Genome-Wide Identification of a Novel Elovl4 Gene and Its Transcription In

Aquaculture 529 (2020) 735666

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Genome-wide identiﬁcation of a novel elovl4 gene and its transcription in response to nutritional and osmotic regulations in rabbitﬁsh (Siganus T canaliculatus) ⁎ ⁎⁎ Zhengyong Wena,b,c, Yang Lid, Chao Bianb,c, Qiong Shib,c, , Yuanyou Lia, a College of Marine Sciences of South China Agricultural University & Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China b BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China c Shenzhen Key Lab of Marine Genomics, Guangdong Provincial Key Lab of Molecular Breeding in Marine Economic Animals, BGI Academy of Marine Sciences, BGI Marine, BGI, Shenzhen 518083, China d Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou 515063, China

ARTICLE INFO ABSTRACT

Keywords: Elongation of very long-chain fatty acids 4 (Elovl4) proteins, belong to the fatty acyl elongase families that are Rabbitfish (Siganus canaliculatus) involved in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis in animals. Thus far, very little is elovl4 known about the roles of Elovl4 in fish. Previously, we reported an elovl4 gene named as elovl4b in rabbitfish Phylogenetic analysis (Siganus canaliculatus). In this study, a novel elovl4-like gene with a 957-bp open reading frame encoding a 318 LC-PUFA amino-acid protein, which shared 67.3% similarity with rabbitfish Elovl4b and more than 80% similarity with Lipid sources Elovl4a from other teleost, was identified in S. canaliculatus, and thus was termed as elovl4a. Multiple protein Salinity adaption sequences alignment, gene synteny and phylogenetic analysis indicated that two obviously different elovl4 genes are widespread in various teleost. Different with elovl4b (mainly expressed in eye and slightly expressed in brain), rabbitfish elovl4a was primarily distributed in brain, slightly examined in eye and gonad tissues while hardly detectable in other tissues. The transcription of cerebral elovl4a was significantly higher in fishes cultured in seawater (32‰) than that of those maintained at a brackish water environment (15‰), when fishes were fed with a vegetable oil (VO) enriched diet. However, the transcriptional pattern was totally inverse when fishes were fed with a fish oil (FO) enriched diet. In the brackish water, diet types had no effect on the transcription of cerebral elovl4a. Whereas in seawater, the transcription of cerebral elovl4a was significantly higher in fishes fed with the VO diet in comparison with those fed with the FO diet. Our findings suggest that elovl4a can be regulated by both nutritional and osmotic factors, and it may play an important role in endogenous biosynthesis of LC-PUFA in the neural system of rabbitfish. This study may help us to understand the effects of nutritional and osmotic factors on the LC-PUFA biosynthesis, as well as contribute to the optimization or enhancement of LC- PUFA biosynthesis in teleost.

1. Introduction occupying high trophic levels, have high content of LC-PUFA, and therefore they have been considered as the main sources of essential Long-chain polyunsaturated fatty acids (LC-PUFA) are unsaturated fatty acids (EFA) of human diet (Kromhout et al., 2012). fatty acids with carbons ≥ C20 and double bonds ≥2. Generally, fish cannot convert oleic acid (18:1n-9) into linoleic acid Eicosapentaenoic acid (EPA, 20:5n-3), arachidonic acid (ARA, 20:4n-6), (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3) (Lin et al., 2018), but and docosahexaenoic acid (DHA, 22:6n-3) are three well-known LC- they can utilize these fatty acids to biosynthesize LC-PUFA for main- PUFA for their important roles in regulating growth, development, re- taining the requirement of normal growth and development, and production, as well as maintaining cell physiological functions in ver- therefore both LA and ALA are essential for fishes. It is usually thought tebrates (Li et al., 2019; Xu et al., 2020). Many fishes, especially species that fish occupying low trophic levels including most freshwater fish

⁎ Corresponding author at: BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China. ⁎⁎ Corresponding author at: College of Marine Sciences of South China Agricultural University & Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China. E-mail addresses: [email protected] (Q. Shi), [email protected] (Y. Li). https://doi.org/10.1016/j.aquaculture.2020.735666 Received 14 April 2020; Received in revised form 27 June 2020; Accepted 27 June 2020 Available online 07 July 2020 0044-8486/ © 2020 Elsevier B.V. All rights reserved. Z. Wen, et al. Aquaculture 529 (2020) 735666 can utilize LA and ALA to biosynthesize the LC-PUFA, whereas fish trout (Oncorhynchus mykiss)(Zhao et al., 2019), Atlantic bluefin tuna occupying high trophic levels including most seawater fish usually lack (Thunnus thynnus)(Betancor et al., 2020), tambaqui (Colossoma mac- this capacity or such ability is very low (Trushenski and Rombenso, ropomum)(Ferraz et al., 2020), gilthead seabream (Sparus aurata) and 2020). And thus, fish oil (FO), which usually come from the marine Senegalese sole (Solea senegalensis)(Torres et al., 2020a, 2020b). Thus fisheries and rich in LC-PUFA, must be added in the compound feed of far, two distinct elovl4 isotypes are widely identified in fish genomes, most marine fish so as to meet the requirement of EFA for normal and their functions are proved to be variable. For example, Elovl4a and growth and development (Li et al., 2019). However, the natural pro- Elovl4b were able to elongate saturated fatty acids, while only Elovl4b duction of marine fishes has declined in recent years due to overfishing, was able to elongate PUFA in zebrafish (Monroig et al., 2010); however, human activity and degradation of the marine environments (Tocher, several recent studies revealed that both Elovl4a and Elovl4b can ef- 2009; Li et al., 2019). As a result, the supplies of fish oil reduced and fectively elongate PUFA substrates in black seabream (Acanthopagrus their price increased, which seriously restricts the healthy development schlegelii)(Jin et al., 2017), African catfish (Clarias gariepinus)(Oboh of aquaculture industry. Therefore, researchers are trying to develop et al., 2017), golden pompano (Trachinotus ovatus)(Zhu et al., 2019) methods to replace fish oil with vegetable oils, which have relatively and tambaqui (C. macropomum)(Ferraz et al., 2020). Moreover, only high resource and low price, and rich in LA and ALA but lack of LC- one elovl4 gene has been characterized in most teleost, and further PUFA. To reach this goal, the regulatory mechanism of fish LC-PUFA analyses suggested these elolv4-like genes were more like to be ortho- biosynthesis has obtained a lot of attention so as to develop methods logues to the fish elovl4b isotype. The functions of Elovl4a are therefore that can optimize the endogenous biosynthesis of LC-PUFA to reduce less known in comparison with Elovl4b. the reliance of the aquaculture industry on fish oil (Li et al., 2019). It is well known that both environmental and nutritional factors can Biosynthesis of LC-PUFA from LA and ALA is catalyzed by two kinds alter the biosynthesis of LC-PUFA by regulating the activity and gene of key rate-limiting enzymes, including fatty acyl desaturases (Fads) expression of key enzymes involved in LC-PUFA biosynthesis in teleost and elongation of very long-chain fatty acids (Elovl) (Tocher et al., (Betancor et al., 2020). In rabbitfish, individuals cultured at lower 2003). The former introduces a stereospecific double bond between the salinity and/or fed with the vegetable oil diet displayed higher capacity defined carbons of fatty acyl chains by conducting dehydrogenation of LC-PUFA biosynthesis, which was consistent with the expression le- reactions, while the latter catalyzes the rate-limiting condensation step vels of related genes involved in the LC-PUFA biosynthesis (Li et al., to elongate the fatty acyl chains (Li et al., 2017b; Zhao et al., 2019). In 2008; Xie et al., 2015). Similarly, diet with low levels of LC-PUFA in- mammals, seven members of elongases, named as ELOVL1-ELOVL7, duced higher expression of fads2 in Atlantic salmon (S. salar) and have been identified, and they are different from each other in their Atlantic bluefin tuna (T. thynnus)(Zheng et al., 2005; Betancor et al., substrate specificity (Castro et al., 2016). Generally, four elongases 2014, 2015). However, the transcriptions of elov4 genes in response to including ELOVL1, ELOVL3, ELOVL6, and ELOVL7 are associated with nutritional regulation were variable in different fishes. Low dietary n-3 elongation of saturated and monounsaturated fatty acids, while the LC-PUFA increased the transcription of elovl4 in orange-spotted grouper other three elongases including ELOVL2, ELOVL4, and ELOVL5 play (E. coioides), large yellow croaker (P. crocea) and rainbow trout (O. important roles in elongation of LC-PUFA (Castro et al., 2016; Oh et al., mykiss), while decreased its expression in Atlantic bluefin tuna (T. 1997). In teleost, six elongases (except for ELOVL3) have also been thynnus)(Li et al., 2017a, 2017b; Zhao et al., 2019; Betancor et al., identified, and they may exhibit similar molecular functions with their 2020). More studies on different fish species are still necessary to elu- counterparts in mammals. However, the roles of these fish elongases are cidate the various regulatory mechanisms of LC-PUFA biosynthesis. rarely examined possibly due to their complex process of evolution. For Rabbitfish (S. canaliculatus) is an economically herbivorous marine example, elovl1, elovl4, and elovl7 genes may commonly contain two fish that was first proved to have the capacity to biosynthesize LC-PUFA isotypes (Monroig et al., 2016). Recently, we reported two novel elovl8 in marine teleost (Li et al., 2008, 2010). Since the LC-PUFA biosynthesis genes in an herbivorous marine fish Siganus canaliculatus (rabbitfish) related genes including a dual-functional Δ6Δ5 fads2, a monofunctional and functional experiments suggested that the Elovl8b but not Elovl8a Δ4 fads2, elovl4 (now renamed as elovl4b), elovl5, elovl8a, elovl8b have is involved in LC-PUFA biosynthesis (Li et al., 2020), which makes the been identified in this fish (Li et al., 2008, 2010; Monroig et al., 2012; Li evolutionary history of elovl genes more complicated in teleost. et al., 2020), rabbitfish has been regarded as a good model for in- Previous studies have suggested that Elovl2 appears to be lost in vestigating the regulatory mechanisms of LC-PUFA biosynthesis in tel- vast marine fish lineages due to marine food chains are full of LC-PUFA, eost. In this study, a novel elovl4 gene was identified in rabbitfish and thus lacking the evolutionary pressure to restrain the capacity of LC- its transcription patterns in response to nutritional and osmotic changes PUFA biosynthesis (Monroig et al., 2010; Morais et al., 2012; Castro were clarified. These data will increase our understanding of the effects et al., 2016). However, this view has been challenged because Elovl2 of nutritional and osmotic factors on the LC-PUFA biosynthesis in was identified in a marine fish European sardine (Sardina pilchardus) various teleost. (Machado et al., 2018), suggesting that rather than habitat (marine or freshwater), the phylogenetic position of fishes accounts for the pre- 2. Materials and methods sence of a elovl2 gene that was believed to be absent in marine teleost genomes (Castro et al., 2016; Li et al., 2017b). Now, it is clear that 2.1. Experimental fishes and samples collection Elovl2 may be lost in Acanthopterygii, a phylogenic group that includes the majority of farmed marine fish species (Xu et al., 2020). Meanwhile, Juvenile rabbitfish (S. canaliculatus) used in present study were Elovl4 and Elovl5 are virtually present in fishes, and the former pos- captured from seawater near the coast of our fishery base at Nan Ao sesses the capacity to elongate C20–22 LC-PUFA, while the latter pri- Marine Biology Station (NAMBS) of Shantou University. Fishes were marily elongates C18 PUFA precursors and C20 LC-PUFA substrates (Li transferred to indoor aquariums and stored at two different salinity et al., 2017b). Thus, Elovl4 has been proposed to compensate the waters, seawater (32‰) and brackish water (15‰), for acclimation. functions of Elovl2 that has been lost in most genomes of marine teleost These experimental fishes were fed (4% body weight) with commercial (Li et al., 2019; Zhao et al., 2019). feeds (containing 32% crude proteins) twice per day for one month. For Elovl4 has been investigated in several teleost species, such as molecular cloning and tissue distribution experiments, six fishes were zebrafish (Danio rerio)(Monroig et al., 2010), Atlantic salmon (Salmo randomly selected from the aquarium with seawater environment and salar)(Carmona-Antonanzas et al., 2011); rabbitfish (S. canaliculatus) 12 tissues (adipose, brain, eye, gill, gonad (testis and ovary), heart, (Monroig et al., 2012), Nibe croaker (Nibea mitsukurii)(Kabeya et al., intestine, kidney, liver, muscle, spleen and stomach) were collected for 2015), orange-spotted grouper (Epinephelus coioides)(Li et al., 2017a), further use. For feeding experiments, fishes with an initial average large yellow croaker (Pseudosciaena crocea)(Li et al., 2017b), rainbow weight of 25.07 ± 1.43 g were assigned to six tanks, with 20 fishes per

2 Z. Wen, et al. Aquaculture 529 (2020) 735666 tank in both salinity waters. Meanwhile, three tanks of fishes were fed construction (Qin et al., 2018; Yang et al., 2018). After calculation, with vegetable oil (VO) enriched diet, while the other three tanks of JTT + G was selected as the best phylogenetic model and the neighbor- fishes were fed with fish oil (FO) enriched diet at both salinity en- joining (NJ) method was chosen to construct the phylogenetic tree. vironments. The feeding experiment lasted two months, and the com- Meanwhile, the robustness of the tree topology was assessed by a positions of experimental diets were provided in our previous report (Li nonparametric bootstrap analysis with 1000 resampling replicates. Fi- et al., 2020). When the experiments were finished, six fishes from each nally, the tree was improved using FigTree (http://tree.bio.ed.ac.uk/). tank were randomly selected and anesthetized, and then their brains GenBank or Ensemble accession numbers of selected Elovls were listed were collected and frozen in liquid nitrogen immediately before storage in Supplemental Table 2. at −80 °C for further utilization. The animal experiments were performed according to the Chinese 2.5. Quantitative real-time PCR Ministry of Science and Technology for Humane Treatment of Laboratory Animals, and approved by the Institutional Animal Care and Tissue samples were manipulated to isolate total RNAs by using Use Committees of Shantou University (Shantou, China) and BGI Trizol reagent (Invitrogen), following the manufacturer's protocol. (Shenzhen, China). Next, agarose gel electrophoresis and spectrophotometer (NanoDrop 2000, Thermo Scientific, USA) were used to assess the quality of the 2.2. Molecular cloning of the elovl4a cDNA isolated RNAs. After assessment, the total RNAs (1 μ g for each sample) were transcribed to cDNAs using QuantiTect Reverse Transcription kit In silico protein similarity-based BLAST search was performed to (Takara Biotech, Dalian, China). Quantitative real-time PCR (qPCR) identify the orthologous gene of fish elovl4a in a rabbitfish genome was conducted to measure the mRNA level of rabbitfish elovl4a on a assembly (data unpublished), using protein sequences of zebrafish LightCycler 480 thermocycler (Roche, Mannheim, Germany) with a Elovl4a (Ensemble accession number: ENSDARG00000006773) and ti- final volume of 20 μL. Meanwhile, relative expression level of mRNA lapia Elovl4a (ENSOABG00000016410) as queries. Subsequently, was normalized with 18S rRNA after assessing the stability of five re- genomic DNA sequences of rabbitfish elovl4a was isolated according to ference genes, including β-actin, GAPDH, EF1α, Tubα1 and 18S RNA,by the high matched scores, and the predicted gene structure and protein GeNorm (Vandesompele et al., 2002; Wen et al., 2015). Moreover, re- sequence were determined using the online software Softberry (http:// lative transcription of elovl4a was calculated using the Pfaffl method www.softberry.com/). Meanwhile, the predicted protein sequence was (Wen et al., 2019, 2020a). Sequences of the specific primers used for blasted online in NCBI (https://blast.ncbi.nlm.nih.gov) to ensure the qPCRs were provided in Supplemental Table 1. accuracy of prediction. In addition, total RNA was isolated from in- dividual brain tissue and then transcribed into cDNA using a 2.6. Statistical analysis SuperScript II RT reverse transcriptase first strand cDNA synthesis kit (Thermo Fisher Scientific, Shanghai, China) following the manufac- Results of the tissue distribution and transcriptional changes of S. turer's instructions, accompanying with a mixture of random primers canaliculatus elov4la were calculated using SPSS 21.0 (IBM, Armonk, and oligo (dT)15 primers (Thermo Fisher Scientific, Shanghai, China). NY, USA) and GraphPad Prism (San Diego, CA, USA), and shown as Two pairs of primers (Supplemental Table 1) were designed to amplify mean normalized values ± SEM (n = 6). Significant differences were the full open reading frame (ORF) sequence using cDNA as the tem- determined using one-way analysis of variance (ANOVA) followed by plate. The PCR procedures were designed as follows: a denaturing stage Tukey's test, and differences were significant when p < .05. at 94 °C for 30 s, gene-specific annealing for 45 s and an elongation stage at 72 °C for 60 s, a total of 34 cycles. The target products were 3. Results purified using TIAN quick mini purification kit (Tiangen Biotech, Beijing, China), cloned into pMD™18-T vector (TaKaRa Biotech, Dalian, 3.1. cDNA cloning of rabbitfish elovl4a China) and subsequently sequenced (Sangon Biotech, Shanghai, China). Two achieved partial cDNA sequences of elovl4a were assembled 2.3. Sequence processing and gene synteny analysis into one complete cDNA sequence, which contained a 957-bp ORF encoding 318 putative amino acid residues (Fig. 1). Based on the si- The obtained sequences were assembled into one complete cDNA milarity with other fish Elovl4, we also identified five transmembrane sequence using BioEdit as described in our previous papers (Wen et al., domains in the rabbitfish Elovl4a, which were underlined in Fig. 1. 2019, 2020a). Subsequently, the ORFfinder (https://www.ncbi.nlm. Meanwhile, the rabbitfish Elovl4a also contained a conserved histidine nih.gov/orffinder/) and Primer Premier 5.0 were applied to predict the dideoxy binding motif (HXXHH) between the second and third trans- ORF region and encoding protein sequence of rabbitfish elovl4a, re- membrane domains (Fig. 1). In addition, a predicted endoplasmic respectively. Functional motifs and domains were identified according to ticulum (ER) retention signal sequence was found at the carboxyl the details in our previous reports (Monroig et al., 2012; Li et al., 2020). terminal of rabbitfish Elovl4a (Fig. 1). The cDNA sequence of rabbitfish Meanwhile, multiple protein sequences alignment was performed using elovl4a has been deposited in GenBank with an accession number ClustalX and BioEdit (Wen et al., 2020b). Additionally, a comparative MT248261. genomic survey was conducted to recognize the genetic loci of rabbitfish elovl4a and elovl4b genes, as well as the corresponding genetic loci 3.2. Multiple protein sequences alignment and elovl4 synteny of other fish elovl4 genes (Li et al., 2020). To verify the elovl4a is a novel, and different gene from the pre- 2.4. Phylogenetic analysis viously reported elovl4 gene in rabbitfish (Monroig et al., 2012), we compared the protein sequence of rabbitfish Elovl4a with other verte- Phylogenetic analysis was conducted to reveal the phylogenetic brate Elovl4 sequences. Our result showed that two distinct isotypes are position and evolutionary history of fish Elovl4s, as well as the phylo- present in various teleost (Fig. 1). Meanwhile, five transmembrane genetic relationship with other fish Elovls. Protein sequences of re- domains, a HXXHH motif, and an ER retention signal were highly presentative fish Elovls were downloaded from NCBI or Ensemble da- conserved in vertebrate Elovl4 (Fig. 1). Notably, the protein sequences tabases, and multiple protein sequences alignment was performed using of teleost Elovl4a and Elovl4b, located between the fifth transmem- ClustalX as described above. Subsequently, the aligned file was up- brane domain and the ER retention signal, were not conserved (Fig. 1). loaded into MEGA 6.0 for further phylogenetic model selection and tree Moreover, the rabbitfish Elovl4a shared 82.0%, 83%, and 96.8%

3 Z. Wen, et al. Aquaculture 529 (2020) 735666

Fig. 1. Multiple protein sequences alignment of vertebrate Elovl4s. Five conserved transmembrane domains are underlined and labeled as I-V, respectively. Predicted histidine dideoxy binding motif (HXXHH) and endoplasmic reticulum (ER) retention signal are highlighted in colorful blocks. Species used in present study include zebrafish (D. rerio; Dr), balck seabream (A. schlegeli; As), loach (M. anguillicaudatus; Ma), rabbitfish (S. canaliculatus; Sc), human (H. sapiens; Hs) and mouse (M. musculus; Mm). The accession numbers of these sequences are provided in Supplemental Table 2. identity with orthologs form zebrafish (D. rerio), loach (Misgurnus an- 3.3. Phylogenetics guillicaudatus) and black seabream (A. schlegelii), while shared 68.3%, 67.3%, and 67.7% identity with its paralog Elovl4b in these fishes Phylogenetic analysis was performed for a better understanding of (Supplemental Table 3). Additionally, Elovl4a was 67.3% identical to the evolutionary process and variable characteristic of fish elovl genes. its paralog Elovl4b in rabbitfish (Supplemental Table 3). The phylogenetic tree was reconstructed using the neighbor joining To further confirm our findings, comparative genomic survey was method and the protein-sequence dataset. We observed that the tree conducted to determine the gene synteny of elovl4 in four re- was divided into two groups, and the group at bottom contained elovl2 presentative teleost species, including zebrafish (D. rerio), Mexican tetra and elovl5 clades, while another group included elovl6, elovl8 and elovl4 (Astyanax mexicanus), tilapia (Oreochromis niloticus), and rabbitfish (S. clades (Fig. 3). Meanwhile, elovl4 and elov8 shared a close relationship, canaliculatus)(Fig. 2). As shown in Fig. 2, fish elovl4a and elovl4b pos- and both contained two distinct isotypes (elovl4a/elovl4b and elovl8a/ sessed significantly different gene orders or locations. The elovl4a gene elovl8b). Consistently, two different rabbitfish elovl4 genes were clus- was linked to soga3a (Fig. 2A), while the elovl4b gene was commonly tered into teleost elovl4a and elovl4b clades, and they shared a close flanked by soga3b and tent5ab (Fig. 2B). Typically, two gene clusters relationship with black seabream elovl4a and elovl4b, respectively including soga3a-elovl4a and rnf146-soga3b-elovl4b-tent5ab were iden- (Fig. 3). tified in these fish genomes. Consistent with taxonomy, the genetic loci of rabbitfish elovl4 genes were highly identical to those of tilapia elovl4 3.4. Tissue distribution of rabbitfish elovl4a genes, but showed some variances in comparison with genetic loci of zebrafish and the Mexican tetra (Fig. 2). Quantitative real-time PCRs were conducted to detect the tissue distribution pattern of rabbitfish elovl4a. A total of 12 tissues including adipose, brain, eye, gill, gonad, heart, intestine, kidney, liver, muscle, spleen and stomach were measured. The transcript of elovl4a was

4 Z. Wen, et al. Aquaculture 529 (2020) 735666

Fig. 2. Comparative synteny analysis of elovl4a (A) and elovl4b (B) genes in four representative ﬁsh genomes. Genes and intergenic regions are highlighted with colorful blocks and solid lines, respectively.

Fig. 3. Phylogenetic relationships of fish Elovls. The tree was constructed with the Neighbor joining method based on a dataset of protein sequences. Numbers up the clades represent the bootstrap percentages from 1000 replicates and the rabbitfish Elovl4s are labeled in red. The Nobel scallop (Mimachlamys nobilis) was used as an outgroup. The accession numbers of these protein sequences are provided in Supplemental Table 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

5 Z. Wen, et al. Aquaculture 529 (2020) 735666

Fig. 4. Relative mRNA tissue distribution of the rabbitﬁsh elovl4a. Twelve tissues including adipose, brain, eye, gill, gonad, heart, intestine, kidney, liver, muscle, spleen and stomach were analyzed. Each error bar represents a standard error of the mean values (n = 6). 18S RNA was used as the reference gene. primarily expressed in brain, eye and gonad tissues, while no expression was detected in adipose, gill, heart, intestine, kidney, liver, muscle, spleen and stomach (Fig. 4). Meanwhile, the highest transcription level of elovl4a was found in brain, following with lower value in eye and the lowest in gonad (Fig. 4).

3.5. Eﬀects of dietary lipid sources and water salinities on the transcription of cerebral elovl4a

Elovl4 has been reported to be sensitive to the nutritional changes. Here, we examined whether dietary lipid sources can regulate the transcription of elovl4a when fishes were cultured in same salinity water. Interestingly, we observed that dietary lipid sources have no effect on the cerebral elovl4a expression when fishes were stored in the brackish water (15‰)(Fig. 5A). However, the transcription of cerebral elovl4a in fishes fed with a VO diet was significantly higher than that in those fed with a FO diet at the seawater environment (32‰)(Fig. 5B). Environmental salinity has also been proved to be involved in reg- Fig. 6. Osmotic regulation of the transcription of cerebral elovl4a in rabbitfish ulating the expressions of LC-PUFA biosynthesis related genes in tel- fed with the vegetable oil (VO) diet (A) and the fish oil (FO) diet (B). Different eost. In order to detect whether salinity can alter the expression of letters above the bars represent significant differences among the examined elovl4a, we compared the transcriptions of cerebral elovl4a in fishes that groups. Each error bar represents a standard error of the mean values (n = 6). were cultured in seawater and brackish water when fed with the same 18S RNA was used as the reference gene. diet. Our results showed that the transcription of cerebral elovl4a in seawater (32‰) was significantly higher than that in brackish water (15‰) when fishes were both fed with the VO diet (Fig. 6A). However, the transcription of cerebral elovl4a in fishes cultured in seawater was significantly lower than that in those cultured in brackish water when fishes were both fed with the FO diet (Fig. 6B).

4. Discussion

Elovl4 is a critical rate-limiting enzyme participating in LC-PUFA biosynthesis in both mammals and teleost (Castro et al., 2016). Pre- vious studies revealed that ﬁsh Elovl4 has the capacity to eﬀectively

elongate C20 and C22 PUFA, which is different with mammalian ELOVL4 that appears to operate only towards longer chain (C26) PUFA (Agbaga et al., 2008; Monroig et al., 2012). This functional property of fish Elovl4 is somewhat similar to the fish Elovl2, suggesting Elovl4 may participate in the DHA biosynthesis and compensate the roles of Elovl2 in Acanthopterygii fishes since elovl2 has lost in their genomes (Morais et al., 2009; Li et al., 2019; Zhao et al., 2019). To date, two Fig. 5. Nutritional regulation of the transcription of cerebral elovl4a in rab- distinct elovl4 isotypes were identified in zebrafish (Monroig et al., bitfish cultured in brackish water (A) and seawater (B). Different letters above 2010), black seabream (Jin et al., 2017), African catfish (Oboh et al., the bars represent significant differences among the examined groups. Each 2017), and tambaqui (Ferraz et al., 2020), and they exhibited different error bar represents a standard error of the mean values (n = 6). 18S RNA was functional properties. Notably, most Elovl4 identified in teleost was used as the reference gene. proved to be orthologs of Elovl4b, hence the exact roles of Elovl4a are

6 Z. Wen, et al. Aquaculture 529 (2020) 735666 less known. In our previous work (Monroig et al., 2012), we reported an (Izquierdo et al., 2008; Morais et al., 2012; Kuah et al., 2015; Xie et al., Elovl4 in rabbitfish, and now we rename it to be Elovl4b due to its high 2015; Li et al., 2016; Li et al., 2017b). However, related studies on fish sequence identity with Elovl4b in other fishes. elovl4 were rarely reported. In orange-spotted grouper and large yellow In the present study, we identified a novel elovl4-like gene in rab- croaker, the expression of elovl4 was down regulated by dietary n-3 LC- bitfish with a name of elovl4a. Its ORF was 957 bp encoding 318 amino PUFA (Li et al., 2017a, 2017b). In rainbow trout, the expression of acids, which shared similar characteristics with the corresponding elovl4b was significant higher in fishes fed with VO oil than those in elovl4a in zebrafish and golden pompano (Monroig et al., 2010; Zhu fishes fed with FO diet (Zhao et al., 2019). In gilthead seabream and et al., 2019), suggesting that elovl4a may be widely existed and play Senegalese sole, diets with insufficient LC-PUFA increased the expres- similar roles in various teleost. Multiple protein sequences alignment sion of elovl4a but did not alter the expression of elovl4b (Torres et al., revealed that the newly identified rabbitfish Elovl4a was significantly 2020a, 2020b). Consistently, we observed the transcription of cerebral different from Elovl4b that was reported earlier in rabbitfish due to elovl4a was significantly higher in fishes fed with the VO diet in com- their low similarity (67.3%); similar results were previously reported in parison with that in fishes fed with the FO diet, suggesting Elovl4a zebrafish (Monroig et al., 2010), black seabream (Jin et al., 2017) and could endogenously biosynthesize LC-PUFA in brain by utilizing the C18 African catfish (Oboh et al., 2017). Moreover, the Elovl4a was com- PUFA of feeds to resist insufficient LC-PUFA in feeds. However, the diet monly 10–20 amino acids longer than its paralog Elovl4b in teleost, and types had no effects on the transcription of cerebral elovl4a when fishes the region at the carbon terminal was extremely variable between were cultured in brackish water, suggesting that the LC-PUFA in fish Elovl4a and Elovl4b. These findings indicate that the two distinct elovl4 brain may be rapidly oxidized to supply the energy for maintaining the genes may have experienced an independent process of evolution, and homeostasis of the osmotic pressure. might play different functional roles in various teleost. Salinity is also considered to affect the expressions of LC-PUFA Comparative genomic analysis showed that two obviously different biosynthesis related genes in aquatic species, and its regulatory effects genetic loci of the elovl4a and elovl4b in four representative fish species, are usually variable in different species (Vagner and Santigosa, 2011; including zebrafish, a cavefish, tilapia, and rabbitfish (Fig. 2). Mean- Fonseca-Madrigal et al., 2012). The genetically improved farmed tilapia while, two gene clusters of soga3a-elovl4a and rnf146-soga3b-elovl4b- (GIFT, O. niloticus) and Mexican silverside (Chirostoma estor) have tent5ab were identified in these fish genomes, indicating that the two higher capacity of LC-PUFA biosynthesis in brackish water than in elovl4 genes are obviously different, and they might be derived from a freshwater, which is consistent with the corresponding expression of specific whole genome duplication event in teleost (Lien et al., 2016; Li hepatic Δ6 fads2 in both waters (Fonseca-Madrigal et al., 2012; You et al., 2020; Wen et al., 2020a). In contrast to mammals, we have re- et al., 2019). Differently, the pathways of LC-PUFA biosynthesis can be ported a novel Elovl elongase in rabbitfish, and which was termed as stimulated by lower salinity in Atlantic salmon (S. salar) and red sea Elovl8 with two distinct isotypes (Li et al., 2020). Consistent with bream (Pagrus major), suggesting their high synthesized capacity in the previous study (Oboh, 2018), we confirm two novel Elovl8 elongases low salinity water (Zheng et al., 2005; Sarker et al., 2011). In our are widely existed in teleost, suggesting that the fish elovl genes may previous studies, we also found that rabbitfish possessed higher capa- have experienced a more complex evolutionary process than that in city of LC-PUFA biosynthesis in brackish water than in seawater (Li mammals (Li et al., 2020). In addition, elovl2 has been reported to be et al., 2008; Xie et al., 2015), according to the expression patterns of lost in the vast majority of marine fish lineages (Castro et al., 2016), related genes (elovl5, Δ65 fads2, and Δ4 fads2) in liver. Here, we ob- which leads to a more complicated situation of fish elovl genes (Monroig served two reverse transcription patterns of cerebral elovl4a in rabbit- et al., 2012; Monroig et al., 2016). Here, our phylogenetic analysis fish that were reared at different salinity waters and fed with different showed that the fish elovl4 clade included two subclades of elovl4a and diets, suggesting that the synthesized ability of LC-PUFA was variable in elovl4b, which is consistent with other findings in several previous different tissues. Meanwhile, osmotic regulation of LC-PUFA biosynth- studies (Jin et al., 2017; Zhu et al., 2019), suggesting that the teleost esis may be explained by the fact that a large amount of energy con- elovl4 genes are commonly including two isotypes. sumption is required to maintain the osmotic equilibrium inside and DHA is one of the most abundant LC-PUFA in tissues such as brain, outside the fish body (You et al., 2019). However, the exact regulatory eye and gonads; it has been proved to play important roles in neuro- mechanisms are still waiting for further investigation. development, vison, and reproduction (Monroig et al., 2011; Li et al., In summary, we identified a novel elovl4-like gene (named as 2019; Xu et al., 2020). Because Elovl4 usually distributes in brain, eye eovl4a) in rabbitfish by using comparative genomic survey and mole- and gonads, it has been considered to possess the capacity to en- cular experiments. Meanwhile, the phylogenetic relationship of fish dogenously biosynthesize DHA and other LC-PUFA in these tissues in elovls was reconsidered, and we confirmed that two elovl4 isotypes were various teleost (Monroig et al., 2011). However, the tissue distribution widely spread in teleost. Moreover, the distribution pattern of elovl4a patterns of elovl4s are variable in different fish species. In zebrafish, was determined, and we found that the elovl4a was primary distributed elovl4a was widely expressed in majority of the examined tissues, while in brain, eye and gonad. Finally, both nutritional and osmotic factors elovl4b presented a more restricted distribution with abundance in eye, could regulate the transcription of elovl4a in brain. Our findings may ovary and testis (Monroig et al., 2010). In orange-spotted grouper and help to understand the effects of nutritional and osmotic factors on the large yellow croaker, elovl4 was extensively distributed with high ex- LC-PUFA biosynthesis, as well as contribute to the optimization or en- pression in eye, brain, and testis (Li et al., 2017a, 2017b). In our pre- hancement of LC-PUFA biosynthesis in various teleost. vious study, we discovered that the elovl4b was mainly distributed in Supplementary data to this article can be found online at https:// eye and brain in rabbitfish, while hardly detectable in other examined doi.org/10.1016/j.aquaculture.2020.735666. tissues (Monroig et al., 2012). Similarly, we observed that elovl4a was primarily expressed in brain, eye and gonad, but not detectable in the Declaration of Competing Interest other tissues. These findings suggest that Elovl4 elongases are variable in different fishes, and they may be involved in endogenous LC-PUFA fi biosynthesis in neural tissues since they are recognized to participate in The authors declare that they have no known competing nancial fl the process of LC-PUFA biosynthesis in a tissue-specific manner (Deak interests or personal relationships that could have appeared to in u- et al., 2019; Ferraz et al., 2020). ence the work reported in this paper. Previous studies indicated that both nutritional and osmotic factors have effects on the expressions of genes involved in LC-PUFA bio- Acknowledgments synthesis (Xie et al., 2015; Li et al., 2017b). Thus far, the nutritional regulation of fads2 and elovl5 have been widely studied in fishes This work was financially supported by National Natural Science

7 Z. Wen, et al. Aquaculture 529 (2020) 735666

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