Commercial shrimp feed with an approximate diameter of 1.0 mm for L. vannamei without Axn supplementation was used as the basal diet. Axn (Bioalgo, Shandong, China) was supplemented into the basal diet at a dose of 0.1 g kg^–1, which was determined as the optimal dose in our pre-experiment. The feed formulation was shown in Table 1.

Juvenile E. carinicauda was obtained from a shrimp hatchery in Rizhao, China. During a week of acclimation, the animals were maintained in an indoor cement pool and fed with first hatched brine shrimp (Artemia salina) three times a day, and approximately half of the seawater in the pool was renewed once a day. A total of 180 healthy shrimps (0.06 ± 0.01 g) were randomly assigned to six tanks filled with 30 L sand-filtered seawater. Each treatment was randomly assigned to three replicated tanks with 30 shrimp each. Shrimp were fed three times a day at 07:00, 12:00, and 18:00 with a daily ration of approximately 5% body mass for 8 weeks. When approximately 50% of seawater in the tanks was renewed, the feces, uneaten feed, and exuviae were removed. Natural illumination was used during the feeding trial, and water quality was maintained at a salinity of 30 ± 1, temperature of 23 ± 0.5°C, pH of 8.2 ± 0.1, and dissolved oxygen of 7.4 ± 0.3 mg L^–1.

All the tanks were used to pool samples that split into two replicate groups of three tanks. The muscles of the two groups were taken after 8 weeks. At each of the two groups, three shrimps were taken from each tank (total = 18 shrimps) for transcriptome analysis and six shrimps were taken from each tank (total = 36 shrimps) for metabonomics analysis. Whole muscles samples were immediately flash frozen in liquid nitrogen, and stored at −80°C until further analysis.

Three muscles per treatment were weighed and homogenized in pre-chilled 0.86% saline solution (1:9, w/v) and centrifuged, and the supernatant was collected to measure total protein content, MDA content, superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and total antioxidant capacity (T-AOC). All biochemical parameters were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Differences in biochemical parameters were considered statistically significant at P < 0.05 using a t-test. Data are expressed as means ± SD (n = 3).

Final body weight (FBW), final body weight (g); WG, weight gain (%) = [(final body weight - initial body weight)/initial body weight] × 100.SGR, specific growth rate (%) = [(loge final body weight - loge initial body weight)/days] × 100.

Total RNA was isolated from muscle tissue using TRIzol reagent (Takara, Japan). The isolated RNA was quantified and qualified using a NanoPhotometer^® spectrophotometer (IMPLEN, Westlake Village, CA, United States) and Qubit^® RNA Assay Kit in Qubit^®2.0 Fluorometer (Life Technologies, Carlsbad, CA, United States), respectively. RNA integrity was detected using 1% agarose gels and an RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, United States). Sequencing libraries were constructed using the NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (New England Biolabs, Ipswich, MA, United States). All experiments were performed in accordance with the manufacturer’s protocol.

Nine differentially regulated mRNAs from the Illumina sequencing results were validated using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Primers were designed using Primer Premier 5 for qRT-PCR. Table 2 lists the primers used. mRNA expression was analyzed by qRT-PCR, and 18S rRNA was used as an internal control. The SYBR Premix Ex Taq on the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, United States) was used to perform qRT-PCR. The PCR program was as follows: 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 30 s. The fold change in the expression levels of the target genes was calculated using the relative quantitative method (2^–ΔΔCt).

Muscle samples (n = 6) from the two treatments were harvested and extracted for metabolomic analysis. The differentially expressed metabolites (DEMs) in the muscle tissues of the two treatments were analyzed using a gas chromatograph system coupled to a Pegasus HT time-of-flight mass spectrometer (GC-TOF-MS).

The resulting three-dimensional data comprising the peak number, sample name, and normalized peak area were inputted into the SIMCA 14.1 software package (V14.1, MKS Data Analytics Solutions, Umea, Sweden) and was used to perform principal component analysis (PCA) and orthogonal projections for latent structure-discriminant analysis (OPLS-DA). PCA revealed the distribution of original data. A higher level of group separation was obtained by supervised OPLS-DA to improve the understanding of the variables responsible for classification. For further model validation, sevenfold cross-validation was used to estimate the model’s robustness and predictive ability. Next, Student’s t-test (P < 0.05) combined with the first principal component of variable importance in projection (VIP) values (VIP > 1) was used to determine the species distribution models (SDMs) among the pairwise comparison groups. The Kyoto Encyclopedia of Genes and Genomes (KEGG)¹ was used to search for metabolic pathways. A free web-based tool, MetaboAnalyst,² was used to conduct pathway analysis, which uses high-quality KEGG metabolic pathways as the backend knowledge base.

To explore the influence of Axn on growth performances and the antioxidant capacity of E. carinicauda, the kits of MDA, T-AOC, GSH, CAT, and SOD were used to prove it. In the muscle, the shrimp in the control group had significantly higher MDA content than those fed with Axn (P < 0.05). The activities of T-AOC, GSH, CAT, and SOD in the control group were significantly lower than those in the Axn group (P < 0.05) (Figure 1).

Following a 56-day feeding trial period, the FBW of shrimp fed an Axn diet was significantly elevated as compared to shrimp fed a diet (P < 0.05; Table 3). Consistently, the control group exhibited lower weight gain (WG) and specific growth rate (SGR) values as compared to those of Axn-fed E. carinicauda.

A total of 1,852 differentially expressed genes (DEGs) were identified in the muscle of the Axn group compared with the control group, with 1,091 genes showing upregulated expression and 761 genes showing downregulated expression (Figure 2A). Raw data were deposited in the Short Read Archive (SRA) of the NCBI with accession numbers of SRX14060554, SRX14060555, SRX14060556, SRX14060557, SRX14060558, and SRX14060559.

Gene Ontology (GO) analysis was used to annotate these DEGs with terms under biological process, cellular component, and molecular function categories for understanding the biological significance of the DEGs. Most of the DEGs were assigned to cell part and membrane part for the cellular component category, the DEGs were mostly associated with cellular and metabolic processes for the biological process category, and most of the DEGs were categorized into binding and catalytic activities for the molecular function category (Figure 2B).

To identify the biochemical pathways influenced by Axn feeding, the KEGG database was used to perform pathway enrichment analysis on the identified DEGs. Of the pathways identified, the most commonly represented class was related to stress and included several subclasses: “Phagosome,” “Protein processing in endoplasmic reticulum,” “Pathogenic Escherichia coli infection,” “Antigen processing and presentation,” “Apoptosis,” “Estrogen signaling pathway,” “Gap junction” and “Protein export” (Figure 2C).

To investigate the metabolic changes in E. carinicauda in response to Axn feeding, an untargeted metabolomic analysis of muscle samples was performed using the UHPLC-Q-TOF-MS platform. A total of 354 negative and 750 positive ion peaks were extracted from the analysis. A total of 136 DEMs were identified in both metabolites, including 62 downregulated metabolites and 74 upregulated metabolites (Table 4). The established OPLS-DA model (model evaluation parameters: positive ion mode: R²Y = 0.98 cum, Q²Y = 0.64 cum; negative ion mode: R²Y = 0.98 cum, Q²Y = 0.59 cum) indicated that the model was stable and reliable (Figures 3A,B). Next, a permutation test was used to establish 200 OPLS-DA models in which the order of the categorical variables Y was changed randomly to obtain the R² and Q² values of the stochastic model (Figures 3C,D). From left to right, all Q² points were lower than the original red Q² points on the right, which indicated a robust and reliable model without overfitting. Thus, it is reliable and stable for the test data and instrument analysis system for the experiment.

To explore the metabolic pathways that might be affected by Axn feeding, KEGG pathway analysis was used to assign these DEMs to metabolic pathways. The pathway analysis results provided details of the changes in metabolic pathways related to Axn feeding. The most relevant pathways were identified based on a p-value < 0.05 and were “Carbohydrate digestion and absorption,” “Inositol phosphate metabolism,” “Valine, leucine and isoleucine biosynthesis,” “Tryptophan metabolism,” “Glutathione metabolism,” “Arginine biosynthesis,” “Biosynthesis of unsaturated fatty acids” (Figure 4).

Kyoto Encyclopedia of Genes and Genomes pathway analysis of genes and metabolomics was performed to determine correlations between the transcriptomic and metabolomic data (Figure 5). The analysis showed that the urea cycle, TCA cycle, amino acid metabolism, fatty acid metabolism, and apoptosis signaling pathways were affected by Axn feeding. These pathways are important components of metabolic pathways. Accordingly, the results showed that downregulation of citrate indicates vigorous metabolism of the TCA cycle. Similarly, it was observed that upregulation of most of the DEGs and DEMs was related to amino acid biosynthesis and fatty acid metabolism. Interestingly, the levels of fatty acids, such as arachidonic acid and palmitic acid, were downregulated while inosine was upregulated. These results indicate the importance of these metabolites in energy replenishment.

To further verify the results of the transcriptome-based quantitative analysis, qRT-PCR was performed. The mRNA transcription levels of nine genes, including six downregulated (cathepsin, eIF2α, Cyt-C, V-ATP, HSP90, and Bcl-XL) and three upregulated (Hsp70, c-jun, and Actin), were measured. The expression levels of the genes showed similar trends with the RNA-sequencing (RNA-seq) results, which indicates the reliability and accuracy of the RNA-seq analysis (Figure 6).

The results showed that Axn feeding triggered a response involving amino acid metabolism. According to the metabolome data, the levels of most amino acids (e.g., alanine, arginine, glutamic acid, leucine, isoleucine, lysine, aspartic acid, valine, serine, threonine, and phenylalanine) were significantly upregulated in the shrimp after Axn feeding. In addition, the RNA-seq data indicated that Axn feeding induced significant changes in the expression levels of amino acid metabolism-associated genes. Citrate, which is the first intermediate of the TCA cycle, and other TCA cycle intermediates, such as arginine and glutamic acid, are important precursors of α-ketoglutarate, acetyl-CoA, and succinyl-CoA (Wu et al., 2018). Other metabolites, such as isoleucine and leucine, participate in immunity, neurotransmission, protein synthesis, and energy production (Zhang et al., 2017). These results suggest that Axn may induce changes in amino acid metabolism.

Glutamine is an important detoxification substance. The significant increase in glutamine content indicates higher antioxidant activity and higher tolerance when the body is injured. L-glutamate, an abundant free amino acid in the body, is derived from the local synthesis of L-glutamine and Krebs cycle intermediates. Glutamate not only enhances the perception of sweetness and saltiness, but also supports muscle protein synthesis and enhances alterations in immunological responses (Tapiero et al., 2002). In this study, the amount of various amino acids in the muscles of E. carinicauda after Axn feeding increased significantly and included not only the amino acids that form the cytoskeleton but also those that enhance flavor. It has been shown that Axn not only improves the anti-apoptotic ability of muscle, but also influences the flavor of E. carinicauda.

Glutamate synthesizes glutamine for detoxification and the cytoskeleton and is also used as a raw material for the synthesis of GSH. The results of this study showed that GSH was upregulated. GSH plays a role in signal transduction, gene expression, and apoptosis. Most of the GSH involved in antioxidant defense in cells is utilized by three members of the GSH peroxidase (GPx) family and one peroxiredoxin (Prdx 6). These enzymes catalyze the reduction of H₂O₂ by GSH into H₂O and GSSG (Forman et al., 2009). Based on the results, it is speculated that the content of glutamate may be increased after Axn feeding, which may also lead to an increase in glutathione content. This also means that the antioxidant capacity of E. carinicauda was greatly improved as a result of Axn feeding.

In terms of lipid metabolism, some DEGs and DEMs were downregulated in response to Axn feeding and were associated with unsaturated fatty acid (UFA) biosynthesis. UFAs are indispensable constituents of cellular membranes and are involved in energy metabolism (Hulbert, 2003). Metabolomic analysis of the muscle indicated that the fatty acid biosynthetic and arachidonic acid metabolic pathways were enriched in the Axn group. Levels of palmitic acid, myristic acid, and arachidonic acid (ARA) were decreased in muscles in response to Axn feeding. Furthermore, the levels of certain fatty acids and energy metabolism-related molecules, such as leukotrienes and glucose-6-phosphate (Tallima and El Ridi, 2018), were also increased in Axn-fed shrimp compared with the control group. The levels of UFAs have been reported to increase under adverse environmental stress in white shrimp and Chinese fleshy shrimp (Fan et al., 2019; Meng et al., 2019). The results of the study indicated that the anti-stress ability of the cell membrane of muscle was improved after feeding with Axn and that it was not necessary for many UFAs to participate in cell membrane synthesis.

The cell membrane has a certain fluidity. The levels of long-chain polyUFAs are generally increased to maintain membrane fluidity, in what is termed homeoviscous adaptation. Phosphoglycerides, including lecithin (PC), cephalin (PE), and phosphatidylserine (PS), are important components of the cell membrane and form the basis of cell metabolism, energy metabolism, and signal transmission. Lysophospholipids, the raw material of biomembranes, also participate in the biological processes of most cells (Schulte, 2015; Wang et al., 2020). After Axn feeding, the levels of almost all PEs, PCs, PSs, and LysoPCs decreased. Therefore, it can be inferred that E. carinicauda activates metabolic pathways, such as ARA metabolism, to protect the muscle from injury. In addition, the DEMs detected in lipid metabolism was less than that detected in amino acid metabolism. This may be because the protein content of the muscle of E. carinicauda is richer than the lipid content of the muscle of E. carinicauda.

Interestingly, it was found that the vitamin A content increased after Axn feeding. It is speculated that Axn is converted into vitamin A and stored in white shrimp. When E. carinicauda was stimulated by an adverse environment, vitamin A had corresponding physiological effects.

Aquatic animals are susceptible to oxidative stress caused by biotic and abiotic factors (Martinez-Alvarez et al., 2005; Lushchak, 2011), which results in the accumulation of reactive oxygen species (ROS) and changes in antioxidant enzymes that lead to tissue damage or cell apoptosis (Lushchak, 2011; Lin et al., 2017). Axn is essential for maintaining proper antioxidant capacity and the health of aquatic organisms to alleviate the negative effects triggered by oxidative stress (Lim et al., 2018). In this study, the activity of antioxidant enzymes was significantly upregulated, which also demonstrates the protective effect of Axn against E. carinicauda.

Among the DEGs, it was shown that ACT1, which participates in cytoskeleton remodeling, was upregulated by Axn feeding. Protection against stress was found to be associated with changes in the expression levels of genes related to cytoskeleton remodeling (Xiao et al., 2019). When the body is adversely stimulated, cytoskeleton-related genes are significantly downregulated. When the expression of these genes increase significantly, the anti-stress ability of the body is significantly improved (Long et al., 2013). Thus, changes in ACT1 expression might be involved in Axn feeding; however, the detailed mechanism of the relationship between remodeling of cytoskeleton structure and Axn should be explored in further studies.

In the present study, the expression of cathepsin was significantly decreased after Axn feeding, indicating its potential role in apoptosis. Moreover, cathepsin, which induces upregulation of proapoptotic genes and downregulation of antiapoptotic genes (Li et al., 2016), may cause Cyt-c to be released into the cytoplasm from mitochondria, leading to apoptosis. Similar results were found in a previous study, in which cathepsin and Cyt-c expression decreased significantly after Axn feeding compared with the control, suggesting that Axn participates in the mitochondrial apoptotic pathway (Ren et al., 2020). The antiapoptotic BCL-XL can inhibit the release of Cyt-c from the mitochondria. Thus, it is interesting to note that BCL-XL decreased after Axn feeding. The reason for this may be that Axn improved the anti-stress ability of the shrimp, which lead to the antiapoptotic gene Bcl-XL not being highly expressed in response to adverse environmental stimuli.