All the experiments were implemented according to the guidelines and regulations of the Animal Research and Ethics Committee of Guangdong Ocean University (NIH Pub. No. 85-23, revised 1996) and the laws and regulations of China on biological research. No endangered or protected species were involved in this study.

Greater amberjack juveniles (body weight: 6.94 ± 0.45 g; body length: 8.40 ± 0.54 cm) were reared in tanks at the temperature of 22 ± 1.0°C in Donghai Island, Guangdong, China. All fish were fed on a commercial diet (Guangdong Yuequn Biotechnology Co., Ltd, Jieyang, China) twice daily. They were randomly divided into 12 cylindrical 1000 L tanks (10 individuals per tank) at different salinities: 40, 30, and 10 parts per thousand (ppt), where all the saline was prepared with commercial seawater salty crystal and aerated tap water. The fish reared in the water with a median salinity of 30 ppt, the natural seawater condition, were set as the control group (CK). The water was changed once a day (every 24 h). All animal experiment protocols were authorized by the Institutional Animal Care and Use Committee of Guangdong Ocean University (Zhanjiang, China). At 72 h treatment, six fish (biological replicates) were randomly selected from each group. All fish were anesthetized using 100 mg/L tricaine methane sulfonate (MS 222; Sigma-Aldrich, St. Louis, MO, USA) and then dissected. The mixed partial gill filament from each gill arch and whole kidney tissues were taken from each individual of each group, collected in centrifuge tubes containing 1 mL RNA stabilization reagent (Accurate Biology, Changsha, China) overnight, and stored at -80°C for further experiments.

For each individual, total RNA was extracted from the gill and kidney tissues of each collected individual using a Trizol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. The cleavage of tissue samples, RNA extraction, RNA purity, degradation, and contamination examinations were performed according to the previous study (Shi et al., 2022). The RNA integrity was examined using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Total RNA with an RNA integrity number (RIN) score >7 was used for sequencing.

According to the manufacturer’s instructions, a total of 1 µg RNA was used for sequencing library construction using NEBNext UltraTM RNA Library Prep Kit for Illumina^® (NEB, Ipswich, MA, USA). The messenger RNA (mRNA) was purified using poly-T oligo-attached magnetic beads (Illumina, San Diego, CA, USA) after treating the RNA samples with DNase I (NEB, Ipswich, MA, USA). A fragmentation buffer reagent (New England Biolabs (NEB), Ipswich, MA, USA) was used to fragment mRNA into short fragments. The library fragments were purified using the AMPure XP Reagent (Beckman Coulter, Beverly, USA). USER Enzyme (NEB, Ipswich, MA, USA) was applied to make the adaptor-ligated complementary DNA (cDNA) for the downstream PCR. Then, polymerase chain reaction (PCR) was carried out using Phusion High-Fidelity DNA polymerase (NEB, Ipswich, MA, USA), with universal PCR primers and index (X) primer. PCR products were purified using the AMPure XP system (NEB, Ipswich, MA, USA). The library quantity and quality were assessed on the Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA) and StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Santa Clara, CA, USA), respectively. The index-coded samples were clustered using the cBot Cluster Generation System with the TruSeq PE Cluster Kit v4-cBot-HS (Illumina, San Diego, CA, USA). All cDNA libraries were sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA).

All the generated raw sequencing data were deposited and submitted to Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI) with an accession number GSE220485 (GSM6805719-GSM6805730). The raw reads were pre-processed for each library, including removing adapter contamination and filtering out the reads containing ploy-N and the low-quality bases through in-house perl scripts. The Q20, Q30, GC-content, and sequence duplication levels of the clean data were assessed to illustrate the data quality after quality control. Then, the clean reads from each sample were mapped to the reference genome of S. dumerili using HISAT2 (Kim et al., 2019).

Gene functions were annotated against the NCBI’s non-redundant (NR; ftp://ftp.ncbi.nih.gov/blast/db/), Swiss-Prot (http://www.uniprot.org/), Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/) and Gene Ontology (GO; http://www.geneontology.org/) databases, using the BLASTx (v. 2.2.26; https://blast.ncbi.nlm.nih.gov/) with an E-value cutoff of 1 × e^-5.

The gene expression level was inferred by the read abundance computed using featureCounts (Liao et al., 2014). The data structure was then assessed by principal component analysis (PCA) using the DESeq2 package (v. 1.38.3) (Love et al., 2014). The expression data were first log2 transformed and the genes were sorted based on their variance across samples. The top 500 genes with the largest variance were selected for PCA using the prcomp function. DESeq2 was used to identify differentially expressed genes (DEGs) under each of salinity stress conditions (10 or 40 ppt) against CK (30 ppt), where Wald test was employed to compute the p-value for each tested gene. Benjamini and Hochberg (BH) method was used to adjust the p-value to correct for multiple testing. Genes with adjusted p-value < 0.05 and |log₂ fold change| > 1.0 were significantly differentially expressed. DEGs showing higher or lower expression levels than CK for each stress condition were denoted as “up-regulated” or “down-regulated” genes, respectively. GO and KEGG pathway enrichment analyses were performed on up- and down-regulated DEGs using clusterProfiler (Yu et al., 2012). The statistical significance (p-value) was measured based on hypergeometric distribution, and the GO terms and KEGG pathways with p-value < 0.05 were significantly over-represented.

Trend analysis was carried out for the gills and kidneys across the salinity conditions (10, 30, and 40 ppt) using the Mfuzz package (Futschik and Carlisle, 2005; Kumar and Futschik, 2007). The gene expression level was normalized within each sample as transcripts per million (TPM). Genes of low variation among conditions were filtered out from the subsequent steps. The retained genes were clustered into six groups of different expression trends across different salinities using the Mfuzz function for each tissue ( Supplementary Figure S1, S2 ). Fisher’s exact test was used to assess the statistical significance of enrichment of each cluster following the strategy as described previously (Qian et al., 2020). Our null hypothesis is that there is no difference in gene numbers across all the trends. To test the hypothesis, all the tested genes were first randomly assigned into six clusters 10,000 times, and the median of the number of genes in each cluster was set as the background. For each cluster, the significance level of the over-representation was tested using one-tail Fisher’s exact test. The clusters with p-value < 0.05 were considered to have a significantly higher proportion of genes than the random assignment.

To validate the DEGs from RNA sequencing (RNA-seq) data, a total of 10 DEGs were randomly selected (five for the gills and five for the kidneys), and their expression levels were assessed by qRT-PCR. The procedure of qRT-PCR is as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles of 20 s denaturation at 94°C, 20 s annealing at 55°C and 20 s extension at 72°C. The primers of all selected genes were designed by Primer Premier software v5.0 (Premier Biosoft International, Palo Alto, CA, USA) and listed in Supplementary Table S1 . A light CyclerTM96 (Roche, Indianapolis, IN, USA) was employed for qRT-PCR using SYBR Green Real-time PCR Master Mix (TaKaRa Biotechnology, Dalian, China). All samples were validated in triplicates. β-actin was used as the reference gene, which was identified to be stably expressed in greater amberjack (Djellata et al., 2021). The relative expression levels of DEGs were estimated using the 2^-ΔΔCt method. Data were expressed as means ± standard error (SE) (n = 3). The statistical analysis was performed in the SPSS v. 16.0 (SPSS Inc., Chicago, IL, USA).

A total of 12 cDNA libraries (G10, G30, G40, K10, K30, and K40; Table 1 ) were constructed to assess the transcriptional responses to salinity stress in the gill and kidney tissues of greater amberjack. After quality control, 134.02 and 136.21 million clean reads were retained for the downstream transcriptomic analyses of the gills and kidneys, respectively. The Q30 values of the clean reads were greater than 91% in all the samples, indicating good data quality. Summary statistics for all the 12 cDNA libraries, including the number of clean reads and bases, Q20 and Q30 values, and GC content, are listed in Table 1 .

PCA was conducted to explore the consistency in gene expression profiles between samples of each treatment. All the biological replicates were aggregated, and different treatments were substantially distinct from each other ( Figure 1A ), where 35% and 23% of the variations were explained by the first and second principal components (PCs), respectively. In the gills, 445 DEGs were identified under 10 ppt salinity stress, including 309 up-regulated and 136 down-regulated genes, compared to the CK ( Figure 1B ; Supplementary Figure S3A ). In the 40 ppt saline treatment group, a total of 320 up-regulated and 103 down-regulated genes were detected ( Figure 1B ; Supplementary Figure S3B ). GO enrichment analysis showed that the down-regulated DEGs were enriched in the lipoprotein metabolic process, inorganic anion transport, chloride, and ammonium transmembrane transport ( Figure 1C ). The up-regulated DEGs under 10 ppt saline were overexpressed in the biological processes involved in cartilage development, oxygen transport, canonical Wnt signaling pathway, transport and microtubule-based process ( Figure 1D ). Under the salinity stress of 40 ppt, the DEGs, involved in the immune response processes, regulation of anion transmembrane transport, and stress response, were substantially inhibited ( Figure 1E ). The up-regulated DEGs were found to participate in extracellular matrix organization, skeletal system development, inorganic anion transport, cartilage development, and oxygen transport ( Figure 1F ).

Compared to the CK, the expression levels of 185 DEGs were enhanced in both 10 and 40 ppt salinity groups, which indicates that these genes participate in the responses to both hypo- and hyper-salinity stress ( Figure 2A ). Of these, the expression levels of 135 and 124 DEGs were specifically induced in 40 and 10 ppt treatment groups, respectively. The significant candidate genes in response to salinity stress in gills are listed in the heatmap, including slc9a3, loxa, mmp14b, col4a5, and so on ( Figure 2B ). To investigate the transcriptional dynamics across different salinities, we grouped the genes into six clusters according to the similarities in their expression profiles. Of these, three clusters (1, 3, and 6) were statistically significant (p-value = 8.55e-06, 4.12e-07, and 6.21e-14; Figures 2C–E ). In cluster 1, gene expression was increased when the salinity varied from 10 to 30 ppt but reduced as the salinity increased from 30 to 40 ppt. GO enrichment analysis suggested that genes in cluster 1 were involved in lipid metabolism and transport, Golgi organization, and response to hypoxia ( Figure 2C ). In contrast, the expression levels of genes in cluster 3 were enhanced when the salinity altered from 30 ppt to either 10 or 40 ppt. These genes were significantly enriched in the immune response, cartilage development, extracellular matrix organization, skeletal system development, and actin cytoskeleton organization ( Figure 2D ). Genes in cluster 6 were substantially down-regulated following the salinity change from 10 to 30 ppt and retained a similar level between 30 and 40 ppt salinity. These genes participated in the mitogen-activated protein kinase (MAPK) cascade, response to stimulus, retrograde transport, endosome to Golgi, cellular iron ion homeostasis, and cytoskeleton organization ( Figure 2E ).

We further compared the gene expression levels between 10 and 40 ppt salinity groups. A total of 118 DEGs were significantly overexpressed in the gills under 40 ppt treatment compared to that of the 10 ppt group ( Supplementary Figure S4A ). Functional enrichment analysis showed that the up-regulated DEGs were enriched in fatty acid metabolism, inorganic anion transport, and lipoprotein metabolic process ( Supplementary Figure S4B ). The down-regulated DEGs were associated with extracellular matri (ECM)-receptor interaction, chloride transmembrane transport, and nucleoside metabolic process ( Supplementary Figure S4C ).

In the kidneys of greater amberjack, a total of 600 (206 up- and 394 down-regulated) and 539 (312 up-regulated and 227 down-regulated) DEGs were detected under the salinity stress of 10 and 40 ppt, respectively ( Figure 3A ; Supplementary Figures S3C, D ). The representative DEGs involved in salinity stress resistance in the kidneys are highlighted in the heatmap, such as hbae5, c5, c4b, itih2, and so on ( Figure 3B ). GO enrichment analysis identified 23 significantly enriched GO terms in 10 ppt saline, where the up-regulated DEGs were involved in oxygen transport and transmembrane transport, while the down-regulated DEGs were highly represented in immune response, negative regulation of endopeptidase activity and lipid transport ( Figure 3C ). Under the salinity stress of 40 ppt, the up-regulated genes were enriched in 16 GO terms, including those related to the regulation of cell growth, microtubule cytoskeleton organization, and ammonium transmembrane transport. In contrast, blood coagulation, lipid metabolic process, and cellular iron ion homeostasis were enriched for the down-regulated DEGs ( Figure 3D ).

In the kidneys, genes exhibited three major trends of expression profiles across different salinities (p-value < 0.05; Figures 3E-G ). In cluster 1 (p-value = 0.016), gene expression was increased when switched from 30 ppt to 10 or 40 ppt salinity, and these genes participate in oxygen transport, pronephros development, and extracellular matrix organization ( Figure 3E ). For cluster 5 (p-value = 4.56e-07), the expression levels of genes showed no apparent difference between 10 and 30 ppt salinities, while they were induced by the increased salinity from 30 to 40 ppt. These genes were significantly enriched in ion transmembrane transport, ionotropic glutamate receptor signaling pathway, lipid metabolic process, and microtubule-based movement ( Figure 3F ). Genes in cluster 6 (p-value = 5.20e-09) exhibited the up-regulated gene expression levels from 10 to 30 ppt salinity, while these were down-regulated from 30 to 40 ppt. GO enrichment analysis displayed that these genes were involved in adenosine triphosphate (ATP) synthesis coupled proton transport, negative regulation of endopeptidase activity, steroid hormone-mediated signaling pathway, and blood coagulation ( Figure 3G ).

In the kidneys, 196 DEGs were detected under hyper-salinity condition (40 ppt) over hypo-salinity scenario (10 ppt) ( Supplementary Figure S4D ). Functional enrichment analysis revealed that the up-regulated DEGs were significantly enriched in the MAPK signaling pathway, Herpes simplex infection, cell growth regulation, and intracellular receptor signaling pathway ( Supplementary Figure S4E ). On the other hand, the down-regulated DEGs were enriched in the cysteine and methionine metabolism, PPAR (peroxisome proliferators-activated receptors) signaling pathway, glycine, serine and threonine metabolism, and lipoprotein metabolic process ( Supplementary Figure S4F ).

To explore the similarity and specificity in stress-response and resistance mechanisms between the different tissues, gills and kidneys, a comparative transcriptome analysis was carried out. When exposed to 10 ppt saline, 24 up-regulated DEGs were overexpressed in both gills and kidneys, which were significantly enriched in oxygen transport, blood vessel development and growth regulatory process ( Supplementary Figure S5A ). A total of 285 and 182 DEGs were up-regulated in gills or kidneys, respectively, mirroring the substantial difference between different tissues. The gill-specific DEGs were enriched in cartilage development, focal adhesion and canonical Wnt signaling pathway, while those specifically up-regulated in kidneys were over-represented in pronephros development, transmembrane transport and nervous system development ( Supplementary Figure S5A ). Similarly, 363 and 105 down-regulated DEGs were specific to gills or kidneys, and only 31 DEGs were overexpressed in both of tissues ( Supplementary Figure S5B ). Functional enrichment analyses illustrated that the DEGs specific to gills were associated with chloride transmembrane transport, ammonium transmembrane transport, and inorganic anion transport, whereas those in the kidneys were enriched in amino acid transmembrane transport, response to stress, and cellular iron ion homeostasis. Those co-expressed DEGs were involved in immune response, cell chemotaxis, and metal ion transport ( Supplementary Figure S5B ).

Under 40 ppt salinity stress, 80 up- and 14 down-regulated DEGs were overlapped in gills and kidneys, while 21 - 73% of the DEGs were identified in the two tissues ( Supplementary Figure S5C, D ). The commonly overexpressed DEGs were enriched in oxygen transport, ECM-receptor interaction, and transport ( Supplementary Figure S5C ), while those of down-regulation were over-represented in immune response, antigen processing, and presentation of peptide antigen via MHC class I and response to stimulus ( Supplementary Figure S5D ). The gill-specific up-regulated DEGs were involved in skeletal system development, inorganic anion transport, and hydrogen ion transmembrane transport, whereas those specific to the kidneys were highly represented in microtubule cytoskeleton organization, microtubule-based process, and AGE-RAGE (advanced glycation end product (AGE)-receptor for AGE (RAGE) signaling) signaling pathway in diabetic complications ( Supplementary Figure S5C ). The gill-specific down-regulated DEGs were enriched in blood coagulation, cellular iron ion homeostasis, and primary bile acid biosynthesis, while those DEGs in kidneys were involved in the regulation of anion transmembrane transport, response to stress and toll-like receptor (TLR) signaling pathway ( Supplementary Figure S5D ).

The expression levels of five randomly selected DEGs were validated in the gills ( Figure 4A ) and kidneys ( Figure 4B ), respectively, by qRT-PCR. In both tissues, the fold changes of the 10 DEGs under the stress of hypo- (10 ppt) and hyper-salinity (40 ppt) were of high consistency between RNA-seq and qRT-PCR ( Figure 4 ), which suggests the reliability of our RNA-seq results.

Insulin-like growth factors (IGFs) signal functions in regulating the cell cycle and cellular repair is essential for surviving salinity stress (Evans and Kültz, 2020). It also plays a key role in cortisol-mediated cell differentiation into mitochondria-rich cells (MRCs) (Gonzalez, 2012). In half-smooth tongue sole, gill igfbp5 (insulin-like growth factors binding protein 5) and igfbp2 were significantly up-regulated under the high and low salinity, responsible for the up-regulation of igf1 expression and regulation of cell growth and proliferation (Zhao et al., 2022). Meanwhile, MRCs were enriched in gills under the high salinity stress. The boosting MRCs would make an important contribution to the osmotic adjustment in teleost fishes upon the increased ambient salinity (Varsamos et al., 2005) by multiple ion channels anchored on the surface. In goby fish (Gillichthys mirabilis), gill igfbp1 mRNA expression is increased in response to the hyper-osmotically (Evans and Somero, 2008). In this study, when exposed to a higher salinity condition (40 ppt), the mRNA abundance of igfbp2 and ifgbp5 showed no change in gills, while ifgbp5 and igfbp6 expression levels were substantially up-regulated in the kidneys. The enhanced expression of Igfbps facilitates cell growth, proliferation, and migration, and increases cellular tolerance to rising ambient salinity (Tipsmark et al., 2007; Seale et al., 2020). In this study, the growth-related processes, cartilage development, skeletal system development and extracellular matrix organization in greater amberjack gills were activated in response to salinity alterations. The cartilage and skeletal development were induced in the gills of greater amberjack by hypo- or hyper-salinity stimuli. The enhanced development of the gill skeletal system may play a key role in maintaining gill structures and improving respiration and osmoregulation under salinity stress, similar to the associations between gill development and the tolerance capacity to hyper-salinity in Persian sturgeon (Acipenser persicus) (Shirangi et al., 2016).

In the kidneys, pronephros development was induced by lower salinity (10 ppt) compared to the optimum growth condition (30 ppt). Pronephros, which is also known as the primordial kidneys, mainly functions in ultrafiltration and urine production before being taken over by the definitive kidneys, and plays a crucial role in osmoregulation throughout the early development stages of fishes (Varsamos et al., 2005). In addition, the expression level of cep70 (centrosomal protein 70) mRNA was significantly increased in response to salinity reduction. Cep70 is involved in cilia assembly and essential for kidney development and homeostasis (Shi et al., 2011; Marra et al., 2016). The expression of ccn2 (cellular communication network factor 2) mRNA was significantly up-regulated in response to the increased salinity. It also known as the connective tissue growth factor that mediates the proliferation and adhesion of vascular endothelial cells. Thus, its enhancement can encourage the growth of blood vessels and reduce tissue damage caused by salinity (Pan et al., 2019). The expression level of mfap2 (microfibril-associated protein 2), a gene associated with blood vessel development, was overexpressed in both gills and kidneys under hypo-salinity stress, indicative of a similar regulatory route for the growth of blood vessels in both tissues. These results indicate that the undesired salinity impacts on the normal kidneys development for adaption to the salinity stress.

Ion transport is important for osmoregulation, which plays a critical role in the adaptive response to changes in external salinity (Deane and Woo, 2006; Tomy et al., 2009; Knowles et al., 2014; Vij et al., 2020). Gills are the main osmotic organs, which face directly to the external aqueous environment, responsible for maintaining the ion-osmotic balance of fish (Alkan and Oğuz, 2021; Zhang et al., 2021). In teleosts, Nkcc1 (Na-K-Cl cotransporter-1, encoded by the gene slc12a2), Nka (Na⁺/K⁺-ATPase), and Cftr (cystic fibrosis transmembrane conductance regulator) are the major ion transport proteins participated in ion secretion and osmoregulation in gills. Nkcc1 is crucial for inward movements of Na⁺, K⁺, and transcellular secretion of Cl⁻ in teleosts (Takvam et al., 2021). Nka is used for the active uptake of K⁺ and extrusion of Na⁺ in cells through protein conformational changes fueled by ATP (Mobasheri et al., 2000; Pierre and Blanco, 2021). Cftr contributes to the across-membrane conduction of Cl^- efflux from the epithelial cell (McCormick et al., 2013). In our study, gill slc12a2/nkcc1 and cftr mRNA expression levels were significantly down-regulated under the hypo-salinity stress at 10 ppt, while these levels were substantially activated in response to the elevated salinity from 30 to 40 ppt at 72 hours in great amberjack, except for the nka gene. The above results suggested that the lower gill permeability to reduce the Na⁺, K⁺ movement and Cl^- ion secretion was induced by hypo-osmotic exposure, while ion excretion upon hyper-salinity stress was boosted and a comparatively steady blood osmolality was maintained (Cao et al., 2022), through Nkcc1 and Cftr ion transport proteins in great amberjack, coincided with Mozambique tilapia gill ion regulation mechanism (Hiroi et al., 2008; Hiroi and McCormick, 2012). In half-smooth tongue sole, gill cftr expression was significantly down-regulated under low salinity but up-regulated under high salinity for 10 days. Gill nkcc and nkain (Na⁺/K⁺ transporting ATPase interacting) mRNA expression showed no significant changes. The results indicate that Cftr is the major ion transporter protein for regulating Cl⁻, Na⁺, and K⁺ ions transport and osmolality balance under the hypo- and hyper-salinity stress (Zhao et al., 2022). Similarly, in freshwater climbing perch (Anabas testudineus), the expression of gill cftr mRNA was up-regulated after exposure to seawater for 6 days (Chen et al., 2018). In European seabass (Dicentrarchus labrax), gill cftr and nkcc1 mRNA expression levels were decreased, while nka mRNA expression level was increased within 4 days post-acute transfer from seawater to freshwater (Maugars et al., 2018), suggesting that Cftr and Nkcc1 were mainly responsible for preventing the ions loses and osmolality out-of-balance under the low-salinity condition. Taken together, gills play a dominant role in osmotic and ionic regulation, but the molecular mechanism was divergent to some degree in different fish species.

Fish kidneys function as the main osmoregulatory organ in integrating ion and water transport to maintain internal homeostasis in osmotic environments. The renal transporters include Nka enzymes, monovalent ion transporters, water transport proteins, and divalent ion transporters. Na⁺/Cl^– co-transporter (Ncc, encoded by the gene slc12a3), is a monovalent ion-absorptive co-transporter, expressed in the kidneys, especially in the mammalian distal convoluted tubule and teleost fish distal nephrons (Gamba, 2005; Hiroi et al., 2008; Hsu et al., 2014). Ncc is essential in producing hypotonic urine in teleost fishes when grown in a hypoosmotic external environment (Takvam et al., 2021). In bull shark (Carcharhinus leucas), kidney ncc mRNA expression in freshwater sharks is higher than in seawater sharks, contributing to NaCl reabsorption from the urine and tolerance to the freshwater environment (Imaseki et al., 2019). In pufferfish (Tetradon nigroviridis) and European eel (Anguilla anguilla), ncc mRNA expression levels and apical location of the Ncc protein in the kidneys were significantly decreased following acclimation to seawater, which demonstrated the essential roles of Ncc in production of hypotonic urine and reabsorbed salts, responded to the freshwater environment (Cutler and Cramb, 2008; Kato et al., 2011). However, in this study, the gene expression level of slc12a3/ncc was significantly down-regulated in the kidneys under the hypo-salinity condition at 10 ppt in great amberjack. We speculated whether some other factors in kidneys were compensable for regulating the water and salt balance under the decreased salinity. Oct2 (organic cation transporter 2, encoded by the gene slc22a2) mediates in the initiation of the renal secretion of organic cations. In great amberjack, slc22a2 was overexpressed at 40 ppt, suggesting an induced secretion of organic cations might be response to the hyper-osmoregulation, to combat the osmotic influx of salts following the drink of seawater upon high salinity stress. When grown in 40 ppt saline, the increased external salinity largely induced the ammonium transmembrane transport gene rhd (Rh blood group, D antigen) and rhag (Rhesus-associated glycoprotein) expression in the kidneys. The up-regulated ammonium transport may mirror an elevated amount of produced ammonia (Wood and Nawata, 2011; Bucking, 2017). It is further supported by the overexpression of the genes encoding solute transport proteins (slc4a3, slc4a5, slc24a2, and slc22a2) under the treatment of 40 ppt saline, which may promote the transmembrane transport of amino acids and other substances. The metabolism of amino acids is specifically enhanced in the kidneys to provide more energy for coping with hyper-salinity stress and function as osmolytes for cell volume regulation (Aragão et al., 2010). Previous studies have demonstrated that amino acid levels are positively correlated with salinity (Aragão et al., 2010; Cheng et al., 2022). Together, our findings offer a molecular insight into the osmoregulatory mechanisms of greater amberjack kidneys against the adverse impacts of ambient salinity alterations.

Major osmotic organs, including gills and kidneys, play critical roles in regulating ion and water transport to maintain internal homeostasis and adapt to the external environment salinity changes. The molecular mechanisms in the gills and kidneys were interconnecting and distinguishing from each other, demonstrated by the expression pattern of the same transporters in different tissues or the different functional transporters, response to the hyper- and hypo-salinity stress.

In the current study, we also found that changes in external salinity would down-regulated the immune function in both the gills and kidneys of greater amberjack juveniles, which is consistent with the previous observations in other marine fishes, spotted scat (Scatophagus argus) (Zhong et al., 2021) and striped eel catfish (Plotosus lineatus) (Hieu et al., 2021). In particular, in the kidneys, the genes involved in immunological responses and blood coagulation were substantially suppressed by hypo- and hyper-salinity stress. In particular, the expression of mhc1 (the major histocompatibility complex (MHC) class I) mRNA, which mainly mediates antigen presentation, was suppressed in the kidneys under 10 or 40 ppt condition at 72 h, suggesting the down-regulated recognition of intracellular pathogen infection and activation of the downstream immune cascade under hypo- and hyper-salinity stress (Jiang et al., 2015). It was different from the observation of Nile tilapia (El-Leithy et al., 2019). High immune-related genes expression levels have indicated the fish is vulnerable to infect agents owing to the immune suppression under hyper-salinity stress (Bowden, 2008). Moreover, the expression levels of genes within or around the complement system, such as those encoding complement component 3 (c3) and vitronectin b (vtnb), were also significantly inhibited in greater amberjack by either decreased or increased ambient salinity at 72 h, indicating the repression of the inflammatory response, impede pathogen clearance, and disrupt cellular homeostasis (Liu et al., 2019; Wang et al., 2019). In puffer (Takifugu fasciatus), the expression of c3 in the gills and kidneys is negatively correlated with salinity (Wang et al., 2019). In this study, the mRNA expression of several genes participating in blood coagulation, such as coagulation factor II (f2) and hemopexin (hpx), were depressed in the greater amberjack when exposed to hyper-salinity stress, which is consistent with the observation in Nile tilapia (Xu et al., 2018). Because of the direct crosstalk between the coagulation and the immune system, the activation of blood coagulation positively contributes to pathogen clearance during infections (Antoniak, 2018). The gene expression of zap70, encoding an enzyme belonging to the protein tyrosine kinase family, was down-regulated in the kidneys of greater amberjack under the hypo-salinity situation. Similarly, in marbled eel (Anguilla marmorata), gill zap70 expression was decreased following the salinity (Chan et al., 1992; Cao et al., 2021). The repression of Zap60 perhaps leads to a disruption in T cell development and lymphocyte activation (Chan et al., 1992; Cao et al., 2021). It is worth noting that the gene fabp4 expression (fatty acid-binding protein-4) was induced in the gills of greater amberjack when exposed to 10 or 40 ppt saline. In fish, Fabp4 can bind to the marine environmental toxins perfluoroalkyl acids (PFAAs), trigger the undesirable uptake and accumulation (Lei et al., 2022), which has been demonstrated to suppress lysozyme activity in marine organisms, then interrupt their resistance to bacterial infection (Betts, 2007). Furthermore, PFAA can easily enter human bodies via the food chain and disturb human hormonal signaling, which poses potential risks to food safety and public health (Betts, 2007; McComb et al., 2020), which highlights the importance of a well-controlled rearing condition during the aquaculture to ensure the food safety of greater amberjack. The immune related genes in teleost gills or/and kidneys were influenced by the environmental salinity, which may impact the immune function, increase the risk of pathogen infection, and reduce the fresh security and yield of greater amberjack. These results help understand the mechanisms of the adaptive immunology response to the environmental salinity stress.