Molecular Characterization, Tissue Distribution and Differential Nutritional Regulation of Three n-3 LC-PUFA Biosynthesis-Related Genes in Hybrid Grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

Simple Summary Polyunsaturated fatty acids, especially DHA and EPA, play crucial roles in fish growth performance, brain and eye development, reproduction and non-specific immune responses. However, considering the environmental unsustainability and the increasing price of fisheries’ by-products, alternative aquafeed ingredients are needed as a source of unsaturated fatty acids for farming marine fish. Here, we isolated and characterized three genes participating in the biosynthesis of n-3 LC-PUFA in hybrid groupers. We found that these genes were expressed in the liver of hybrid groupers in response to dietary fatty acid levels. Our findings contribute to a better understanding of the n-3 LC-PUFA biosynthesis process in this marine fish species. Abstract Elongases of very long-chain fatty acids (Elovls) and fatty acid desaturases (Fads) are crucial enzymes involved in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs). In this paper, we report the molecular cloning and characterization of three genes from the marine teleost Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂, and analyzed tissue distribution and their expression in response to dietary n-3 LC-PUFA levels after a 42-day feeding experiment. The elovl5, elovl8 and fads2 genes encoded 294, 263 and 445 amino acids, respectively, which exhibited all the characteristics of the Elovl and Fads family. Tissue distribution analysis revealed that elovl5, elovl8 and fads2 were widely transcribed in various tissues, with the highest level in the brain, as described in other carnivorous marine teleosts. The transcript levels of elovl5, elovl8 and fads2 in the liver were significantly affected by dietary n-3 LC-PUFA, and higher LC-PUFA levels repressed their expression. These results demonstrated, for the first time, the presence and nutritional modulation of elovl5, elovl8 and fads2 cDNA in the juvenile hybrid grouper. Further studies are needed to determine the functional characterization of these genes and explore the mechanism of these genes when regulated by dietary fatty lipid profiles in this species.


Introduction
Long-chain polyunsaturated fatty acids (LC-PUFA, ≥C20 and ≥2 double bonds) are widely present in organisms and play vital roles in maintaining cell membrane fluidity, regulating fat metabolism, enhancing immunity and reducing inflammation [1]. Among these, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are the most critical LC-PUFAs, with beneficial effects on human health particularly in the prevention of cardiovascular and neuro developmental disorders [2]. At present, fish, particularly marine species, represent a rich source of EPA and DHA for human consumers, due to the high content of LC-PUFAs acquired from the feed [3]. However, the raw materials for

Fish and Sampling
Juvenile hybrid groupers (4 months old, approximate 20 g) were selected in our trial as the first few months of life are the most vulnerable period and crucial for fish growth. They were obtained from a local commercial hatchery (Wenchang, Hainan province, China), and were acclimated in circular tanks at room temperature (28 • C) for two days. The fish were provided a natural photoperiod and adequate dissolved oxygen (>6.0 mg L −1 ). Four fish were collected and anesthetized with MS-222 (Sigma, St. Louis, MO, USA); then, tissues including liver, heart, intestine, muscle, kidney, stomach, pyloric cease and brain were snap-frozen by liquid nitrogen and then stored at −80 • C until use.

Gene Cloning 2.2.1. Primers Design
According to the highly conserved domains of the elovl5, elovl8 and fads2 gene sequence obtained by DNAssist software (Version 1.0) from other species, including cobia (Rachycentron canadum), Atlantic salmon, golden pompano (Trachinotus ovatus), orangespotted grouper and giant grouper in the GenBank database (http://www.ncbi.nlm.nih. gov/genbank/ (accessed on 4 January 2022)), specific PCR primers were designed using Primer 5.0 based on the consensus sequences to amplify and confirm the full length of these cDNAs (Table 1) ( Figure S1). Total RNA was extracted from the liver, heart, intestine, muscle, kidney, stomach, pyloric cease and brain using Trizol Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Quantification of RNA was performed using NanoDrop ND-1000 spectrophotometer (Wilmington, DE, USA), and the quality was measured by electrophoresis on a 1.0% denaturing agarose gel. The RNA (1 µg) from all the above tissues was reverse-transcribed into cDNA by PrimeScript™ RT kit and gDNA Eraser (Perfect Real Time) (Takara, Japan) according to the manufacturer's instructions. To obtain the first partial fragment of gene cloning, RNA (1 µg) from liver samples was reverse-transcribed into cDNA using SMARTer®RACE5 /3 kit (Takara, Japan) and GoScript™ Reverse Transcription System kit (Promega, USA), according to the manufacturer's instructions. The first partial fragment was sequenced by Sangon Biotech Co., Ltd (Shanghai, China), and was verified by NCBI BlastN.

RACE Amplification
The 5 and 3 rapid amplification of cDNA ends (RACE) PCR template were obtained according to the manufacturer's instructions (SMARTer™ RACE cDNA Amplification Kit, Clontech, San Jose, CA USA). For 3 and 5 RACE of elovl8, gene-specific primers, Elovl8-3F and Elovl8-5R, and Universal Primer (provided in the kit) were used in gradient PCR. Amplification was performed in a total volume of 20 µL, containing 10 µL 2×Taq PCR Master Mix II, 0.5 µL of each primer (10 µM), 7 µL nuclease-free water, and 2 µL cDNA (50 ng/µL). For 3 and 5 RACE of elovl5, Elovl5-3F and Elovl5-5R, and Universal Primer were used in gradient PCR. For 3 and 5 RACE of fads2, Fads2-3F1 and Fads2-5R1, and Universal Primer were used in gradient PCR. The PCR product was purified, cloned into a pMD19-T vector (Takara, Japan), then transformed and sequenced. The full lengths of the grouper elovl5, elovl8 and fads2 cDNA were obtained by aligning the first partial sequence with the corresponding RACE PCR products using DNAssist software (Version 1.0). The cloned PCR fragments were then sequenced by Sangon Biotech Co. Ltd. (Shanghai, China).

Sequence and Phylogenetic Analysis
The nucleotide sequence and the amino acid sequence of Elovl5, Elovl8 and Fads2 were analyzed by the NCBI blast program (http://www.ncbi.nlm.nih.gov/blast/Blast. cgi (accessed on 4 January 2022)). The conserved domains of Elovl5, Elovl8, and Fads2 sequences were predicted by the SMART and CDD programs. The molecular weight, isoelectric point, and amino acid content were identified by online software ExPASy (http: //web.expasy.org/protparam/ (accessed on 4 January 2022)). The Elovl5, Elovl8 and Fads2 amino acids' multiple sequence alignments were found using the CLUSTALX software (Version 1.8.1). The sequences alignment of Elovl4, Elovl5 and Elovl8 were uploaded into MEGA 10.0 and all columns containing 75% gaps were stripped from the alignment, leaving a total of 232 positions for phylogenetic analysis. The sequences alignment of Fads2 were uploaded into MEGA 10.0 and all columns containing 4.3% gaps were stripped from the alignment, leaving a total of 438 positions for phylogenetic analysis. The evolutionary model was determined using the MODELS option in MEGA 10.0 resulting in a LG + G, and branch support was calculated using maximum likelihood ( Figure S2). The number of booststrap replications for phylogeny test are 1000.2.2. Feeding trial with different n-3 LC-PUFA levels Three isoenergetic (339 kcal/100 g), isonitrogenous (53%) and isolipidic (7%) experimental diets ( Table 2) were formulated containing graded levels of n-3 LC-PUFA (0.53%, 1.19% and 2.69%) based on our previous study (Table 3) ( Figure S3). Juvenile hybrid groupers were obtained from a local commercial hatchery (Wenchang, Hainan province, China). Fish were acclimated with commercial diets for 2 weeks before the experiment. A total of 108 fish (average initial weight, 20.8 ± 0.03 g) were selected and randomly distributed into 9 glass tanks (L 60 cm × W 45 cm × H 50 cm) connected to a water-recycling system. The water was oxygenated through air stones at the bottom of each tank. Triplicate groups of fish were fed to apparent satiation by hand twice daily (8:00 am and 4:30 pm). Water temperature (29 ± 0.5 • C), total ammonia (0-0.15 mg/L) and dissolved oxygen (5.9 ± 0.1 mg/L) were monitored daily. Fish were exposed to a 12 h: 12 h light: dark cycle. The feeding trial lasted 42 days. At the end of the experiment, fish were counted and weighed after being subjected to 16 h fasting. Two fish per tank were randomly collected and anesthetized with MS-222 at 80 mg L -1 (Sigma, St. Louis, MO, USA): Liver was sampled and snap-frozen by liquid nitrogen and then stored at −80 • C for the RNA extraction.

Quantitative Real-Time PCR
RT-PCR was carried out in a quantitative thermal cycler (Roche Light Cycler®480, Switzerland). The amplification was performed in a total volume of 20 µL containing 10 µL power SYBR®Green PCR Master Mix (Takara, Japan), 0.8 µL of each primer (10 µM), 6.4 µL nuclease-free water and 2 µL of cDNA (50 ng/µL). The real-time RT-PCR program was as follows: 95 • C for 30 s, followed by 40 cycles of 95 • C for 5 s, 60 • C for 20 s, and 65 • C for 15 s. After the amplification phase, a melt curve of 0.5 • C increments from 65 • C to 90 • C was performed, enabling the confirmation of the amplification of a single product in each reaction for the melting curve. Standard curves were made with five different dilutions (in triplicate) of the cDNA samples and the amplification efficiency was analyzed according to the following equation E = 10 (−1/slope) −1. Real-time PCR efficiencies of elovl8, elovl5, fads2 and elongation factor 1 (ef1alpha) ranged between 96.2% and 104.7%, 96.5% and 105.5%, 97.1% and 106.1%, 95.6% and 106.2%, respectively. Ef1alpha was used as the housekeeping gene to normalize of the results (as our previous study). The expression levels of the target genes were calculated followed by the 2− ∆∆Ct method.

Statistical Analysis
The normality and homogeneity of the data were explored using Hartley's test. Then, data were subjected to one-way analysis of variance (ANOVA) and Duncan's multiple range test (SPSS 22.0 for Windows, Chicago IL, USA) to determine if significant differences occurred between different treatments. Differences were considered significant at p < 0.05. All data are presented as mean ± S.E.M.
The cloned elovl8 cDNA from a hybrid grouper (uploaded GenBank Accession Number MZ713364) was 1228 bp, which contained a 792 bp ORF that encoded a polypeptide of 263 amino acids with a 111 bp 5 UTR and a 325 bp 3 UTR ( Figure S5). The encoded Elovl8 protein has a calculated molecular mass of 30.

Tissue Distribution of elovl5, elovl8 and fads2 mRNA Hybrid Grouper
As shown in Figure 6, the elovl5, elovl8 and fads2 genes were widely expressed in all the detected tissues of the hybrid grouper, including intestine, liver, muscle, brain, kidney, stomach, heart and pyloric caeca, with the highest expression in the brain (p < 0.05). Furthermore, moderate expression of elovl5 and elovl8 genes was found in the liver and stomach, while the fads2 gene most highly expressed in liver and muscle. The results also showed that the heart and kidney were the tissues with the lowest gene expression of elovl5 and fads2 (p < 0.05), while the lowest value for elovl8 gene expression was found in the kidney (p < 0.05). Our Epinephelus fuscoguttatus ♀× Epinephelus lanceolatus ♂sequence was marked " ".

Tissue Distribution of elovl5, elovl8 and fads2 mRNA Hybrid Grouper
As shown in Figure 6, the elovl5, elovl8 and fads2 genes were widely expressed in all the detected tissues of the hybrid grouper, including intestine, liver, muscle, brain, kidney, stomach, heart and pyloric caeca, with the highest expression in the brain (p < 0.05). Furthermore, moderate expression of elovl5 and elovl8 genes was found in the liver and stomach, while the fads2 gene most highly expressed in liver and muscle. The results also showed that the heart and kidney were the tissues with the lowest gene expression of elovl5 and fads2 (p < 0.05), while the lowest value for elovl8 gene expression was found in the kidney (p < 0.05).

Nutritional Regulation of Hybrid Grouper elovl5, elovl8 and fads2 Genes Expression
The gene expression of elovl8, elovl5 and fads2 in the liver of hybrid grouper were significantly affected by the dietary n-3 LC-PUFA levels (p < 0.05) (Figure 7). Elovl5 and elovl8 relative mRNA expressions were significantly decreased at 1.19 and 2.69 of dietary n-3 LC-PUFA levels compared with group 0.53 (p < 0.05). In contrast, a significantly downregulated gene expression of fads2 was recorded in group 2.69 compared with other treatments (p < 0.05).

Nutritional Regulation of Hybrid Grouper elovl5, elovl8 and fads2 Genes Expression
The gene expression of elovl8, elovl5 and fads2 in the liver of hybrid grouper were significantly affected by the dietary n-3 LC-PUFA levels (p < 0.05) (Figure 7). Elovl5 and elovl8 relative mRNA expressions were significantly decreased at 1.19 and 2.69 of dietary n-3 LC-PUFA levels compared with group 0.53 (p < 0.05). In contrast, a significantly downregulated gene expression of fads2 was recorded in group 2.69 compared with other treatments (p < 0.05).

Discussion
Similar to other cultured marine fish species, fish oil is the primary lipid source for hybrid groupers. Considering the non-sustainable reliance on fish oil to farm this species, several trials have investigated the impact of the dietary replacement of fish oil with alternatives [30][31][32][33]. However, studies related to the molecular basis of LC-PUFA biosyn-

Discussion
Similar to other cultured marine fish species, fish oil is the primary lipid source for hybrid groupers. Considering the non-sustainable reliance on fish oil to farm this species, several trials have investigated the impact of the dietary replacement of fish oil with alternatives [30][31][32][33]. However, studies related to the molecular basis of LC-PUFA biosynthesis and regulation, which could optimize the pathway to the efficient use of alternatives, are still limited in hybrid groupers. Thus, we first cloned and characterized the main enzymes participating in the LC-PUFA biosynthesis in the present study, including Elovl5, Elovl8 and Fads2, from this fish species. To date, elovl5 cDNA has been identified in numerous marine fish species, including Epinephelinae, and has been demonstrated to effectively elongate C18 and C20 PUFA [34]. In the present study, the isolated hybrid grouper elovl5 cDNA sequence had 294 amino acids and showed high identity with other teleost elovl5, particularly Perciformes including Epinephelus coioides (99%), Epinephelus lanceolatus (99%), Acanthopagrus schlegelii (95%) and Larimichthys crocea (95%). The hybrid grouper Elovl5 possessed standard features for Elovl protein family members, including the so-called histidine box (HXXHH), the canonical C-terminal ER retention signal, several predicted transmembrane regions and other highly conserved motifs [35].
Elovl8a and elovl8b were first identified and functionally characterizated in rabbitfish from Li's lab. Multiple sequences alignment showed that elovl8 shared a closer relationship with elovl4 [18]. Afterwards, Sun et al. conducted a phylogenetic analysis to establish the orthology of the newly identified elovl8 gene and revealed that the similar orthologs from some fish species which were all annotated as elovl4 or elovl4-like could be misidentified due to the sequence similarity to elovl4 genes [9]. Thus, the previously similar elovl genes that have been annotated as "elovl4" (or "elovl4-like") in many fish species were considered to be elovl8 [9]. Similarly, amino acid sequence alignment with elovl4, elovl5 and elovl8 from other species showed that the new isolated "elovl4" gene from hybrid grouper was highly similar to those of elovl8b, and exhibited high identity with other teleost elovl8b, particularly Perciformes, including Siganus canaliculatus Elovl8b (86%), Salmo salar Elovl8b (83%), Oncorhynchus mykiss Elovl8b (83%), Ictalurus punctatus Elovl8b (81%) and Danio rerio Elovl8b (80%). Hybrid grouper Elovl8 possessed all the features of Elovl8 protein family members, including motifs (KXXEXXDT, QXXFLHXYHH, NXXXHXXMYXYY and TXXQXXQ), endoplasmic reticulum (ER) retention signal, multiple membrane-spanning regions and a histidine box (HXXHH), which is involved in the coordination of electron reception during fatty acid elongation [36]. It has been shown that the two Elovl8 isoforms may play various roles during the n-3 PUFA synthesis in different species. For instance, Elovl8b could elongate C18 (18:2n-6, 18:3n-3 and 18:4n-3) and C20 (20:4n-6 and 20:5n-3) polyunsaturated fatty acids (PUFAs) to longer-chain polyunsaturated fatty acids (LC-PUFAs), whereas Elovl8a lacked this ability in rabbitfish. However, in the study of zebrafish, Elovl8a activity was specific to C18-C20 PUFAs, just as Elovl4, Elovl5 and Elovl8b activity was specific to C18:0 and C20:1 MUFAs similar to Elovl1, Elovl3, and Elovl7 [9,19]. Thus, further studies need to explore Elovl8 functions in LC-PUFA synthesis in this hybrid grouper.
Fads2 (∆6 desaturase) catalyzes the first desaturation step in LC-PUFA synthesis and has been widely studied as the rate-limiting enzyme in the classical "∆6 desaturation-Elovl5-∆5 desaturation" LC-PUFA biosynthetic pathway [37]. Like all teleost fish species, phylogenetic analysis confirmed that the identified hybrid grouper cDNA was fads2 rather than fads1, while both Fads1 and Fads2 desaturases were found in Elopomorpha species such as Japanese eel (Anguilla japonica), and it was considered that Fads1 was retained in Chondrichthyes and early ray-finned fish before its subsequent loss in Osteoglossomorpha and Clupeocephala [38]. As it is compensating for the lack of Fads1 in teleost genomes, Fads2 have functionally diversified during their evolution, such that ∆6, ∆4, ∆5, ∆8, or bifunctional desaturation abilities have been recorded in different fish species. Moreover, it has been demonstrated that the low expression of the fads2 gene in carnivorous marine species was due to the lack of a binding site for stimulatory protein 1 (Sp1) [39].
The analysis of tissue distribution patterns for genes is helpful to better understand their physiological roles. Our study showed that the gene expression of elovl5, elovl8, and fads2 was detected in all the examined tissues, which was consistent with the ubiquitous expression previously reported in fish [18,19,28]. Furthermore, these three genes were predominantly expressed in the brains of hybrid groupers, as reported in other carnivorous marine fish species, including groupers and other species, such as cobia [40], Asian seabass (Lates calcarifer) [41], Nibe croaker (Nibea mitsukurii) [22], and Northern pike (Esox lucius) [42]. However, inconsistently with the elovl8b distribution pattern in the tissues, the elovl8a was highly expressed in the heart and spleen of rabbitfish, which demonstrates their various roles in the LC-PUFA synthesis process. DHA is one of the most abundant LC-PUFA in the brain and is vital for brain function and development; this is the likely reason for elovl5, elovl8 and fads2 being highly expressed, to supply the high requirements for this tissue. However, studies in freshwater and marine herbivorous species such as rabbitfish and Atlantic salmon [11,18] showed that the liver and intestine, as the significant metabolic sites for LC-PUFA biosynthesis, exhibited the highest expression of elovl5 and fads2 genes. In addition, our study also showed that relatively higher gene expressions of elovl5 were found in pyloric ceca compared with intestine and muscle, which indicated that it could be an important site of LC-PUFA, as reported in rainbow trout [43]. However, the gene expression in the kidney was the lowest among all the measured tissue, which was inconsistent with the orange-spotted grouper findings [44]. These results suggest that the diversification of fish LC-PUFA synthesis in different tissues could be associated with factors such as feeding habits, ecological habits, and species-specific evolutionary history [45].
Marine fish species' requirement for LC-PUFA firstly depends on daily intake from the diet. As alternative oil sources lacking in LC-PUFA are gradually being used in the diet, it is essential to clarify the patterns of dietary fatty acids in regulating genes encoding desaturases and elongases in farmed fish. As the liver serves as the primary site for FA metabolism, including de novo synthesis of FA, we evaluated the gene expression of elovl5, elovl8 and fads2 in the liver of hybrid groupers, and they were down-regulated by higher dietary n-3 LC-PUFA levels, which was consistent with the findings in the orange-spotted grouper [28,29,44] and other fish species such as the large yellow croaker [46]. These observations reflect the negative feedback regulation of the LC-PUFA synthetic pathway. Atlantic salmon elovl5 reporter activities were induced by the overexpression of LXRα, but not by the overexpression of sterol regulatory element-binding protein 1 (SREBP-1) [47]. In a study of Japanese seabass, CpG methylation of the fads2 promoter was observed as hepatic fads2 expression decreased in response to high dietary n-3 LC-PUFA, but a similar result was not recorded in European seabass [48]. Thus, further investigation is required to explore the regulation mechanism related to the different genes involved in LC-PUFA for hybrid groupers.

Conclusions
In summary, elovl5, elovl8 and fads2 genes were identified from hybrid groupers. The characteristics of the above genes were similar to other marine teleost fish species. Moreover, elovl5, elovl8 and fads2 were broadly expressed in most tissues, with the highest levels being found in the brain and liver, followed by the stomach, and the lowest levels being found in the kidney. Furthermore, the hepatic gene expression of elovl5, elovl8 and fads2 was downregulated by high dietary n-3 LC-PUFA. However, future studies are needed to determine the functional characterization of these genes and to explore the mechanism of these genes when regulated by the dietary fatty lipid profiles to better understand the process of n-3 LC-PUFAs biosynthesis in this fish species.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ani12030234/s1. Figure S1: Multiple Sequence Alignment of elovl5, elovl8 and fads2 genes used for primer design. The design sequence of the primers is marked with square, Figure S2: Maximum likelihood method parameters, Figure S3: Effect of dietary n-3 LC-PUFA levels (g/100 g) on weight gain (%) of hybrid grouper, Figure S4: Nucleotide and deduced amino acid sequence of Elovl5 in hybrid grouper (GenBank accession number MZ713365). The nucleotide sequence is numbered from the first base at 5 end, started in codon (ATG) and stopped in codon (TGA). Four conserved motif of elongases are in gray; the putative histidine-richdomain (HXXHH) is in bold; the predicted seven (I-VII) putative membrane-spanning domains are underlined; the ER retrieval signal is wavy underlined, Figure S5: Nucleotide and deduced amino acid sequence of Elovl8 in hybrid grouper (GenBank accession number MZ713364). The nucleotide sequence is numbered from the first base at 5 end, started in codon (ATG) and stopped in codon (TGA). The putative transmembrane regions are in gray; the putative histidine-richdomain (HXXHH) is in bold; the predicted seven (I-VII) putative membrane-spanning domains are underlined; the ER retrieval signal is wavy underlined, Figure S6: Nucleotide and deduced amino acid sequence of Fads2 in hybrid grouper (GenBank accession number MZ713366). The nucleotide sequence is numbered from the first base at the 5 end, started in codon (ATG) and stopped in codon (TGA). The Cytochrome b5-like Heme/Steroid binding domain is in gray; the putative histidine-rich domain (HXXHH, HDXGH and QXXHH) is in bold; the predicted four (I-IV) putative membrane-spanning domains are underlined.
Author Contributions: Q.W. manuscript preparation; C.W. and Y.W. investigation; Z.Z. and Y.S. formal analysis; Y.G. conceptualization and writing-review and editing. All authors have read and agreed to the published version of the manuscript.