9.8 g.
Table 2. Primers used for qRT-PCR.
Table 2. Primers used for qRT-PCR.
Gene NamePrimer Sequence (5′-3′)Accession Number Efficiency
TLR1F-CTTTGTCAAGAGCGAGTGGT
R-GGTTCATCATGGCCTTCAGC		KF318376.1 98
TLR2AF-GTCCGACAACCTGCTGACTGA
R-CAGGTGGGTGAGTTTGGAGAG 		KKF22682.1 101
TLR2BF-ATGATGTGCTATGGCGAGGG
R-TCGGCAAACATGTGGTCACT 		KKF15865.1 97
TLR5AF-GGCACAGTGAGGAAAGGT
R-TAGCAAGCGTCCACATAC		XM_019267725.2 98
TLR22F-AGCACCGACTTCATCTGCTTTG
R-TGGTCTTCCTGCTCGCATAGATG		GU324977 102
IL-1βF-CAGCTGTTCTCAAGTATGTGGC
R-GTTGTAAATAGTGGGTGTGTCG		XM_010736551.3 98
IL-4/13AF-TGGTACTGCTGGTCAATCCG
R-TTTTGCCTTCAGCCAGATGT		KU885454 98
IL-4/13BF-AGTTCTTCTGTCGCGCTGAG
R-GCTATGTATGTGCGGTTGCTG		KU885453 98
IL-6F-GCTGTTCTCAAGTATGTGGCG
R-TGTTGTAAATAGTGGGTGTGTCG		XM_010734753.399
IL-10F-AGTCGGTTACTTTCTGTGGTG
R-TGTATGACGCAATATGGTCTG		XM_010738826.3102
TNF-αF-ACACCTCTCAGCCACAGGAT
R-CCGTGTCCCACTCCATAGTT		XM_010745990 102
TGF-βF-AGCAACCACCGTACATCCTG
R-AGGTATCCCGTTGGCTTGTG		XM_027280465.1 99
β-ActinF-GACCTGACAGACTACCTCATG
R-AGTTGAAGGTGGTCTCGTGGA		GU584189 98
Table 3. Growth performance of L. crocea fed with different experimental diets.
Table 3. Growth performance of L. crocea fed with different experimental diets.
FMSBM15SBM30SBM45
IBW (g)47.9 ± 1.948.6 ± 1.548.4 ± 1.648.7 ± 1.7
FBW (g)103.11 ± 2.54 a100.80 ± 4.88 ab93.53 ± 2.75 bc85.30 ± 6.65 c
WGR (%)112.60 ± 5.23 a107.84 ± 10.05 ab92.85 ± 5.66 bc75.88 ± 13.70 c
SGR (%/d)1.40 ± 0.05 a1.35 ± 0.09 ab1.22 ± 0.05 bc1.05 ± 0.15 c
PER1.28 ± 0.07 a1.38 ± 0.04 a1.22 ± 0.07 a1.14 ± 0.02 b
FE0.58 ± 0.03 a0.60 ± 0.02 a0.55 ± 0.03 ab0.51 ± 0.01 b
FCR1.49 ± 0.08 b1.46 ± 0.06 b1.55 ± 0.07 b1.64 ± 0.05 a
Values are means ± SD of four replicates. The superscript small letters in the same row mean a significant difference at p < 0.05.
Table 4. Digestive enzyme activities in the gut of L. crocea fed with different experimental diets.
Table 4. Digestive enzyme activities in the gut of L. crocea fed with different experimental diets.
FMSBM15SBM30SBM45
α-Amylase (U/mg protein)0.83 ± 0.06 ab1.03 ± 0.17 a0.90 ± 0.14 ab0.77 ± 0.04 b
Lipase (U/g protein)149.08 ± 13.45 b193.82 ± 8.00 a143.55 ± 9.28 b109.30 ± 8.32 c
Chymotrypsin (U/mg protein)2.50 ± 0.01 a1.81 ± 0.04 b1.76 ± 0.34 b1.39 ± 0.42 c
Values are means ± SD of four replicates. The superscript small letters in the same row mean a significant difference at p < 0.05.
Table 5. The antioxidant parameters in the liver of L. crocea fed with different experimental diets.
Table 5. The antioxidant parameters in the liver of L. crocea fed with different experimental diets.
FMSBM15SBM39SBM45
MDA (nmol/mg protein)2.96 ± 0.98 c4.85 ± 0.19 b5.32 ± 0.42 b9.27 ± 0.71 a
CAT (nmol/mg protein)268.50 ± 39.29 a185.91 ± 19.30 b149.17 ± 8.53 b109.56 ± 2.57 c
SOD (U/mg protein)76.67 ± 3.34 a77.76 ± 6.39 a60.59 ± 5.00 b54.76 ± 6.53 b
Values are means ± SD of four replicates. The superscript small letters in the same row mean a significant difference at p < 0.05.
Table 6. Alpha diversity index of the gut microbiota of fish fed the experimental diets.
Table 6. Alpha diversity index of the gut microbiota of fish fed the experimental diets.
EstimatorsFMSBM15SBM30SBM45
Ace487.86 ± 212.15537.52 ± 283.03418.12 ± 231.15231.8 ± 66.75
Chao1488.93 ± 213.18538.16 ± 283.49422.92 ± 230.65232.67 ± 66.91
Shannon2.37 ± 0.862.28 ± 1.143.84 ± 0.734.00 ± 0.57
Values are means ± SD of three replicates.
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Zheng, L.; Zeng, C.; Zhu, W.; Zhang, J.; Wang, L.; Shao, J.; Zhao, W. TLR2/TLR5 Signaling and Gut Microbiota Mediate Soybean-Meal-Induced Enteritis and Declined Growth and Antioxidant Capabilities in Large Yellow Croaker (Larimichthys crocea). J. Mar. Sci. Eng. 2024, 12, 2016. https://doi.org/10.3390/jmse12112016

AMA Style

Zheng L, Zeng C, Zhu W, Zhang J, Wang L, Shao J, Zhao W. TLR2/TLR5 Signaling and Gut Microbiota Mediate Soybean-Meal-Induced Enteritis and Declined Growth and Antioxidant Capabilities in Large Yellow Croaker (Larimichthys crocea). Journal of Marine Science and Engineering. 2024; 12(11):2016. https://doi.org/10.3390/jmse12112016

Chicago/Turabian Style

Zheng, Lei, Chao Zeng, Wanqin Zhu, Jiaonan Zhang, Lei Wang, Jianchun Shao, and Wei Zhao. 2024. "TLR2/TLR5 Signaling and Gut Microbiota Mediate Soybean-Meal-Induced Enteritis and Declined Growth and Antioxidant Capabilities in Large Yellow Croaker (Larimichthys crocea)" Journal of Marine Science and Engineering 12, no. 11: 2016. https://doi.org/10.3390/jmse12112016

APA Style

Zheng, L., Zeng, C., Zhu, W., Zhang, J., Wang, L., Shao, J., & Zhao, W. (2024). TLR2/TLR5 Signaling and Gut Microbiota Mediate Soybean-Meal-Induced Enteritis and Declined Growth and Antioxidant Capabilities in Large Yellow Croaker (Larimichthys crocea). Journal of Marine Science and Engineering, 12(11), 2016. https://doi.org/10.3390/jmse12112016

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