An Investigation on the Effects of Dietary Vitamin E on Juvenile Sea Urchin (Strongylocentrotus intermedius): Growth, Intestinal Microbiota, Immune Response, and Related Gene Expression.

A 90 d feeding experiment was conducted to investigate the effects of vitamin E (VE) on growth, intestinal microbiota, immune response, and related gene expression of juvenile sea urchin (Strongylocentrotus intermedius). Six dry feeds were made to contain graded levels of VE (78, 105, 152, 235, 302, and 390 mg/kg); these were named E78, E105, E152, E235, E302, and E390, respectively. Dry feed E50 and fresh kelp (HD) were used as the control diets. There were six replicates of cages in each dietary group, and each cage held 20 sea urchins with an initial body weight of approximately 1.50 g. Results exhibited that weight gain rate and gonadosomatic index (GSI) of the sea urchins were not significantly affected by dietary VE ranging from 78 to 390 mg/kg. Sea urchins in the dry feed groups showed poorer growth performance, but significantly higher GSI than those in the fresh kelp groups. The pepsin and lipase activities were not significantly promoted by low or moderate VE, but were inhibited by a high level of VE (302-390 mg/kg), while amylase and cellulase activities were significantly increased by low or moderate VE, with the highest values observed in the E105 and E235 groups, respectively. VE addition at a low dosage (105-152 mg/kg) showed inhibitory effects on immune and antioxidant enzyme activities and expression of inflammation-related genes, but showed no beneficial effects at moderate or high dosage (235-390 mg/kg), while a moderate or relatively higher level of VE (235-302 mg/kg) significantly increased the expression of several immune-related genes. The relative abundance of Proteobacteria, Actinobacteria, Ruegeria, and Maliponia in the intestine of the sea urchins increased with the increase in VE in the dry feeds. On the contrary, the relative abundance of the Firmicutes, Bacteroidetes, Escherichia-Shigella, Bacteroides, and Clostridium sensu stricto 1 gradually decreased as VE content increased. These results indicated that a moderate level of VE (172.5-262.4) can achieve ideal digestive enzyme activities and growth performance, but a relatively higher level of VE (235-302 mg/kg) was beneficial for maintaining the immune and antioxidant capacity of juvenile S. intermedius by regulating the expression of inflammation- and immune-related genes and abundance of some bacteria to a healthy state.

Effects of Supplementary Kelp Feeding on the Growth, Gonad Yield, and Nutritional and Organoleptic Quality of Subadult Sea Urchin (Strongylocentrotus intermedius) with Soya Lecithin Intake History.

A 23-week feeding experiment was conducted to investigate the effects of supplementary kelp feeding on the growth, gonad development, and nutritional and sensory properties of sea urchin (Strongylocentrotus intermedius) with soya lecithin (SL) intake history. The feeding experiment was divided into experimental phase I and phase II. During phase I, 48 subadult sea urchins (initial weight: 6.28 ± 0.07 g) were fed one of the feeds with different levels of SL (0%, 1.6%, 3.2%) or kelp (Saccharina japonica) for 12 weeks. Then, all sea urchins were fed kelp for the next 11 weeks during the phase II. Each diet was randomly allocated to six cages of sea urchins. The results of phase I showed that weight gain rate (WGR), gonadosomatic index (GSI), gonad sensory properties (color and texture), and essential amino acid (EAA) contents were not significantly affected by SL level in the feed groups. High level (3.2%) of SL suppressed gonad development of S. intermedius with retarded gametogenesis in the 3.2% SL group (stage Ⅱ) compared to those fed 0% and 1.6% SL groups (stage Ⅲ). Sea urchins fed dry feeds exhibited significantly lower WGR and values of color (redness and yellowness) and texture (hardness and gumminess) but higher contents of EAA in the gonads than those fed kelp. The n-3/n-6 polyunsaturated fatty acid (PUFA) and eicosapentaenoic acid (EPA) of gonads in the groups fed with dry feeds showed no significant differences, but were significantly lower than that of kelp group. At the end of phase II, the gonad yellowness and EPA content of gonads in all dry feed groups were significantly increased by supplementary kelp feeding, with a higher increase observed in S. intermedius with SL intake history, while arachidonic acid (ARA) content was significantly improved by supplementary kelp feeding in S. intermedius with SL intake history. Gonad texture was improved to some extent by supplementary kelp feeding. These results indicated that S. intermedius fed dry feeds showed significantly higher GSI and EAA but poorer organoleptic quality and lower n-3/n-6 PUFA and EPA than those fed kelp. Kelp supplementary feeding improved the fatty acid value and organoleptic quality of gonads, especially for the sea urchins with SL intake history.

Application of SNP in Genetic Sex Identification and Effect of Estradiol on Gene Expression of Sex-Related Genes in Strongylocentrotus intermedius.

Sea urchin (Strongylocentrotus intermedius) is an economically important mariculture species in Asia, and its gonads are the only edible part. The efficiency of genetic breeding in sea urchins is hampered due to the inability to distinguish gender by appearance. In this study, we first identified a sex-associated single nucleotide polymorphism (SNP) by combining type IIB endonuclease restriction site-associated DNA sequencing (2b-RAD-seq) and genome survey. Importantly, this SNP is located within spata4, a gene specifically expressed in male. Knocking down of spata4 by RNA interference (RNAi) in male individuals led to the downregulation of other conserved testis differentiation-related genes and germ cell marker genes. We also revealed that sex ratio in this validated culture population of S. intermedius is not 1:1. Moreover, after a 58-day feeding experiment with estradiol, the expression levels of several conserved genes that are related to testis differentiation, ovary differentiation, and estrogen metabolism were dynamically changed. Taken together, our results will contribute toward improving breeding efficiency, developing sex-controlled breeding, and providing a solid base for understanding sex determination mechanisms in sea urchins.

Comparative lipidomics profiling of the sea urchin, Strongylocentrotus intermedius.

Strongylocentrotus intermedius is an edible sea urchin and well-known for its nutritional value, such as a high content of polyunsaturated fatty acids (PUFAs). We carried out an untargeted lipidomics via high-resolution ultra-high-performance liquid chromatography - mass spectrometry (UPLC-MS) to highlight the features of the lipids profile of sea urchin gonad, which allowed for a more detailed interpretation of the accumulation of PUFAs with different abundances among sea urchins. For the first time, lipidomics profiling of lipid abundances in S. intermedius was demonstrated. We detected 11 PUFAs in sea urchin gonads, which represented >54.13% of the total fatty acid content. A total of 1552 lipid molecular species belonging to 36 lipid classes were identified. Lipidomics profiles data were analyzed using orthogonal partial least squares discriminant analysis (OPLS-DA) model and distinguished the PUFA abundances in both sexes of sea urchins. The significant differences in lipid molecules were highlighted and the major lipid classes identified were phosphatidylcholine (PC [19 species]) among females and triglycerides (TG [11 species]) among males. PC (42: 11) may be used as a potential marker for distinguishing high levels of PUFAs in sea urchin individuals, which as the result of the high level of PC (42:11). These data enrich the lipid profile library of aquatic products and provide a more reliable and refined biomarkers for the further research on fatty acid synthesis and metabolism in aquatic animals.

Molecular characterization and expression of SiFad1 in the sea urchin (Strongylocentrotus intermedius).

Fatty acid desaturases (Fads) are a key enzyme in the process of biosynthesis of highly unsaturated fatty acids (HUFAs). In this study, we cloned the full-length sequence of the SiFad1 gene (SiFad1) and analyzed its expression profiles during different developmental stages and in different tissues of Strongylocentrotus intermedius. The full-length cDNA of SiFad1 is composed of 1086 bp, with a putative open reading frame of 885 bp encoding a polypeptide of 294 amino acid (AA) residues. The predicted molecular mass of SiFad1 is 34.67 kDa and its theoretical pI is 8.41. The presence of conserved motifs including three histidine boxes (HXXXH, HXXHH, XXXHH), a FA_desaturases domain and three transmembrane domains suggests that SiFad1 belongs to the microsomal fatty acid desaturases family. Its tissue distribution showed that the highest expression of SiFad1 is in the intestine and the weakest expression is in Aristotle's lantern of S. intermedius. Time-course expression measurements in different developmental stages showed the highest expression of SiFad1 occurs in the gastrula and the weakest expression in the juvenile sea urchin. Knock-down of SiFad1 by specific siRNA revealed that the significantly depressed expression of Elovl5 had decreased in the coelomocytes, intestines and gonads at 24 h post transfection, indicating that the downstream target gene of SiFad1 is Elovl5 and SiFad1 and Elovl5 have positive regulatory effects. When we examined the changes in fatty acids in the gonads before and after interference, the results showed that after 24 h of interference, the content of C20:4n-6 produced by SiFad1 had decreased. Taken together, these results will enable us to understand the role of SiFad1 in fatty acid anabolism, which will help us to understand the fatty acid synthesis pathways and regulatory mechanisms of Strongylocentrotus intermedius and provide a theoretical experimental basis for improving the ability of sea urchins to synthesize fatty acids and cultivating sea urchins of higher quality and nutritional value.

Regulation of FADS2 transcription by SREBP-1 and PPAR-α influences LC-PUFA biosynthesis in fish.

The present study was conducted to explore the mechanisms leading to differences among fishes in the ability to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs). Replacement of fish oil with vegetable oil caused varied degrees of increase in 18-carbon fatty acid content and decrease in n-3 LC-PUFA content in the muscle and liver of rainbow trout (Oncorhynchus mykiss), Japanese seabass (Lateolabrax japonicus) and large yellow croaker (Larimichthys crocea), suggesting that these fishes have differing abilities to biosynthesize LC-PUFAs. Fish oil replacement also led to significantly up-regulated expression of FADS2 and SREBP-1 but different responses of the two PPAR-α homologues in the livers of these three fishes. An in vitro experiment indicated that the basic transcription activity of the FADS2 promoter was significantly higher in rainbow trout than in Japanese seabass or large yellow croaker, which was consistent with their LC-PUFA biosynthetic abilities. In addition, SREBP-1 and PPAR-α up-regulated FADS2 promoter activity. These regulatory effects varied considerably between SREBP-1 and PPAR-α, as well as among the three fishes. Taken together, the differences in regulatory activities of the two transcription factors targeting FADS2 may be responsible for the different LC-PUFA biosynthetic abilities in these three fishes that have adapted to different ambient salinity.

Molecular cloning and functional characterization of arachidonate 5-lipoxygenase (Alox5), and its expression in response to the ratio of linolenic acid to linoleic acid in diets of large yellow croaker (Larmichthys crocea).

This study was conducted to clone and functionally characterize a full-length cDNA encoding arachidonate 5-lipoxygenase (Alox5) from large yellow croaker (Larmichthys crocea) and investigate its gene expression in response to graded dietary ratio of linolenic acid (ALA) to linoleic acid (LNA) (0.03, 0.06, 0.45, 0.90 and 1.51). An isolated 2372bp cDNA clone of Alox5 contained an open reading frame spanning 2025bp encoding a protein with the ability to modify arachidonate acid (AA) to 5-hydroxyeicosatetraenoic (5-HETE). In the liver, the Alox5 mRNA expression levels significantly increased to the maximum when the dietary ALA/LNA increased from 0.03 to 0.06, and then significantly decreased with dietary ALA/LNA increased to 1.51 (P<0.05). In the kidney, the expression levels of Alox5 of fish fed diets with low dietary ALA/LNA (0.03-0.06) were significantly higher than those of fish fed diets with high dietary ALA/LNA (0.45-1.51) (P<0.05). The dual-luciferase reporter assays showed that the nuclear factor kappa B (NF-κB) could act on cognate cis-acting elements in the promoter of Alox5 and increased the transcription of Alox5. Results of the present study suggested that the expression of Alox5 is higher in croakers fed high concentrations of LNA compared to those fed high concentrations of ALA, which might be regulated by NF-κB and contribute to the inflammation process by catalyzing the dioxygenation of AA.

Effects of oxidised dietary fish oil and high-dose vitamin E supplementation on growth performance, feed utilisation and antioxidant defence enzyme activities of juvenile large yellow croaker (Larmichthys crocea).

This study was conducted to elucidate the effects of oxidised dietary lipids and high-dose vitamin E (VE) on growth performance and immune responses of large yellow croaker. Juvenile fish (initial average body weight of 7·82 (sem 0·68) g) were fed diets containing either fresh fish oil (fresh diet, peroxide value (POV)=1·72 mEq/kg) or fish oil oxidised to varying degrees (oxidised diets, POV=28·29-104·21 mEq/kg), with or without supplementary 600 mg VE/kg diet, for 10 weeks in floating cages. Growth was significantly lower and feed intake (g/100 g body weight per d) was higher in fish fed the oxidised diet. Supplementation with VE increased the growth of fish fed the oxidised diets, but significantly decreased the growth of fish fed the fresh diet. Hepatosomatic index increased with increasing dietary POV and decreased with VE supplementation. Hepatic catalase activity, superoxide dismutase (SOD) activity and malondialdehyde content were significantly higher in fish fed the oxidised diets, and these values decreased significantly following VE supplementation. However, hepatic SOD activity was enhanced by VE supplementation in fish fed the fresh diet. Air-exposure mortality was significantly increased by dietary POV, and this effect was inhibited by VE supplementation. These results suggest that dietary oxidised fish oil could stimulate the activities of antioxidant defence enzymes in stressed large yellow croaker. High-dose VE supplementation can alleviate oxidative stress of large yellow croaker fed oxidised fish oil, but can exert deleterious effects on fish in the absence of oxidative stress.

Dietary ALA, but not LNA, increase growth, reduce inflammatory processes, and increase anti-oxidant capacity in the marine finfish Larimichthys crocea: dietary ALA, but not LNA, increase growth, reduce inflammatory processes, and increase anti-oxidant capacity in the large yellow croaker.

Whilst aquaculture feed is increasingly formulated with the inclusion of plant oils replacing fish oil, and increasing research effort has been invested in understanding the metabolic effects of reduced dietary n-3 long chain poly unsaturated fatty acids (n-3 LC-PUFA), relatively little information is available on the potential direct metabolic roles of dietary alpha-linolenic acid (ALA, 18:3n-3) and alpha-linolenic acid/linoleic acid (LNA, 18:2n-6) ratio in cultured marine finfish species. In this study, four plant oil based diets, with varying ALA/LNA ratio (0.0, 0.5, 1.0 and 1.5) were fed to juvenile large yellow croakers (Larimichthys crocea) and compared to a fish oil-based control diet (CD) to evaluate the resulting effects on growth, nonspecific immunity, anti-oxidant capacity and related gene expression. High dietary LNA negatively impacted fish growth performance, nonspecific immunity and antioxidant capacity, but growth and immunity were maintained to levels comparable to CD by increasing the ratio of dietary ALA/LNA. The over-expression of genes associated with inflammation (cyclooxygenase-2 and interleukin-1ß) and fatty acid oxidation (carnitine palmitoyl transferase I and acyl CoA oxidase) in croakers fed high concentrations of LNA were reduced to levels comparable to those fed CD by increasing dietary ALA/LNA. This study showed that dietary ALA, by increasing the overall n-3/n-6 PUFA ratio, exerts direct anti-inflammatory and antioxidant effects, similar to those exerted by dietary n-3 LC-PUFA.

Regulation of tissue LC-PUFA contents, Δ6 fatty acyl desaturase (FADS2) gene expression and the methylation of the putative FADS2 gene promoter by different dietary fatty acid profiles in Japanese seabass (Lateolabrax japonicus).

The present study was conducted to evaluate the influences of different dietary fatty acid profiles on the tissue content and biosynthesis of LC-PUFA in a euryhaline species Japanese seabass reared in seawater. Six diets were prepared, each with a characteristic fatty acid: Diet PA: Palmitic acid (C16:0); Diet SA: Stearic acid (C18:0); Diet OA: Oleic acid (C18:1n-9); Diet LNA: α-linolenic acid (C18:3n-3); Diet N-3 LC-PUFA: n-3 LC-PUFA (DHA+EPA); Diet FO: the fish oil control. A 10-week feeding trial was conducted using juvenile fish (29.53 ± 0.86 g). The results showed that Japanese seabass had limited capacity to synthesize LC-PUFA and fish fed PA, SA, OA and LNA showed significantly lower tissue n-3 LC-PUFA contents compared to fish fed N-3 LC-PUFA and FO. The putative gene promoter and full-length cDNA of FADS2 was cloned and characterized. The protein sequence was confirmed to be homologous to FADS2s of marine teleosts and possessed all the characteristic features of microsomal fatty acid desaturases. The FADS2 transcript levels in liver of fish fed N-3 LC-PUFA and FO were significantly lower than those in fish fed other diets except LNA while Diet PA significantly up-regulated the FADS2 gene expression compared to Diet LNA, N-3 LC-PUFA and FO. Inversely, fish fed N-3 LC-PUFA and FO showed significantly higher promoter methylation rates of FADS2 gene compared to fish fed the LC-PUFA deficient diets. These results suggested that Japanese seabass had low LC-PUFA synthesis capacity and LC-PUFA deficient diets caused significantly reduced tissue n-3 LC-PUFA contents. The liver gene expression of FADS2 was up-regulated in groups enriched in C16:0, C18:0 and C18:1n-9 respectively but not in the group enriched in C18:3n-3 compared to groups with high n-3 LC-PUFA contents. The FADS2 gene expression regulated by dietary fatty acids was significantly negatively correlated with the methylation rate of putative FADS2 gene promoter.

Effects of conjugated linoleic acid on growth, non-specific immunity, antioxidant capacity, lipid deposition and related gene expression in juvenile large yellow croaker (Larmichthys crocea) fed soyabean oil-based diets.

The effects of conjugated linoleic acid (CLA) on growth performance, non-specific immunity, antioxidant capacity, lipid deposition and related gene expression were investigated in the large yellow croaker (Larmichthys crocea). Fish (7·56 (SEM 0·60) g) were fed soyabean oil-based diets with graded levels of CLA (0, 0·42, 0·83, 1·70%) for 70 d. Quantitative PCR was used to assess the effects of CLA on the transcription of inflammation- and fatty acid oxidation-related genes. Growth in fish fed the diet with 0·42% CLA was significantly higher. Also, phagocytic index and respiratory burst activity were significantly higher in fish fed the diets containing 0·42 and 0·83% CLA, respectively. Hepatic total antioxidative capacity and catalase activities increased significantly when CLA increased from 0 to 0·83%, and then decreased with further increase of CLA. However, hepatic malondialdehyde content decreased significantly as dietary CLA increased. Lipid concentration in the whole body and muscle increased significantly with increasing dietary CLA. Transcription of genes related to inflammation (cyclo-oxygenase-2 and IL-b) in the liver and kidney and fatty acid oxidation (carnitine palmitoyl transferase I and acyl CoA oxidase) in the kidney decreased significantly as dietary CLA increased. PPAR alpha and acyl CoA oxidase expression in the liver decreased significantly as CLA increased from 0·42 to 1·70%. These results strongly suggest that dietary CLA could significantly affect growth performance, non-specific immunity, antioxidant capacity, lipid deposition and transcription of inflammation- and fatty acid oxidation-related genes of the large yellow croaker. This may contribute to our understanding of the mechanisms related to the physiological effects of dietary CLA in fish.

Effects of dietary n-3 highly unsaturated fatty acids on growth, nonspecific immunity, expression of some immune related genes and disease resistance of large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans).

The study was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acid (n-3 HUFA) on growth, nonspecific immunity, expression of some immune related genes and disease resistance of juvenile large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans). Six isoproteic and isolipidic diets were formulated with graded levels of n-3 HUFA ranging from 0.15% to 2.25% of the dry weight and the DHA/EPA was approximately fixed at 2.0. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.0 × 1.0 × 1.5 m), and each cage was stocked with 60 fish (initial average weight 9.79 ± 0.6 g). Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 58 days. Results showed that moderate n-3 HUFA level (0.98%) significantly enhanced growth compared with the control group (0.15% HUFA) (P < 0.05), while higher n-3 HUFA levels (1.37%, 1.79% and 2.25%) had detrimental effects on the growth though no significance was found (P > 0.05). Nitro blue tetrazolium (NBT) positive leucocytes percentage of head kidney and serum superoxide dismutase (SOD) activity increased with increasing n-3 HUFA from 0.15% to 0.60%, and decreased with further increase of n-3 HUFA from 0.60% to 2.25% (P < 0.05). Serum lysozyme activity increased significantly as n-3 HUFA increased from 0.15% to 1.37%, and then decreased with n-3 HUFA from 1.37% to 2.25% (P > 0.05). There were no significant differences in phagocytosis index (PI) of head kidney leucocytes among dietary treatments (P > 0.05). The hepatic mRNA expression of Toll-like receptor 22 (TLR22) and Myeloid differentiation factor 88 (MyD88) was significantly up-regulated in fish fed the diets with low or moderate levels, while in kidney this increment was only found at specific sampling time during the natural infestation of parasites. The 13 d cumulative mortality rate following natural infestation of parasites decreased with n-3 HUFA increased from 0.15% to 0.60% (P < 0.05), and significantly increased with n-3 HUFA from 0.60% to 2.25% (P < 0.05). Results of this study suggested that fish fed low or moderate dietary n-3 HUFA had higher growth, nonspecific immune responses, expression levels of some immune related genes and disease resistance of large yellow croaker following natural infestation of parasites and dietary n-3 HUFA may regulate fish immunity and disease resistance by altering the mRNA expression levels of TLR22 and MyD88.