Taurine metabolism and effects of inclusion levels in rotifer (Brachionus rotundiformis, Tschugunoff, 1921) on Atlantic bluefin tuna (Thunnus thynnus, L.) larvae

Accepted Manuscript Taurine metabolism and effects of inclusion levels in rotifer (Brachionus rotundiformis, Tschugunoff, 1921) on Atlantic bluefin tuna (Thunnus thynnus, L.) larvae Mónica B. Betancor, Guillaume R. Laurent, Aurelio Ortega, Fernando de la Gándara, Douglas R. Tocher, Gabriel Mourente PII: S0044-8486(19)30320-5 DOI: https://doi.org/10.1016/j.aquaculture.2019.05.040 Reference: AQUA 634155 To appear in: aquaculture Received date: 7 February 2019 Revised date: 19 April 2019 Accepted date: 18 May 2019 Please cite this article as: M.B. Betancor, G.R. Laurent, A. Ortega, et al., Taurine metabolism and effects of inclusion levels in rotifer (Brachionus rotundiformis, Tschugunoff, 1921) on Atlantic bluefin tuna (Thunnus thynnus, L.) larvae, aquaculture, https://doi.org/10.1016/j.aquaculture.2019.05.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Taurine metabolism and effects of inclusion levels in rotifer (Brachionus rotundiformis, Tschugunoff, 1921) on Atlantic bluefin tuna (Thunnus thynnus, L.) larvae Mónica B. Betancora*, Guillaume R. Laurenta, Aurelio Ortegab, Fernando de la Gándarab, Douglas R. Tochera and Gabriel Mourentec a Institute of Aquaculture, Faculty of Natural Sciences, University of Stirling, FK9 4LA Stirling, Scotland, UK b Planta Experimental de Cultivos Marinos, Instituto Español de Oceanografía (IEO), 30860 Puerto de Mazarrón (Murcia), Spain c Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain * Corresponding author: Tel.: +44-1786-467892 E-mail address: [email protected] ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2 Abstract Taurine appears to be a crucial nutrient for teleosts, especially top predator species such as Atlantic bluefin tuna (Thunnus thynnus, L.; ABT). While dietary taurine supplementation has been highly recommended, there is a lack of studies on taurine assimilation and biosynthesis for this iconic species. The present study aims to provide insight into the molecular mechanisms involved in taurine biosynthesis and transport in ABT by studying tissue distribution and ontogenetic development of expression of cysteine dioxygenase (cdo), cysteine sulfinic acid decarboxylase (csad), 2aminoethanethiol dioxygenase (ado) and taurine transporter (tauT) in response to graded levels of dietary taurine supplementation. The full open reading frame (ORF) for cdo and partial sequences for csad, ado and tauT were obtained, with the translated polypeptides being 202, 176, 166 and 324 amino acids, respectively. All three showed characteristics such as cupin motifs in Cdo and predicted N-glycosylation sites in Taut that are common to these genes in other species. Phylogenetic analysis showed that the ABT sequences clustered with sequences of other teleosts, and separately from mammals and molluscs. Tissue distribution varied, with adipose tissue, kidney, white muscle and testis/brain showing highest expression of cdo, csad, ado and tauT, respectively. Whole larvae expression of csad peaked at 15 dah, whereas the other genes generally increased throughout development to show highest expression at 25 dah. The nutritional trial was carried out by feeding ABT larvae from mouth opening to 14 days after hatching (dah) with rotifers (Brachionus rotundiformis) enriched with 4 different levels of taurine: 0.0 (tau0), 0.5 (tau0.5), 1.0 (tau1), and 2.0 g taurine per 106 rotifers (tau2). Rotifers effectively accumulated taurine with ABT larvae fed on treatment tau2 attaining the highest concentration of taurine. However, ABT larvae fed tau1 displayed higher growth and survival, and flexion index at 14 dah, than larvae fed the other taurine levels. Larvae fed tau1 also showed generally higher expression of tauT and cdo and digestive and antioxidant enzyme genes. While this study showed that larval ABT express taurine metabolism genes, suggesting possible synthesis that could contribute to the taurine pool in the fish, larval ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 3 performance was enhanced by a level of dietary taurine (3.7 mg taurine g-1 rotifer) supplied by enrichment of rotifers at 1 g taurine per 106 rotifers. Keywords: bluefin tuna, larvae, taurine, gene expression, rotifer enrichment, cDNA Abbreviations: aa, amino acids; ABT, Atlantic bluefin tuna (Thunnus thynnus); alp, alkaline phosphatase; amy, amylase; anpep, amino peptidase; bactin, beta actin; bal1, bile salt activated lipase 1; bal2, bile salt activated lipase 2; cat, catalase; cdo, cysteine dioxygenase; csad, cysteine sulfinic acid decarboxylase; dah, days after hatch; ef1α, elongation factor 1 alpha; FC, fold change; gpx1, glutathione peroxidase 1; gpx4, glutathione peroxidase 4; myhc, myosin heavy chain; ORF, open reading frame; pl, pancreatic lipase; pla2, phospholipase A2; qPCR, quantitative real time PCR; sod, superoxide dismutase; tauT, taurine transporter; tropo, tropomyosin; tryp, trypsin; ubiq, ubiquitin; UTR, untranslated region. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 4 Introduction Atlantic bluefin tuna (ABT, Thunnus thynnus, L.) is a species with high market value although its closed aquaculture is currently inefficient and far from large-scale commercial production with low survival of larval stages, (De la Gandara et al., 2016; Van Beijnen, 2017). In order to optimize the ABT production cycle, further knowledge of the nutritional requirements of the species is pivotal, and understanding biological mechanisms of nutrient assimilation in larvae is a key area. Although some studies have been performed on different aspects of ABT nutrition (Morais et al., 2011; Betancor et al., 2017a,b; Koven et al., 2018) there is limited information regarding requirements for many nutrients that can be critical for larval and juvenile stages of this species. Taurine is the common name for 2-aminoethanesulfonic acid, an amino sulfonic acid which is not incorporated into proteins but, rather, resides in the free amino acid pool (Hamre et al., 2013). Despite this, taurine is not considered an amino acid since it contains a sulphonyl acid group rather than a carboxyl acid group (Pinto et al., 2012). However, taurine plays a critical role in many major biological functions and, in teleosts, is involved in bile salt conjugation, osmoregulation, membrane stabilization, modulation of neurotransmitters, antioxidant function and early development of visual, neural and muscular systems (Huxtable, 1992; Salze and Davis, 2015). In vertebrates, there are two main pathways for biosynthesizing taurine from cysteine with the final step in both pathways being the oxidation of hypotaurine to taurine, with the production of hypotaurine varying (Salze and Davis, 2015). One pathway involves the participation of two enzymes, cysteine dioxygenase (Cdo; EC 1.13.11.20) and cysteine sulfinate decarboxylase (Csad; EC 4.1.1.29), which produce hypotaurine from cysteine. A second route for hypotaurine production is through the action of the enzyme 2aminoethanethiol dioxygenase (Ado; EC 1.13.11.19), which converts cysteamine, derived from coenzyme A degradation, to hypotaurine. In addition to these enzymes, taurine transporter (Taut), a highly conserved membrane transporter is critical for the transport and recycling of taurine and plays crucial roles in intestinal functions (O’Flaherty et al., 1997; Shimizu and Satsu, 2000). Fish have varied taurine biosynthesis capability, possibly reflecting differences in the expression ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 5 levels/activities of the key biosynthetic enzymes and the taurine transporter (Liu et al., 2017). For instance, Csad activity has been reported to differ among different teleost species (El-Sayed, 2014; Salze and Davis, 2015) and an apparent lack of Csad activity has been reported in fish families such as the Labridae, Scombridae and Soleidae (Salze and Davis, 2015) and ABT (Yokoyama et al., 2001). So far, it is unknown if the metabolic pathway for biosynthetizing taurine using enzymes to transform methionine-derived cysteine is active in ABT. Therefore, if ABT is unable to synthesize taurine by endogenous metabolism, dietary input would be essential especially for larval stages where biosynthetic functions in general are still developing and incomplete (De la Rosa and Stipanuk, 1985). In the wild, ABT larvae can assimilate taurine from natural food, mainly copepods (Uotani et al., 1990; Catalan et al., 2011) that contain high levels of taurine (Van der Meeren et al., 2008; Karlsen et al., 2015). In farming, taurine would have to be supplied by feed and, given the present trend in aquafeed production, with fish meal and oil being replaced by terrestrial plant sources that are devoid of taurine, it is crucial to determine the taurine biosynthetic capacity of ABT, as a deficiency in this nutrient could appear (Gatlin et al., 2007; Barrows et al., 2008; Takagi et al., 2008). This is particularly important in ABT, a top predator in the trophic chain, suggesting that taurine enrichment of feed might be essential. Some previous studies have indicated the positive effect that dietary taurine can have on teleost larvae, such as enhancement on growth (Matsunari et al., 2005a,b, 2008, 2013; Karlsen et al., 2015; Kim et al., 2016), feed conversation ratio and lipid metabolism (Chatzifotis et al., 2007), digestive enzyme activities (Salze et al., 2012), and metamorphosis (Pinto et al., 2010). Indeed, a recent study in Pacific bluefin (Thunnus orientalis) and yellowfin tuna (T. albacares) larvae demonstrated that feeding rotifers enriched with 800 mg taurine L-1 promoted larval growth and total protein content (Katagiri et al., 2017), suggesting that taurine is an important nutrient for the early stages of rapidly growing teleost species. The aim of the present study was to provide insight into the molecular mechanisms involved in taurine biosynthesis and transport in ABT by studying the tissue distribution, ontogenetic development and response to graded dietary taurine supplementation of cdo, csad, ado and tauT genes ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 6 For this purpose, the open reading frames (ORF) of the genes were sequenced and their expression determined by real time quantitative PCR (qPCR) in tissues and during development. Additionally, a dose-response nutritional trial was performed by feeding ABT larvae from mouth opening to 14 days after hatching (dah) with rotifers enriched with four increasing levels of taurine (0.0 g taurine per 106 rotifers, tau0; 0.5 g taurine per 106 rotifers, tau05; 1.0 g taurine per 106 rotifers, tau1 or 2.0 g taurine per 106 rotifers, tau2). Moreover, the effects of graded taurine inclusion in rotifers on the expression of larval ABT genes related to antioxidant and digestive enzymes was also investigated. 2. Materials and Methods 2.1. Isolation of genes of taurine metabolism Sequences of genes encoding for taurine metabolism (tauT, cdo, ado and csad) were obtained by identifying the sequences from Sequence Read Archives (SRA) SRX2255758, ERX555873 and ERX555874. The set of contiguous sequences were assembled using CAP3 (Huang and Madan, 1999) and identity of the deduced amino acid (aa) sequences confirmed using the BLASTp sequence analysis service of the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). Primers were designed in order to sequence the open reading frames (ORF) of each gene (Supplementary Table) using cDNA from whole ABT larvae (see below) as template. PCR products obtained were purified using the Illustra GFX PCR DNA and Gel Band Purification kit (GE Healthcare, Little Chalfont, UK) and sequenced to confirm identity (Sanger ABI3730xl, Eurofins Genomics, Konstanz, Germany). Subsequently, primers for qPCR were designed on these PCR fragments using the online software Primer3 (Untergasser et al., 2012; Supplementary Table). The deduced aa sequences of the newly sequenced ABT tauT, cdo, ado and csad and sequences of these genes of a variety of species across vertebrate and invertebrate lineages were aligned with the ClustalW tool (BioEdit v7.0.9, Tom Hall, Department of Microbiology, North Carolina State University, USA). Phylogenetic analysis was performed using the neighbour-joining ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 7 method with MEGA 5.1 (http://www.megasoftware.net/) (Saitou and Nei, 1987). Confidence in the resulting tree branch topology was measured using bootstrapping through 1,000 replications. 2.2. Tissue RNA extraction and cDNA synthesis Samples of 100 mg of larvae or tissue were homogenized in 1 mL of TRI Reagent (SigmaAldrich, Dorset, UK) using a bead tissue disruptor (BioSpec, Bartlesville, OK, USA) before being mixed with 100 µL BCP (Phase separation reagent, 1–bromo–3–chloropropane, Sigma-Aldrich). The upper aqueous phase was transferred to a fresh tube and mixed with RNA precipitation solution (sodium chloride + sodium citrate sesquihydrate, Sigma-Aldrich) and isopropanol. After centrifugation, the RNA pellet was washed twice with ethanol and resuspended in molecular biology grade water. Quantity and quality of the RNA were determined by spectrophotometry using a NanoDrop ND-1000 (Labtech Int., East Sussex, UK), and integrity determined by electrophoresis using 200 ng of total RNA in 1 % agarose gel. cDNA was synthesized using 2 μg of total RNA and random primers in 20 μL reactions and the high capacity reverse transcription kit without RNase inhibitor according to the manufacturer’s protocol (Applied Biosystems, Warrington, UK). 2.3. Quantitative PCR (qPCR) analysis of gene expression Primers for qPCR were designed on the above PCR fragments for taurine metabolism genes using the online software Primer3 (Untergasser et al., 2012), and were available for ABT genes related to antioxidant enzymes, digestive enzymes and housekeeping from previous studies (Betancor et al., 2017a,b) (see Supplementary Table). Three housekeeping genes were tested (elongation factor1α, elf1α, ubiquitin, ubiq and β-actin, bactin), with elf1α and ubiq selected as being more stable according to geNorm (Vandesompele et al., 2002; M stability value = 0.165 for both genes). The efficiency of primers for each gene was evaluated by serial dilutions of cDNA pooled from the samples to confirm it was > 85 % for all primer pairs. qPCR was performed using a Biometra TOptical Thermocycler (Analytik Jena, Goettingen, Germany) in 96-well plates in duplicate 20 μL reaction ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 8 volumes containing 10 μL of Luminaris Color HiGreen qPCR Master Mix (Thermo Scientific, Hemel Hempstead, UK), 1 μL of the primer corresponding to the analyzed gene (10 pmol concentration), 3 μL of molecular biology grade water, and 5 μL of cDNA (1/20 diluted). In the case of housekeeping genes only 2 μL of cDNA were used increasing the molecular biology grade water to 6 μL. In addition, amplifications were carried out with a systematic negative control (NTC, no template control) containing no cDNA. Standard amplification parameters contained a UDG pre-treatment at 50 °C for 2 min and an initial denaturation step at 95 °C for 10 min, followed by 35 cycles: 15 s at 95 °C, 30 s at the annealing temperature (Supplementary Table 1) and 30 s at 72 °C. At the end of the qPCR run, a melt curve of 0.5 °C increments from 75 °C to 90 °C was performed, enabling confirmation of the amplification of a single product in each reaction. For gene expressions in ontogenesis and the dietary trial, the expression levels (gene expression fold change) of the target genes were calculated following the method described by Pfaffl (Pfaffl, 2001). The relative expression of each gene among the tissues was calculated as the logarithm of arbitrary units after normalization against the expression level of the housekeeping gene elf1α. One arbitrary unit was equal to the lowest expression level of the gene in each dataset. 2.4. Tissue distribution of taurine metabolism genes Samples of tissues including brain, gills, heart, kidney, spleen, liver, intestine, white muscle, red muscle, adipose tissue, ovary and testis were obtained from broodstock tuna (n = 4; 2 males and 2 females; between 200 - 250 kg total weight and 10 to 15 years old) that were being sacrificed as part of the normal operating procedures to check for maturation stage and gonadal development. Additionally, ovaries and testis from a futher two females and males were collected in order to have an adequate sample size (n = 4). All tissue samples (~ 100 mg) were placed in RNALater® (SigmaAldrich, Dorset, UK), left overnight at 4 °C and subsequently stored at -70 °C prior to RNA extraction. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 15 The taurine metabolism genes showed varied tissue distributions (Fig. 2). The highest number of transcripts of cdo was found in adipose tissue, followed by liver and intestine. In contrast, the expression level of csad was highest in kidney followed by intestine with liver showing the lowest value. The highest number of mRNA copies of tauT were found in red muscle, followed by white muscle ≥ spleen, with only a low level found in liver. With ado, testis and brain were the tissues with the higher numbers of transcripts whereas expression was much lower in all the other tissues. 3.4. Dietary trial 3.4.1. Taurine content in ABT larvae ABT larvae effectively accumulated taurine in their bodies as a strong and positive correlation was found between dietary taurine and larval taurine levels (Tables 1 and 2). This relationship was found to be linear with an R2 value of 0.95 (y = 5.3x – 4.3) (Table 2). 3.4.2. Growth, development and survival of ABT larvae Growth performance of ABT larvae 14 dah and fed on rotifers B. rotundiformis enriched with Algamac 3050 Bio Marine® and different doses of taurine (0.0, 0.5, 1.0 and 2.0 g taurine.10-6 rotifer) is shown in Table 3. Total length and weights were significantly highest when ABT larvae were fed diet tau1 (rotifers enriched with 0.5 g taurine per106 rotifers), which corresponded to 3.7 mg taurine g-1 rotifer dry mass based on the measured taurine content of the rotifers (Table 1), and numerically lowest in those fed tau0. Flexion index was significantly higher in ABT larvae fed tau1 compared to larvae fed tau0 and tau0.5, with larvae fed tau2 showing an intermediate value. While ABT larvae fed the tau1 diet showed the numerically highest average survival, there were no statistically significant differences in survival among ABT larvae fed the different taurine doses largely due to variations within treatments. 3.4.3. Gene expression in ABT larvae ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 16 The expression levels of both cdo and csad were both significantly higher in larvae fed diet tau1 compared to larvae fed tau0 and the other levels of dietary taurine (Fig. 3). In contrast, the expression of ado showed the opposite pattern to this with expression being lower in larvae fed tau1 compared to larvae fed the other diets. The tauT expression levels showed a decreasing trend as dietary taurine increased with expression in larvae fed tau0 being significantly higher than in larvae fed the diets supplemented with taurine (Fig. 3). The expression of all the digestive genes measured showed a similar pattern with highest expression in ABT larvae fed tau1 (Fig. 4). The expression of both bile salt-activated lipase 1 (bal1) and phospholipase A2 (pla2) was significantly higher in ABT larvae fed tau1 compared to larvae fed tau0. While a similar pattern in expression was observed with bile salt-activated lipase 2 (bal2) the differences did not reach statistical significance. All the genes of the antioxidant system that were measured showed a similar pattern with the highest expression in ABT larvae fed the tau1 diet (Fig.5). While this was significant for superoxide dismutase (sod), glutathione peroxidase 1 (gpx1) and glutathione peroxidase 4 (gpx4), the differences in expression of catalase (cat) were not statistically significant. 4. Discussion The present study aimed to investigate the impacts of dietary taurine level via enrichment of rotifer on growth and metabolism of first feeding ABT larvae. Firstly, key genes of taurine metabolism were cloned, with the full ORF sequence obtained for cdo, and partial sequences achieved for tauT, csad and ado. For tauT the partial ORF (324 aa) contained potential N-glycosylation sites and six transmembrane domains, which was in agreement with tauT of other species (Wang et al., 2017). Phylogenetic analyses showed a clear distinction between teleost and mammal clusters with similarity scores of more than 90 % and 81 %, respectively. Furthermore, molluscs were clearly separated from both mammals and teleosts, which may indicate that taurine transporter developed earlier in evolution as previously suggested (Hui et al., 2012). In agreement the phylogenetic trees ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 17 for the three genes grouped ABT together with other teleost species indicating high evolutionary conservation. The full mRNA sequence for Cdo was obtained with an ORF coding for a protein of 202 aa, whereas a partial ORF sequence of 166 aa was acquired for Ado. Alignment of aa from both genes revealed cupin motifs 1 and 2 separated by an intermotif region, which are common characteristics for cupin proteins (Dunwell et al., 2001; Stipanuk et al., 2011; Wang et al., 2016). The partial ORF sequence coding for 176 aa found for csad contained the important pyridoxal-dependent decarboxylase conserved domain, an enzyme group which is also present in csad of Pagrus major, Seriola quinqueradiata, Oreochromis niloticus, and Oryzias latipes (Haga et al., 2015). The phylogenetic analyses also revealed high similarity scores for the ABT genes with genes of other teleosts other than salmonids in the case of Csad, and Salmo salar and Anguilla japonica for Cdo. This highlights interesting differentiation in taurine metabolism genes, on one hand, between freshwater and marinewater species and, on the other hand, between anadromous and catadromous fish. Thus, evolutionary adaptations to different lifestyles, including migrations and transfer between freshwater and marine environments with associated different requirements of osmoregulation may have generated differentiation in genes for taurine assimilation and/or biosynthesis. The expression levels of the four ABT taurine metabolism genes was evaluated during early ontogenesis from 1 dah to 25 dah. Results showed that, during early larval development, the expression level of the csad gene peaked earlier than the expression levels of cdo, ado and tauT. In general, expression of the genes was low 1 dah and increased during development suggesting increasing biosynthesis of taurine, which may reflect that taurine is necessary for larval development of ABT. As the transcript copies could be detected at 1 dah, it is possible that maternal mRNA is present in the egg, as has been observed in zebrafish embryos (Chang et al., 2013). The peak of tauT transcript copy number at 25 dah was similar to results found in Senegalese sole at 30 dah by Pinto et al. (2010), which may indicate that during the intermediate larval stage (18-25 dah), marine fish larvae including ABT have increased capacity to transport taurine. Although the ontogenic analysis ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 18 of gene expression was carried out on whole larvae, muscle is the main tissue and, given that tauT expression was greatest in ABT muscle tissues, it is likely that the peak in tauT expression reflects the enhanced transport of taurine in muscle, where growth potential is very high at this stage of development. In agreement with this, Ado, an enzyme that produces hypotaurine by the oxidation of cysteamine through a pathway different to that of Csad and Cdo (Salze and Davis, 2015), also peaked at 25 dah. However, the highest fold change (FC) for these genes is relatively low (1.8 for tauT and 2.4 for ado), whereas a FC of 18.3 and 33.3 was observed for csad and cdo, respectively, both enzymes participating in the same biosynthetic pathway. These high FC indicate that the Csad/Cdo combination is the main pathway for taurine biosynthesis and that csad is the rate limiting enzyme for taurine biosynthesis in both mammals (De La Rosa and Stipanuk, 1985) and fish (Chang et al., 2013). The four taurine metabolism genes were expressed to some extent in all tissues of ABT examined, in agreement with other fish species (Pinto et al., 2012; Haga et al., 2015; Plasus et al., 2019). However, in the present study, tauT was predominantly expressed in muscle tissue (white > red), which is consistent with fish muscle containing relatively high levels of taurine (Huxtable, 1992). Therefore, the high expression levels observed in this tissue might reflect the physiological function of tauT, inducing the uptake of taurine into skeletal muscle cells. Adipose tissue displayed the highest cdo transcript copy number, indicating a high potential for taurine biosynthesis in this tissue, as found previously in mice (Ueki and Stipanuk, 2008). However, taurine also plays an important role in osmoregulation and this may be reflected in the high mRNA copy numbers of csad in kidney, which has also been observed in other teleost species (Haga et al., 2015). In the present study, the highest expression levels of ado in ABT were observed in testis and brain. The high level of expression of these genes in gonads is related to the high concentration of taurine in these tissues (Plante et al., 2008). Little information is currently available regarding the cysteamine pathway involving ado, although a recent study in carp (Cyprinus carpio) reported brain to be the main tissue expressing the enzyme, although testis was not included in that study (Plasus et al., 2019). Studies in ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 19 different animal species have also shown that activities of the taurine metabolism enzymes vary among tissues (Kuo and Stipanuk, 1984; Stipanuk and Ueki, 2011). Therefore, it seems that the pattern of tissue expression of the taurine metabolism genes in ABT is related to the biochemical functions each enzyme and the role of different tissues. On the other hand, it should noted that high mRNA levels of these genes have not been correlated to higher enzyme activity (Higuchi et al., 2012). This could explain why, for instance, the expression levels of csad in kidney were elevated whereas cdo levels were quite low, suggesting that regulation might be at the protein level as opposed to the transcriptional level. Overall though, the presence and expression of these genes indicates that, despite being a top predator, ABT has some capacity to biosynthesize taurine, and does not rely entirely upon dietary intake. However, no taurine was detected in larvae fed tau0, which indicates that although they contain the enzymatic machinery, it is not efficient. In contrast, neither mRNA nor enzyme activity for some of the taurine metabolism enzymes have been identified in some fish species such as cobia (Rachycentron canadum; Goto et al., 2001a; Watson et al., 2014). In order to confirm an active role for taurine metabolism including biosynthesis in ABT, a trial was carried out by feeding larvae from mouth opening to 14 dah with different levels of taurine supplied via rotifers enriched with increasing levels of taurine. Taurine concentration in larvae was strongly correlated to the level of taurine enrichment in rotifer in agreement with previous trials (Matsunari et al., 2007; Katagiri et al., 2017; Koven et al., 2018). This confirms that ABT larvae are able to assimilate dietary taurine into their tissues and may reflect a taurine requirement. The lack of taurine in the enrichment media (tau0) led to poor growth in terms of total length and total dry mass and impaired development indicated by reduced flexion index. In contrast, the highest growth and most rapid development was obtained in larvae fed tau1 that corresponded to 3.7 mg taurine per g rotifer dry mass. These results are consistent to what has been observed in larvae of other tuna (Katagiri et al., 2017) and teleost species (Matsunari et al., 2005a.b, 2013; Pinto et al., 2010; Hawkyard et al., 2015; Kim et al., 2016), where enrichment of rotifers with taurine promoted larval growth. Nonetheless, the increase of dietary taurine from 3.7 to 9.0 mg g-1 rotifers did not further ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 20 promote larvae growth, similarly to a study in humpback grouper (Cromileptes altivelis), where increasing the levels from 2.7 to 8.5 mg taurine g-1 rotifer did not lead to increased larval total length (Ridwan and Haryati, 2017). These results indicate that levels of taurine of around 3.8 mg g-1 may satisfy the requirements of ABT larvae for this nutrient. In contrast, survival of larval ABT was not significantly affected by dietary taurine in the present study in contrast to several previous studies in Pagrus major and Paralichthys olivaceus (Chen et al., 2004a,b), Seriola dumerili (Matsunari et al., 2013), Nibea albiflora (Xie et al., 2015) or Seriola lalandi (Rotman et al., 2017). This is likely due to the large inter-tank variability observed in the present trial, although a lack of effect of dietary taurine has also been reported in other species such as Atractoscion nobilis (Rotman et al., 2017) and Solea Senegalensis (Pinto et al., 2010). While the above confirmed a role for dietary taurine in larval ABT, the present trial also demonstrated a role for endogenous taurine metabolism. The mRNA copy number of tauT was regulated by dietary taurine in a dose dependent manner, with the gene being down-regulated as dietary levels of taurine increased. This indicates that when substrate (taurine) levels are low, tauT expression is up-regulated to promote and enhance the absorption and transport of taurine. Similar results were observed in turbot (Scophtalmus maximus) both in vitro (Wang et al., 2017) and in vivo (Wei et al., 2018) as well as in Atlantic salmon smolts (Zarate and Bradley, 2007). Aside from tauT, other genes in teleosts have been speculated to take part in taurine homeostasis, participating in the biosynthesis of this amino acid. In this respect, the regulation of taurine biosynthesis is complicated, as it is not only regulated by the product taurine but also the levels of substrate sulfur amino acids, with differential regulation of csad and cdo (Wang et al., 2016). It would be expected that both enzymes would be up-regulated when taurine levels were low/deficient, but this was not the case as peak mRNA copy numbers were observed in larvae fed tau1 with 3.7 mg taurine per g rotifers. Several studies in teleosts have reported the lack of regulation by taurine of cdo expression/activity, which was mainly regulated by cysteine and methionine (Gaylord et al., 2006; Wang et al., 2015, 2016). Therefore, the consistent pattern of expression of both cdo and csad in ABT could be influenced by ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 21 the combination/ratio of sulphur amino acids rather than solely by the levels of dietary taurine. Additionally, the lack of regulation by dietary taurine could indicate a low capacity to biosynthesize taurine in ABT, given that in the wild these fish usually consume taurine-rich prey, such as smaller fish. Consistent with this, no Csad activity was found in Pacific bluefin tuna (Yokoyama et al., 2001). There is another pathway to produce taurine in teleosts using cysteamine, produced from the breakdown of coenzyme A, which is then the substrate for cysteamine dioxygenase (Ado). Most of the studies in teleosts have focussed on the cysteine sulfinic acid pathway, and paid little attention to the expression and/or activity of ado. In the present study, a partial ado mRNA was reported for the first time in tuna, and it was shown that its transcript copy number was modulated by dietary taurine level. A dietary taurine level of 3.7 mg g-1 rotifer (tau1) lead to down-regulation of ado expression although the levels were not statistically different to those in fish fed tau0 or tau2. Previous studies showed no regulation of ado expression by taurine in a zebrafish cell line, which could indicate that, similar to csad and cdo, ado could be regulated post-transcriptionally (Liu et al., 2017). These results suggest that the cysteamine pathway is not very active in ABT, as has been shown for other carnivorous marine teleosts (Goto et al., 2001b). In addition to promoting growth, taurine has also been shown to enhance digestibility in fish (Lunger et al., 2007). The digestive enzymes, bile salt-dependant lipases 1 and 2 (bal1 and bal2), have been reported to be the main enzymes involved in lipid digestion in Pacific bluefin tuna (Murashita et al., 2014). In the present trial, both bal1 and bal2 showed a similar pattern of expression, with highest expression levels in larvae fed tau1 (3.7 mg g-1 rotifers). Furthermore, pla2, an enzyme involved in intestinal phospholipid digestion (Tocher, 2003), showed the same pattern as bal1, again with highest expression level in larvae fed tau1. Taken together these results indicate a digestive promoting effect of taurine at an enrichment level of 3.7 mg taurine g-1 rotifer, which was entirely consistent with the impact of dietary taurine on ABT larval growth. However, it is worth noting that the expression levels of the digestive genes could be influenced by growth rather than dietary taurine levels, as previously suggested (Betancor et al., 2017b). Indeed, similar results were ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 22 found by Sæle et al. (2010), where a relationship between bal genes and cod (Gadus morhua) larvae body size was shown. Taurine is known to have antioxidant properties, and can serve as a scavenger of some reactive oxygen species (Metayer et al., 2008). Indeed, taurine deficiency can have an impact on red-ox balance that can, consequently, result in mitochondrial oxidative stress in vitro (Jong et al., 2012). A previous study found that Cat, Sod and Gpx activities increased with dietary taurine level in several fish species (Li et al., 2016). In agreement, the expression levels of sod, gpx1 and gpx4 in ABT in the present study were highest in larvae fed tau1, these larvae also showing the highest growth and rate of development. Indeed, a strong correlation was found between larval total length, dry weight and gpx1 expression levels (r = 0.6 and 0.5, respectively), which corroborates the role of taurine as an antioxidant. In contrast, another study showed decreased expression of antioxidant enzymes when sea bream larvae were fed increased dietary taurine levels (Izquierdo et al., 2019). In summary, the present study indicated that ABT larvae possess enzymes necessary to biosynthesize taurine through the two main pathways. The three enzymes and the taurine transporter showed differential tissue expression and could be detected before the onset of external feeding. Expression of the biosynthesis enzymes was not obviously regulated by dietary taurine level, possibly indicating a nutritional requirement for this nutrient. In contrast, tauT expression was upregulated when dietary levels of taurine were low, indicating a role for this gene in maintaining taurine levels in muscle and taurine homeostasis in ABT. Rotifers supplemented with taurine at 1 g per 106 rotifers improved the growth of ABT larvae, without affecting final survival. In conclusion, despite the presence of taurine biosynthesis genes, ABT larvae required a supply of dietary taurine at around 3.7 mg g-1 feed (rotifer) in order to ensure adequate growth and development. Acknowledgements We wish to thank the technical staff at Laboratory of Marine Aquaculture (IEO), Puerto de Mazarrón (Murcia), Spain and Nutritional Analytical Services (NAS), Institute of Aquaculture, University of ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 23 Stirling, UK that contributed to this work. This work was supported by the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía, Proyecto de Excelencia de Promoción General del Conocimiento [Ref. RNM 733, 2012), and Programa Estatal de Investigación del Ministerio de Economía y Competitividad [Ref. AGL2014-52003-C2-1-R, 2014]. 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Chang, Y.C., Ding, S.T., Lee, Y.H., Wang, Y.C., Huang, M.F., Liu, I.H., 2013. Taurine homeostasis requires de novo synthesis via cysteine sulfinic acid decarboxylase during zebrafish early embryogenesis. Amino Acids 44, 615-629. Chatzifotis, S., Polemitou, I., Divanach, P., Antonopoulou, E., 2007. Effect of dietary taurine supplementation on growth performance and bile salt activated lipase activity of common dentex, Dentex dentex, fed a fish meal/soy protein concentrate-based diet. Aquaculture 275, 201-208 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 24 Chen, J.N., Takeuchi, T., Takahashi, T., Tomoda, T., Koiso, M., Kuwada, H., 2004. Effect on rotifers enriched with taurine on growth and survival activity of red sea bream Pagrus major larvae. Nippon Suisan Gakkaishi 70, 542-547. Chen, J.N., Takeuchi, T., Takahashi, T., Tomoda, T., Koiso, M., Kuwada, H., 2004. Effect on rotifers enriched with taurine on growth in larvae of Japanese flounder Paralichthys olivaceus. Nippon Suisan Gakkaishi 71, 342-347. De la Gandara, F., Ortega, A., Buentello, A., 2016. Tuna aquaculture in Europe. Advances in Tuna Aquaculture 6, 115-157. De la Rosa, J., Stipanuk, M.H., 1985. The effect of taurine depletion with guanidinoethane sulfonate on bile acid metabolism in the rat. Life Sci. 36, 1347-1351. Dunwell, J.M., Culham, A., Carter, C.E., Sosa-Aguirre, C.R., Goodenough, P.W., 2001. Evolution of functional diversity in the cupin superfamily. Trends Biochem. Sci. 26, 740-746. El-Sayed, A.F.M., 2014. Is dietary taurine supplementation beneficial for farmed fish and shrimp? A comprehensive review. Rev. Aquac. 6, 241-255. Gatlin, D., Barrows, F.T., Brown, P., Dabrowski, K., Gaylord, T.G., Hardy, R.W., Herman, E., Hu, G., Krogdahl, A, Nelson, R., Overturf, K., Rust, M., Sealey, W., Skonberg, D., Souza, E.J, Stone, D, Wilson, R., Wurtele, E., 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquacult. Res. 38, 551-579. Gaylord, T.G., Teague, A.M., Barrows F.T., 2006. Taurine supplementation of all-plant protein diets for rainbow trout (Oncorhynchus mykiss). J. World. Aquac. Soc. 37, 509-517. Goto, T., Tiba, K., Sakurada, Y., Takagi, S., 2001a. Determination of hepatic cysteinesulfinate decarboxylase activity in fish by means of OPA-prelabelling and reverse-phase high-performance liquid chromatographic separation. Fisheries Sci. 67, 553-555. Goto, T., Matsumoto, T., Takagi, S., 2001b. Distribution of the hepatic cysteamine dioxygenase activities in fish. Fisheries Science 67, 1187-1189. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 31 Figure Legends Figure 1. Expression of cysteine dioxygenase (cdo), cysteine sulfinic acid decarboxylase (csad), taurine transporter (tauT) and 2-aminoethanethiol dioxygenase (ado) during development of Atlantic bluefin tuna (Thunnus thynnus) larvae (1 dah-25 dah) reared under standard procedures. Results represent means ± standard error (n = 4) of relative expression normalized with two housekeeping genes (ubiquitin and elongation factor 1 alpha). Different letters show significant differences for the expression of each gene during development. Figure 2. Tissue distribution of cdo, csad, tauT and ado transcripts in Atlantic Bluefin tuna broodstock. Transcript expression level was determined by qPCR in 12 tissues with values denoting the log-normalized (ef1  ) relative expression of the target genes in each tissue. Data represent the average of four individuals (n = 4) with standard errors (SEM). B, brain; G, gills; H, heart; K, kidney; S, spleen; L, liver; I, intestine; R, red muscle; W, white muscle; A, adipose tissue; O, ovary; T, testis. Figure 3. Nutritional regulation of taurine metabolism genes, cysteine dioxygenase (cdo), cysteine sulfinic acid decarboxylase (csad), taurine transporter (tauT) and cysteamine dioxygenase (ado) in larvae of Atlantic bluefin tuna (T. thynnus). Larvae were fed rotifers (Brachionus rotundiformis) enriched with 4 levels of taurine: 0.0 (tau0); 0.5 (tau0.5); 1.0 (tau1); 2.0 (tau2) g taurine.10-6 rotifers. Values are normalized expression ratios, corresponding to an average of 6 pools of larvae (n = 6) with standard errors (SEM). Letters denote significate differences as determined by one-way ANOVA (p < 0.05). Figure 4. Nutritional regulation of digestive enzymes, bile salt-activated lipase 1 (bal1), bile saltactivated lipase 2 (bal2) and phospholipase A2 (pla2) in larvae of Atlantic bluefin tuna (T. thynnus). Larvae were fed rotifers (Brachionus rotundiformis) enriched with 4 levels of taurine: 0.0 (tau0); 0.5 (tau0.5); 1.0 (tau1); 2.0 (tau2) g taurine.10-6 rotifers. Values are normalized expression ratios, ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 32 corresponding to an average of 6 pools of larvae (n = 6) with standard errors (SEM). Letters denote significate differences as determined by one-way ANOVA (p < 0.05). Figure 5. Nutritional regulation of antioxidant enzymes, glutathione peroxidase 1 (gpx1), glutathione peroxidase 4 (gpx4), catalase (cat), superoxide dismutase (sod) in larvae of Atlantic bluefin tuna (T. thynnus). Larvae were fed rotifers (Brachionus rotundiformis) enriched with 4 levels of taurine: 0.0 (tau0); 0.5 (tau0.5); 1.0 (tau1); 2.0 (tau2) g taurine.10-6 rotifers. Values are normalized expression ratios, corresponding to an average of 6 pools of larvae (n = 6) with standard errors (SEM). Letters denote significate differences as determined by one-way ANOVA (p < 0.05). ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 33 Table 1. Total amino acid content including taurine (mg/g dry mass) of rotifers B. rotundiformis enriched with Algamac 3050® and increasing doses of taurine (0.0 g/106 rotifers (tau0), 0.5 g/106 rotifers (tau0.5), 1.0 g/106 rotifers (tau1) and 2.0 g/106 rotifers (tau2). tau0 tau0.5 tau1 tau2 Taurine 0.0 ± 0.0e 2.5 ± 0.2d 3.7 ± 0.1c 9.0 ± 0.1a EAA Valine 18.5 ± 3.4 22.8 ± 0.8 20.4 ± 1.2 22.2 ± 3.0 Isoleucine 1.7 ± 0.3 2.1 ± 0.1 1.9 ± 0.1 2.0 ± 0.3 Leucine 26.8 ± 1.8b 30.3 ± 0.6a 26.4 ± 1.6b 31.3 ± 0.1a Phenylalanine 17.3 ± 1.2b 19.5 ± 0.5a 16.9 ± 1.0b 20.2 ± 0.3a Histidine 6.1 ± 0.8b 7.1 ± 0.2a 6.0 ± 0.4b 7.4 ± 0.6a Lysine 24.0 ± 1.8b 28.0 ± 0.6a 23.0 ± 2.1b 30.1 ± 0.1a Arginine 17.5 ± 3.2b 22.1 ± 0.4a 18.6 ± 1.7ab 23.0 ± 0.3a Threonine 11.3 ± 1.6b 14.7 ± 0.6a 11.5 ± 0.6b 14.3 ± 0.3a Methionine 7.2 ± 0.1b 8.4 ± 0.1a 7.1 ± 0.6b 8.4 ± 0.1a NEAA Aspartic acid 33.9 ± 2.1b 38.1 ± 0.8a 32.5 ± 1.9b 38.2 ± 0.2a Glutamic acid 42.4 ± 2.8b 49.0 ± 1.4a 42.3 ± 2.4b 49.5 ± 0.3a Serine 12.1 ± 0.4bc 16.1 ± 0.4a 10.4 ± 0.6c 13.3 ± 0.7b Proline 17.9 ± 1.2ab 19.7 ± 0.7a 16.7 ± 1.0b 19.8 ± 0.3a Glycine 15.7 ± 1.5b 17.2 ± 0.4ab 16.2 ± 1.1b 18.9 ± 0.3a Alanine 15.4 ± 1.0bc 17.1 ± 0.5ab 15.5 ± 0.7bc 18.1 ± 0.2a Tyrosine 13.4 ± 0.8b 15.6 ± 0.7a 12.5 ± 0.8bc 15.3 ± 0.2a Cysteine 3.4 ± 0.1ab 4.0 ± 0.1a 3.4 ± 0.3ab 3.0 ± 0.2b Data are means ± SD (n = 3). Means within a row bearing different superscript letters are significantly different as determined by one-way analysis of variance (ANOVA), and Tukey’s multiple comparison test (P < 0.05). EAA, essential amino acids; NEAA, non-essential amino acids. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 34 Table 2. Total amino acid content including taurine (mg/g dry mass) of Atlantic bluefin tuna (T. thynnus L.) larvae 14 days after hatch fed on rotifers B. rotundiformis enriched with Algamac 3050 ® and increasing doses of taurine; 0.0 g/106 rotifers (tau0), 0.5 g/106 rotifers (tau05), 1.0 g/106 rotifers (tau1) and 2.0 g/106 rotifers (tau2). tau0 tau0.5 tau1 tau2 Taurine 0.0 ± 0.0d 1.8 ± 0.1c 3.8 ± 0.1b 6.4 ± 0.2a EAA Valine 35.6 ± 0.6 35.9 ± 0.1 32.5 ± 4.6 36.8 ± 0.6 Isoleucine 25.8 ± 0.4a 26.1 ± 0.2a 25.7 ± 0.4a 26.3 ± 0.5a Leucine 42.4 ± 0.3bc 43.1 ± 0.2ab 42.8 ± 0.5b 44.4 ± 0.4a Phenylalanine 23.9 ± 0.7 24.2 ± 0.7 24.1 ± 0.4 25.1 ± 0.8 Histidine 3.2 ± 0.4 3.3 ± 0.2 3.2 ± 0.2 3.1 ± 0.6 Lysine 45.6 ± 0.5b 46.5 ± 0.2b 46.6 ± 0.6b 48.5 ± 0.7a Arginine 8.5 ± 0.5 9.1 ± 0.2 8.9 ± 0.2 9.1 ± 0.3 Threonine 10.2 ± 0.4c 11.7 ± 0.4ab 11.5 ± 0.3b 12.7 ± 0.5a Methionine 22.0 ± 0.8ab 22.4 ± 0.3ab 20.9 ± 1.1b 23.8 ± 1.3a NEAA Aspartic acid 9.4 ± 0.8 9.7 ± 0.2 9.2 ± 0.5 9.7 ± 0.2 Glutamic acid 18.2 ± 0.6 19.4 ± 0.9 19.6 ± 0.6 20.1 ± 1.7 Serine 3.3 ± 0.2b 3.9 ± 0.5ab 4.7 ± 0.6a 4.6 ± 0.3a Proline 15.1 ± 0.6ab 15.8 ± 0.5ab 14.8 ± 0.3b 16.0 ± 0.3a Glycine 10.2 ± 0.5 10.4 ± 0.4 9.5 ± 0.8 9.2 ± 1.0 Alanine 13.7 ± 0.6 14.8 ± 0.5 14.0 ± 0.5 14.5 ± 0.2 Tyrosine 17.4 ± 0.5 17.9 ± 0.8 17.2 ± 0.6 18.3 ± 0.8 Cysteine 4.1 ± 0.4 3.6 ± 0.4 3.2 ± 0.8 4.1 ± 0.8 Data are means ± SD (n = 3). Means within a row bearing different superscript letters are significantly different as determined by one-way analysis of variance (ANOVA), and Tukey’s multiple comparison test (P < 0.05). EAA, essential amino acids; NEAA, non-essential amino acids. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 35 Table 3. Growth performance of 14 days after hatch ABT larvae fed on rotifers Brachionus rotundiformis enriched with Algamac 3050 Bio Marine® and different doses of taurine (0.0, 0.5, 1.0 and 2.0 g of taurine per 106 rotifers). Results for growth performance are presented as means ± SD (n = 25 per replicate for total length, total weight and flexion index, and n = 3 for survival rates. An SD of 0.0 implies an SD of < 0.05. Means within a row bearing different superscript letters are significantly different as determined by one-way analysis of variance (ANOVA), and Tukey’s multiple comparison test (P < 0.05). tau0 tau0.5 tau1 tau2 Total length (mm) 6.6 ± 0.4c 6.7 ± 0.1bc 6.9 ± 0.3a 6.8 ± 0.3b Dry weight (mg) 0.41 ± 0.04c 0.45 ± 0.01bc 0.55 ± 0.06a 0.46 ± 0.08bc Flexion index 38.7 ± 16.2b 40.0 ± 7.2b 51.0 ± 10.4a 45.7 ± 9.7ab Survival (%) 12.4 ± 1.8 9.6 ± 2.8 14.7 ± 7.8 10.5 ± 8.5 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 36 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 37 Highlights - Tissue distribution, ontogenetic development and expression in response to graded levels of dietary taurine supplementation of genes involved in taurine metabolism were evaluated in Atlantic Bluefin tuna. - Tissue distribution varied, with adipose tissue, kidney, white muscle and testis/brain showing highest expression of cysteine dioxygenase (cdo), cysteine sulfinic acid decarboxylase (csad), 2aminoethanethiol dioxygenase (ado) and taurine transporter (tauT), respectively. - Whole larvae expression of csad peaked at 15 dah, whereas the other genes generally increased throughout development to show highest expression at 25 dah. - Atlantic Bluefin tuna larvae fed 1 g taurine per 106 rotifers (3.7 mg 3 taurine g-1 rotifer) displayed the best growth and expression levels of tauT, cdo, digestive and antioxidant enzymes. ACCEPTED MANUSCRIPT Figure 1 Figure 2 Figure 3