Molecular and functional characterisation of two elovl4 elongases involved in the biosynthesis of very long-chain (>C24) polyunsaturated fatty acids in black seabream Acanthopagrus schlegelii

Elongation of very long-chain fatty acid (Elovl) 4 proteins are important fatty acyl elongases that participate in the biosynthesis of long-chain (C20-24) and very long-chain (˃C24) polyunsaturated fatty acids (LC-PUFA and VLC-PUFA, respectively) in teleost fish, especially in marine species. Moreover, knowledge of Elovl4 and other elongases such as Elovl2 has contributed to an advanced understanding of the LC-PUFA biosynthetic pathway in marine fish. In the present study, elovl4a and elovl4b were cloned from black seabream Acanthopagrus schlegelii and functionally characterised using recombinant expression in yeast. The elovl4a and elovl4b cDNA sequences included open reading frames (ORF) of 969 and 918 base pairs (bp), encoding proteins of 322 and 315 amino acids (aa), respectively. The functional characterisation of A. schlegelii Elovl4 proteins showed they were able to utilise all assayed C18-22 PUFA substrates except 22:6n-3. Moreover, it was particularly noteworthy that both A. schlegelii Elovl4a and Elovl4b proteins had the ability to elongate 20:5n-3 and 22:5n-3 to 24:5n-3, which can be potentially desaturated and β-oxidised to 22:6n-3. Tissue transcript abundance analysis showed the highest expression of elovl4a and elovl4b in brain and eye, respectively, suggesting these tissues were major sites for VLC-PUFA biosynthesis in black seabream. The functions of the A. schlegelii Elovl4-like elongases, Elovl4a and Elovl4b, characterised in the present study, along with those of the Elovl5 and fatty acyl desaturase (Fads2) proteins of A. schlegelii characterised previously, provided evidence of the biosynthetic pathways of LC-PUFA and VLC-PUFA in this teleost species

Effects of dietary zinc level on growth performance, lipolysis and expression of genes involved in the calcium/calmodulin-dependent protein kinase kinase-β/AMP-activated protein kinase pathway in juvenile Pacific white shrimp

The present study evaluated the effects of dietary zinc level on growth performance, serum and hepatopancreas metabolites, and expression of genes involved in lipid and energy metabolism, and the signal pathway of dietary Zn-induced lipolysis. Five isonitrogenous and isolipidic diets were formulated to contain different zinc levels: 46.4 (basal diet), 77.2, 87.0, 117.1, and 136.8 mg kg-1, respectively. The results indicated that shrimp fed the diet containing zinc at 117.1 mg kg-1 had higher weight gain and specific growth rate, and the lowest feed intake and feed conversion rate, than shrimp fed the other diets. The deposition rate of Zn in whole body significantly decreased with increasing dietary zinc level. Dietary Zn prevented the accumulation of free radicals and improved antioxidant activities by increasing Cu/Zn superoxide dismutase and reducing malonaldehyde in hepatopancreas. Dietary Zn supplementation enhanced lipase activity and adiponectin, which could promote triglyceride breakdown and fatty acid oxidation and lead to reduced lipid in hepatopancreas. The mRNA expressions of ob-rb, adipor, camkkβ, ampk, cd36, mcd, cpt1 involved in Zn-induced lipid catabolism were up-regulated, and expressions of srebp, acc, fas and scd1 were down-regulated. The mRNA levels of SLC39 family genes (zip3, zip9, zip11, zip14) in hepatopancreas were up-regulated with increasing dietary Zn level. The results demonstrated that dietary Zn level could significantly affect growth performance, tissue deposition of zinc, lipid metabolites and expression of genes involved in lipogenesis and lipolysis in Litopenaeus vannamei

Dietary DHA/EPA ratio affected tissue fatty acid profiles, antioxidant capacity, hematological characteristics and expression of lipid-related genes but not growth in juvenile black seabream (Acanthopagrus schlegelii)

An 8-week feeding trial was conducted to investigate the effects of dietary docosahexaenoic to eicosapentaenoic acid ratio (DHA/EPA) on growth performance, fatty acid profiles, anti- oxidant capacity, hematological characteristics and expression of some lipid metabolism related genes of juvenile black seabream (Acanthopagrus schlegelii) of initial weight 9.47 ± 0.03 g. Five isonitrogenous and isolipidic diets (45% crude protein and 14% crude lipid) were formulated to contain graded DHA/EPA ratios of 0.65, 1.16, 1.60, 2.03 and 2.67. There were no differences in growth performance and feed utilization among treatments. Fish fed higher DHA/EPA ratios had higher malondialdehyde (MDA) contents in serum than lower ratios. Serum triacylglycerol (TAG) content was significantly higher in fish fed the lowest DHA/EPA ratio. Tissue fatty acid profiles reflected the diets despite down-regulation of LC- PUFA biosynthesis genes, fatty acyl desaturase 2 (fads2) and elongase of very long-chain fatty acids 5 (elovl5), by high DHA/EPA ratios. Expression of acetyl-CoA carboxylase alpha (accα) and carnitine palmitoyl transferase 1A (cpt1a) were up-regulated by high DHA/EPA ratio, whereas sterol regulatory element-binding protein-1 (srebp-1) and hormone-sensitive lipase (hsl) were down-regulated. Fatty acid synthase (fas), 6-phosphogluconate dehydro- genase (6pgd) and peroxisome proliferator-activated receptor alpha (pparα) showed highest expression in fish fed intermediate (1.16) DHA/EPA ratio. Overall, this study indicated that dietary DHA/EPA ratio affected fatty acid profiles and significantly influenced lipid metabo- lism including LC-PUFA biosynthesis and other anabolic and catabolic pathways, and also had impacts on antioxidant capacity and hematological characteristics

Dietary choline supplementation attenuated high-fat diet-induced inflammation through regulation of lipid metabolism and suppression of NFKB activation in juvenile black seabream (Acanthopagrus schlegelii)

The present study aimed to investigate whether dietary choline can regulate lipid metabolism and suppress NFκB activation and, consequently, attenuate inflammation induced by a high-fat diet in black sea bream (Acanthopagrus schlegelii). An 8-week feeding trial was conducted on fish with an initial weight of 8·16 ± 0·01 g. Five diets were formulated: control, low-fat diet (11 %); HFD, high-fat diet (17 %); and HFD supplemented with graded levels of choline (3, 6 or 12 g/kg) termed HFD + C1, HFD + C2 and HFD + C3, respectively. Dietary choline decreased lipid content in whole body and tissues. Highest TAG and cholesterol concentrations in serum and liver were recorded in fish fed the HFD. Similarly, compared with fish fed the HFD, dietary choline reduced vacuolar fat drops and ameliorated HFD-induced pathological changes in liver. Expression of genes of lipolysis pathways were up-regulated, and genes of lipogenesis down-regulated, by dietary choline compared with fish fed the HFD. Expression of nfκb and pro-inflammatory cytokines in liver and intestine was suppressed by choline supplementation, whereas expression of anti-inflammatory cytokines was promoted in fish fed choline-supplemented diets. In fish that received lipopolysaccharide to stimulate inflammatory responses, the expression of nfκb and pro-inflammatory cytokines in liver, intestine and kidney were all down-regulated by dietary choline compared with the HFD. Overall, the present study indicated that dietary choline had a lipid-lowering effect, which could protect the liver by regulating intrahepatic lipid metabolism, reducing lipid droplet accumulation and suppressing NFκB activation, consequently attenuating HFD-induced inflammation in A. schlegelii

Biosynthesis of long-chain polyunsaturated fatty acids in the razor clam Sinonovacula constricta: characterization of four fatty acyl elongases and a novel desaturase capacity

As an unusual economically important aquaculture species, Sinonovacula constricta possesses high levels of long-chain polyunsaturated fatty acids (LC-PUFA). Previously, our group identified fatty acyl desaturases (Fad) with Δ5 and Δ6 activities in S. constricta, which was the first report of Δ6 Fad in a marine mollusc. Here, we further successfully characterize elongases of very long-chain fatty acids (Elovl) in this important bivalve species, including one Elovl2/5, two Elovl4 isoforms (a and b) and a novel Elovl (c) with Elovl4 activity. In addition, we also determined the desaturation activity of S. constricta Δ6 Fad toward 24:5n-3 to give 24:6n-3, a key intermediate in docosahexaenoic acid (DHA) biosynthesis. Therefore, S. constricta is the first marine mollusc reported to possess all Fad and Elovl activities required for LC-PUFA biosynthesis via the ‘Sprecher pathway’. This finding greatly increases our understanding of LC-PUFA biosynthesis in marine molluscs. Phylogenetic analysis by interrogating six marine molluscan genomes, and previously functionally characterized Elovl and Fad from marine molluscs, suggested that DHA biosynthetic ability was limited to a few species, due to the general lack of Δ4 or Δ6 Fad in most molluscs

Influence of dietary zinc on growth, zinc bioaccumulation and expression of genes involved in antioxidant and innate immune in juvenile mud crabs (Scylla paramamosain)

The aim of present study was to investigate the effects of dietary Zn level on growth performance, Zn bioaccumulation, antioxidant capacity and innate immunity in juvenile mud crab (Scylla paramamosain). Six semi-purified diets were formulated to contain dietary Zn levels of 44.5, 56.9, 68.5, 97.3, 155.6 or 254.7 mg·kg-1, respectively. Dietary Zn level significantly influenced percent weight gain (PWG), with highest observed in crab fed the diet containing 97.3 mg·kg-1 Zn. Tissue Zn concentrations significantly increased as dietary Zn levels increased from 44.5 to 254.7 mg·kg-1. Retention of Zn in hepatopancreas increased with dietary Zn levels up to 68.5 mg·kg-1 and then significantly decreased. Moreover, inadequate dietary Zn (44.5 and 56.9 mg·kg-1) reduced anti-oxidation markers including total superoxide dismutase and copper/zinc superoxide dismutase activities and total anti-oxidant level. Crabs fed the diet with 44.5 mg·kg-1 Zn also showed significantly lower expression of genes involved in antioxidant status, such as Cu/Zn sod, glutathione peroxidase, catalase and thioredoxin than those fed diets containing 68.5 and 97.3 mg·kg-1 Zn. Highest activities of phenoloxidase and alkaline phosphatase were recorded in crab fed the diets containing 68.5 and 97.3 mg·kg-1 Zn. Expression levels of prophenoloxidase and toll-like receptor 2 were higher in crab fed the 97.3 mg·kg-1 Zn diet compared to crab fed the other diets. Based on PWG alone, the optimal dietary Zn level was estimated to be 82.9 mg·kg-1, with 68.5 to 97.3 mg·kg-1 recommended for maintaining optimal Zn bioaccumulation, oxidation resistance and innate immune response of juvenile mud crab

Effects of dietary lipid level on growth, fatty acid profiles, antioxidant capacity and expression of genes involved in lipid metabolism in juvenile swimming crab, Portunus trituberculatus

The regulation of lipogenesis and lipolysis mechanisms related to consumption of lipid has not been studied in swimming crab. The aims of present study were to evaluate the effects of dietary lipid levels on growth, enzymes activities, and expression of genes of lipid metabolism in hepatopancreas of juvenile swimming crab. Three isonitrogenous diets were formulated to contain crude lipid levels at 5.8 %, 9.9 % and 15.1 %, respectively. Crabs fed the diet containing 15.1 % lipid had significantly lower weight gain, specific growth rate and survival, and higher feed conversion ratio than those fed the 5.8 % and 9.9 % lipid diets. Crabs fed 5.8 % lipid had lower malondialdehyde concentrations in the hemolymph and hepatopancreas than those fed the other diets. Highest glutathione peroxidase in hemolymph and superoxide dismutase in hepatopancreas were observed in crabs fed 5.8 % lipid. The lowest fatty acid synthase and glucose 6-phosphate dehydrogenase activities in hepatopancreas were observed in crabs fed 15.1 % lipid, whereas crabs fed 5.8 % lipid had lower carnitine palmitoyltransferase-1 activity than those fed the other diets. Crabs fed 15.1 % lipid showed lower hepatopancreas expression of genes involved in LC-PUFA biosynthesis, lipoprotein clearance, fatty acid uptake, fatty acid oxidation, lipid anabolism and lipid catabolism than those fed the other diets, whereas expression of some genes of lipoprotein assembly and fatty acid oxidation were up-regulated compared with crabs fed 5.8 % lipid. Overall, high dietary lipid level can inhibit growth, reduce feed utilization and reduce antioxidant enzyme activities. Moreover, dietary lipid influenced enzyme activities and gene expression involved in lipid metabolism of juvenile swimming crab

Transcriptomic and physiological analyses of hepatopancreas reveal the key metabolic changes in response to dietary copper level in Pacific white shrimp Litopenaeus vannamei

All living organisms require copper for growth and development, but the gene expression profiles and molecular mechanisms underpinning dietary copper are poorly investigated. Therefore, the present study aimed to determine the potential metabolic changes in response to dietary copper based on analysis of hepatopancreas transcriptome in Litopenaeus vannamei. Three practical diets were formulated to supplement 0 (control diet; Csingle bondCu) and 40 mg kg−1 inorganic Cu (CuSO4·5H2O; Isingle bondCu) and copper amino acid chelate (Osingle bondCu), with analyzed Cu being 12.4, 49.8 and 50.0 mg kg−1, respectively. Shrimp fed Isingle bondCu and Osingle bondCu diets had higher percent weight gain and Cu concentration in tissues. Some essential amino acids (lysine, methionine, isoleucine, leucine, valine) and non-essential amino acids (tyrosine, glycine, aspartic acid, proline and serine) in hepatopancreas significantly increased in shrimp fed the copper supplemented diets. Transcriptome analysis indicated a total of 742 and 912 genes were differentially expressed (q < 0.001; log2fold change ≥2) in shrimp fed the Isingle bondCu and Osingle bondCu diets, respectively, in comparison to shrimp fed the control diet. Five and eight significantly changed pathways were annotated in the Csingle bondCu vs. Isingle bondCu and Csingle bondCu vs. Osingle bondCu comparisons, with metabolism the leading category for both. Similarly, the proportion of differentially expressed genes revealed that most were enriched in the category of metabolism. Further analysis revealed that dietary copper mainly affected amino acid and glycerophospholipid metabolism. Moreover, two significantly changed pathways (phagosome and IL-17 signaling pathway) related to the immune system were identified in shrimp fed the Osingle bondCu diet. The present study analyzing the hepatopancreas transcriptome identified potential roles of dietary copper on amino acid and glycerophospholipid metabolism and provided new insight that will be valuable in future studies to further elucidate the nutritional molecular basis of copper

Modification of nutritional values and flavor qualities of muscle of swimming crab (Portunus trituberculatus): Application of a dietary lipid nutrition strategy

Lipid sources as alternatives to fish oil could alter the nutritional value and flavor quality of crab meat affecting consumer preferences. Herein, an 8-week nutritional trial was designed to investigate the effects of dietary lipid sources including fish oil (FO), krill oil (KO), palm oil, rapeseed oil, soybean oil and linseed oil on profiles of amino acids, fatty acids and volatiles in muscle of swimming crab (Portunus trituberculatus). Volatiles of crab muscle were characterized by headspace solid-phase microextraction and gas chromatography-tandem mass spectrometry. Results revealed that crabs fed FO and KO had significantly higher levels of protein, indispensable amino acids, eicosapentaenoic acid and docosahexaenoic acid in muscle. Principal component analysis and hierarchical cluster analysis demonstrated that muscle volatiles of crabs fed different dietary oils exhibited significant variations. Dietary FO and KO significantly increased the relative levels of 3-methylbutanal, heptanal, benzaldehyde and nonanal in muscle, which may produce more pleasant flavors

Dietary DHA/EPA ratio affects growth, tissue fatty acid profiles and expression of genes involved in lipid metabolism in mud crab Scylla paramamosain supplied with appropriate n-3 LC-PUFA at two lipid levels

An 8-week feeding trial was conducted to determine the optimal dietary docosahexaenoic acid/eisosapentaenoic acid (DHA/EPA) ratio of mud crab (Scylla paramamosain) supplied with optimal n-3 LC-PUFA at two dietary lipid levels. Eight isonitrogenous diets were formulated to contain 7% and 12% crude lipid, each with DHA/EPA ratios of 0.6, 1.2, 2.3 and 3.2, respectively. Each diet was randomly assigned to triplicate groups of 30 juvenile mud crabs (initial weight 20.9 ± 0.6g) that were stocked in single crab cells. In crabs fed 7% lipid, the diet with a DHA/EPA ratio of 2.3 showed significantly higher weight gain than crabs fed the other ratios while in crabs fed 12% lipid, lower weight gain and specific growth rate were observed in crabs fed the diet with a DHA/EPA ratio of 0.6 than crabs fed the other ratios. Lipid content in hepatopancreas significantly increased as dietary DHA/EPA ratio increased from 1.2 to 2.3 in crabs fed 7% lipid, while no differences were observed among crabs fed the diets with DHA/EPA ratios higher than 0.6 when fed 12% lipid. Total fatty acid and DHA contents and DHA/EPA ratio showed increasing, and EPA decreasing, trends in muscle and hepatopancreas with increased dietary DHA/EPA ratio, at both dietary lipid levels. The hemolymph triacylglycerol and total cholesterol contents were higher in crabs fed dietary DHA/PA ratios of 1.2 and 2.3 than those fed ratios of 0.6 and 3.2 at 7% dietary lipid, and lowest low and high-density lipoprotein cholesterol contents were observed in crabs fed DHA/EPA dietary ratios of 0.6 and 3.2 at 7% and 12% lipid, respectively. The expression levels of fas, aco3 and fatp4 were significantly up-regulated, and cptⅠ, hsl and ldlr were down-regulated, with increased dietary DHA/EPA ratio in crabs fed 7% lipid. In crabs fed 12% lipid, the expression levels of g6pd, 6pgd, srebp-1, aco1 and fatp4 were down-regulated, and fabp-1 was up-regulated, with increased dietary DHA/EPA ratio. The expression levels of elovl4 and Δ6 fad initially increased and then decreased as dietary DHA/EPA ratio increased from 0.6 to 3.2 in crabs fed both 7% and 12% lipid. Based on analysis of weight gain versus dietary DHA/EPA ratio, the optimal dietary DHA/EPA ratios of mud crab S. paramamosa were estimated to be 2.2 and 1.2 when supplied with optimal n-3 LC-PUFA at 7% and 12% lipid, respectively