A complete biosynthetic pathway of the long-chain polyunsaturated fatty acids in an amphidromous fish, ayu sweetfish Plecoglossus altivelis (Stomiati; Osmeriformes).

The biosynthetic capability of the long-chain polyunsaturated fatty acids (LC-PUFA) in teleosts are highly diversified due to evolutionary events such as gene loss and subsequent neo- and/or sub-functionalisation of enzymes encoded by existing genes. In the present study, we have comprehensively characterised genes potentially involved in LC-PUFA biosynthesis, namely one front-end desaturase (fads2) and eight fatty acid elongases (elovl1a, elovl1b, elovl4a, elovl4b, elovl5, elovl7, elovl8a and elovl8b) from an amphidromous teleost, Ayu sweetfish, Plecoglossus altivelis. Functional analysis confirmed Fads2 with Δ6, Δ5 and Δ8 desaturase activities towards multiple PUFA substrates and several Elovl enzymes exhibited elongation capacities towards C18-20 or C18-22 PUFA substrates. Consequently, P. altivelis possesses a complete enzymatic capability to synthesise physiologically important LC-PUFA including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) from their C18 precursors. Interestingly, the loss of elovl2 gene in P. altivelis was corroborated by genomic and phylogenetic analyses. However, this constraint would possibly be overcome by the function of alternative Elovl enzymes, such as Elovl1b, which has not hitherto been functionally characterised in teleosts. The present study contributes novel insights into LC-PUFA biosynthesis in the relatively understudied teleost group, Osmeriformes (Stomiati), thereby enhancing our understanding of the complement of LC-PUFA biosynthetic genes within teleosts.

Improved Flesh Pigmentation of Rainbow Trout (Oncorhynchus mykiss) by Feeding Z-Isomer-Rich Astaxanthin Derived from Natural Origin.

The use of Paracoccus carotinifaciens-derived natural astaxanthin as an alternative to synthetic astaxanthin has attracted considerable attention from the aquaculture industry. Furthermore, to enhance the bioavailability of astaxanthin, its "Z-isomerization" has been actively studied in recent years. This study investigated the effects of feeding a diet containing astaxanthin rich in the all-E- or Z-isomers derived from P. carotinifaciens on the pigmentation and astaxanthin concentration in rainbow trout (Oncorhynchus mykiss) flesh. Z-Isomer-rich astaxanthin was prepared from the P. carotinifaciens-derived all-E-isomer by thermal treatment in fish oil, and the prepared all-E-isomer-rich astaxanthin diet (E-AST-D; total Z-isomerratio = 9.1%) and Z-isomer-rich astaxanthin diet (Z-AST-D; total Z-isomer ratio of astaxanthin = 56.6%) were fed to rainbow trout for 8 weeks. The feeding of Z-AST-D resulted in greater pigmentation and astaxanthin accumulation efficiency in the flesh than those fed E-AST-D. Specifically, when E-AST-D was fed to rainbow trout, the SalmoFan score and astaxanthin concentration of the flesh were 22.1±1.4 and 1.36±0.71 µg/g wet weight, respectively, whereas when Z-AST-D was fed, their values were 26.0±2.5 and 5.33±1.82 µg/g wet weight, respectively. These results suggest that P. carotinifaciens-derived astaxanthin Z- isomers prepared by thermal isomerization are more bioavailable to rainbow trout than the all-E-isomer.

Determination of dietary essential fatty acids in a deep-sea fish, the splendid alfonsino Beryx splendens: functional characterization of enzymes involved in long-chain polyunsaturated fatty acid biosynthesis.

The splendid alfonsino Beryx splendens is a commercially important deep-sea fish in East Asian countries. Because the wild stock of this species has been declining, there is an urgent need to develop aquaculture systems. In the present study, we investigated the long-chain polyunsaturated fatty acid (LC-PUFA) requirements of B. splendens, which are known as essential dietary components in many carnivorous marine fish species. The fatty acid profiles of the muscles, liver, and stomach contents of B. splendens suggested that it acquires substantial levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from its natural diet. The functional characterization of a fatty acid desaturase (Fads2) and three elongases (Elovl5, Elovl4a, and Elovl4b) from B. splendens confirmed their enzymatic capabilities in LC-PUFA biosynthesis. Fads2 showed Δ6 and Δ8 bifunctional desaturase activities. Elovl5 showed preferential elongase activities toward C18 and C20 PUFA substrates, whereas Elovl4a and Elovl4b showed activities toward various C18-22 substrates. Given that Fads2 showed no Δ5 desaturase activity and no other fads-like sequence was found in the B. splendens genome, EPA and arachidonic acid cannot be synthesized from C18 precursors; hence, they can be categorized as dietary essential fatty acids in B. splendens. EPA can be converted into DHA in B. splendens via the so-called Sprecher pathway. However, given that fads2 is only expressed in the brain, it is unlikely that the capacity of B. splendens to biosynthesize DHA from EPA can fulfill its physiological requirements. These results will be useful to researchers developing B. splendens aquaculture methods.

Impact of purine nucleotide on fatty acid metabolism and expression of lipid metabolism-related gene in the liver cell of rainbow trout Oncorhynchus mykiss.

Studies have suggested that dietary purine nucleotides (NT) affect the muscle and liver fatty acid composition of rainbow trout. To examine the direct regulation of liver fatty acid metabolism by purine NT in rainbow trout, the liver cells were cultured in the presence of 500 µmol/L inosine, adenosine, or guanosine monophosphate (IMP, AMP, or GMP). The expression of pparα was significantly decreased in the liver cells cultured with purine NT for 24 h, whereas the expression of fads2 (Δ5) was increased. Docosahexaenoic acid (DHA) content in the liver cells was significantly higher after culturing with GMP. To determine the dose-dependent effects of NT, 50, 100, and 500 µmol/L GMP was added to the liver cells cultured in L-15 medium. At 48 h, 20:4n - 6, 22:5n - 3, 22:6n - 3, Æ© PUFA, and Æ© n - 3 PUFA content in the 50 µM GMP-containing medium was significantly higher compared with the other medium. The expression of Δ5 fads2, elovl2, and elovl5 in the liver cells was significantly higher in the 500 µmol/L GMP-containing medium at 48 h along with increased srebp-1 expression. These results suggest that purine NT directly affect fatty acid composition through modification of fatty acid metabolism-related genes in the liver of rainbow trout.

A complete enzymatic capacity for biosynthesis of docosahexaenoic acid (DHA, 22 : 6n-3) exists in the marine Harpacticoida copepod Tigriopus californicus.

The long-standing paradigm establishing that global production of Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) derived almost exclusively from marine single-cell organisms, was recently challenged by the discovery that multiple invertebrates possess methyl-end (or ωx) desaturases, critical enzymes enabling the biosynthesis of n-3 LC-PUFA. However, the question of whether animals with ωx desaturases have complete n-3 LC-PUFA biosynthetic pathways and hence can contribute to the production of these compounds in marine ecosystems remained unanswered. In the present study, we investigated the complete enzymatic complement involved in the n-3 LC-PUFA biosynthesis in Tigriopus californicus, an intertidal harpacticoid copepod. A total of two ωx desaturases, five front-end desaturases and six fatty acyl elongases were successfully isolated and functionally characterized. The T. californicus ωx desaturases enable the de novo biosynthesis of C18 PUFA such as linoleic and α-linolenic acids, as well as several n-3 LC-PUFA from n-6 substrates. Functions demonstrated in front-end desaturases and fatty acyl elongases unveiled various routes through which T. californicus can biosynthesize the physiologically important arachidonic and eicosapentaenoic acids. Moreover, T. californicus possess a Δ4 desaturase, enabling the biosynthesis of docosahexaenoic acid via the 'Δ4 pathway'. In conclusion, harpacticoid copepods such as T. californicus have complete n-3 LC-PUFA biosynthetic pathways and such capacity illustrates major roles of these invertebrates in the provision of essential fatty acids to upper trophic levels.

Flatfishes colonised freshwater environments by acquisition of various DHA biosynthetic pathways.

The colonisation of freshwater environments by marine fishes has historically been considered a result of adaptation to low osmolality. However, most marine fishes cannot synthesise the physiologically indispensable fatty acid, docosahexaenoic acid (DHA), due to incomplete DHA biosynthetic pathways, which must be adapted to survive in freshwater environments where DHA is poor relative to marine environments. By analysing DHA biosynthetic pathways of one marine and three freshwater-dependent species from the flatfish family Achiridae, we revealed that functions of fatty acid metabolising enzymes have uniquely and independently evolved by multi-functionalisation or neofunctionalisation in each freshwater species, such that every functional combination of the enzymes has converged to generate complete and functional DHA biosynthetic pathways. Our results demonstrate the elaborate patchwork of fatty acid metabolism and the importance of acquiring DHA biosynthetic function in order for fish to cross the nutritional barrier at the mouth of rivers and colonise freshwater environments.

Cloning and functional characterization of fads2 desaturase and elovl5 elongase from Japanese flounder Paralichthys olivaceus.

Japanese flounder Paralichthys olivaceus has an essential requirement for long-chain polyunsaturated fatty acids (LC-PUFA), particularly docosahexaenoic acid and eicosapentaenoic acid, but the enzymes involved in LC-PUFA biosynthesis are thought to be absent or to have low activity. Teleost fish, in particular, have quite diversified substrate preference of these enzymes even among closely related species, implying that each species could have different LC-PUFA biosynthetic capabilities. Therefore, in the present study, we characterized Japanese flounder fatty acid desaturase 2 (Fads2) and elongation of very long-chain fatty acids protein 5 (Elovl5) in order to precisely characterize the LC-PUFA biosynthesis pathway. Fads2 has Δ6 and Δ8 desaturase activity and Elovl5 has elongase activity toward C18 and C20 PUFA, suggesting that Japanese flounder is capable of synthesizing 20:4n-3 and 20:3n-6 from 18:3n-3 and 18:2n-6, respectively. Expression analysis showed that the fads2 was highly expressed in the brain and eye, while the elovl5 was highly expressed in the eye and pyloric caeca. This information will be beneficial for developing an ideal feed to support the aquaculture of Japanese flounder.

Polyunsaturated fatty acid metabolism in a marine teleost, Nibe croaker Nibea mitsukurii: Functional characterization of Fads2 desaturase and Elovl5 and Elovl4 elongases.

To reduce the requirement for fish oil in marine aquaculture, it would be advantageous to endow marine fish species with the capability for the endogenous biosynthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). For this purpose, we have previously produced transgenic Nibe croaker (Nibea mitsukurii) carrying an elongase of very-long-chain fatty acids 2 (elovl2) gene isolated from Masu salmon (Oncorhynchus masou). However, fatty acid analysis revealed that 24:5n-3 accumulated in the liver of the transgenic fish, whereas the DHA level did not differ between non-transgenic and transgenic fish. Therefore, to select more effective enzymes for successful transgenic synthesis of DHA, understanding the endogenous DHA biosynthetic pathway in the Nibe croaker is considered to be important. The present study aimed to investigate the biochemical functions of the Elovl5, Elovl4 and Fads2 enzymes involved in the DHA biosynthetic pathway in the Nibe croaker. The results showed that both Elovl5 and Elovl4 were able to elongate C18 fatty acids to C22 fatty acids and that Fads2 had Δ6 desaturase activity toward C18 fatty acids and weak Δ8 desaturase activity toward C20 fatty acids. On the other hand, Fads2 was found to lack the ability to convert 24:5n-3 to 24:6n-3, a fatty acid that can directly be converted to DHA via ß-oxidation.

Modification of the n-3 HUFA biosynthetic pathway by transgenesis in a marine teleost, nibe croaker.

Marine fishes are generally unable to produce sufficient quantities of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) for their normal growth and survival, as the key fatty acid-metabolizing enzymes in the EPA and DHA biosynthetic pathway are limited. It is therefore necessary to supplement cultured marine fish species diets with fish oils in order to supply EPA and DHA. Given that freshwater fishes are capable of synthesizing both EPA and DHA, they presumably express all of the enzymes required for this biosynthetic pathway. Thus, we hypothesize that transgenic marine species carrying these fatty acid-metabolizing enzymes could be reared without the dietary supplementation of fish oil. As the first step toward this goal, we used marine fish, nibe croaker to produce a transgenic line carrying the elongase gene isolated from masu salmon. Fatty acid analysis revealed that the liver EPA (20:5n-3) content in the transgenic fish was lower (3.3% vs. 7.7%). However, docosapentaenoic acid (22:5n-3) content in the transgenic fish was 2.28-fold (4.1% vs. 1.8%) higher than in non-transgenic fish. Further, tetracosapentaenoic acid (24:5n-3) was specifically detected in the transgenic fish. We therefore conclude that the development of transgenic fish lines with these fatty acid-metabolizing enzymes could be a powerful tool for manipulating fatty acid metabolic pathways in fish.

Microarray analysis of hepatic gene expression in juvenile Japanese flounder Paralichthys olivaceus fed diets supplemented with fish or vegetable oils.

Gene expression profiling was performed in Japanese flounder Paralichthys olivaceus fed diets supplemented with fish oil (FO), linseed oil (LO), or olive oil (OO) for 6 weeks. The LO and OO groups showed significantly retarded growth, lower feed intake, lower protein efficiency ratio, and lower hepatosomatic index (P < 0.05). Liver fatty acid composition reflected the dietary fatty acid composition. Microarray analysis revealed that dietary n - 3 highly unsaturated fatty acid (HUFA) deficiency affected 169 transcripts. In the LO group, 57 genes were up-regulated and 38 genes were down-regulated, whereas in the OO group nine genes were up-regulated and 87 genes were down-regulated. Analysis of the functional annotations suggested that dietary n - 3 HUFA affected genes involved in signal transduction (23.2 %), cellular processes (21.1 %), metabolism (including glucose, lipid, and nucleobase; 15.5 %), transport (11.3 %), regulation of transcription (10.5 %), and immune response (4.2 %). Several genes encoding serine/threonine kinases such as protein kinase C and calmodulin-dependent kinase and nuclear hormone receptors such as vitamin D receptor, retinoic acid receptor, and receptors for cytokines (bone morphogenic protein and transforming growth factor ß) were affected. Among 169 transcripts, 22 genes were affected in both LO and OO groups. The present study identified several genes involved in n - 3 HUFA deficiency-sensitive pathways, which will be useful for selective breeding of flounder strains able to adapt to n - 3 HUFA deficiency.

Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model.

To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.

Differentiation of chondrocytes and scleroblasts during dorsal fin skeletogenesis in flounder larvae.

In teleosts, the embryonic fin fold consists of a peridermis, an underlying epidermis and a small number of mesenchymal cells. Beginning from such a simple structure, the fin skeletons, including the proximal and distal radials and lepidotrichia (finrays), develop in the dorsal fin fold at the larval stage. Their process of skeletogenesis and embryonic origin are unclear. Using flounder larvae, we report the differentiation process for chondrocytes and scleroblasts prior to fin skeletogenesis and the effects of retinoic acid (RA) on it. In early larvae, the mesenchymal cells grow between the epidermis and spinal cord to form a line of periodical condensations, which are proximal radial primordia, to produce chondrocytes. The prescleroblasts, which ossify the proximal radial cartilages, differentiate in the mesenchymal cells remaining between the cartilages. Then, mesenchymal condensations occur between the distal ends of the proximal radials, forming distal radial primordia, to produce chondrocytes. Simultaneously, condensations occur between the distal radial primordia and peridermis, which are lepidotrichia primordia, to produce prescleroblasts. Exogenous RA specifically inhibits the mesenchymal condensation prior to the proximal radial formation together with the down-regulation of sonic hedgehog (shh) and patched (pta) expression, resulting in the loss of proximal radials. Thus, it was indicated that differentiation of the precursor cells of radials and lepidotrichia begins in the proximal part of the fin fold and that the initial mesenchymal condensation prior to the proximal radial formation is highly susceptible to the effects of RA. Lepidotrichia formation does not occur where proximal radials are absent, indicating that lepidotrichia differentiation requires interaction with the radial cartilages. To examine the suggestion that neural crest cells contribute to the medial fin skeletons, we localized the HNK-1 positive cells in flounder embryos and slug and msxb-positive cells in pufferfish, Fugu rubripes, embryos. That the positive cells commonly arrive at the proximal part of the fin fold does not contradict the suggestion, but their final destiny as radial chondrocytes or lepidotrichia scleroblasts, should be further investigated.

Retinoic acid isomers produce malformations in postembryonic development of the Japanese flounder, Paralichthys olivaceus.

We previously reported that characteristic deformities were induced by retinoic acid (RA) treatment of the Japanese flounder, Paralichthys olivaceus, at 6-9 days post-hatching (dph). To evaluate the toxic potency of nuclear retinoid receptors in induction of deformities by RA, we here investigated the effects of retinoic acid isomers on postembryonic development of this species. Larvae were exposed to either 25 nM of all-trans RA (atRA), 9-cis RA (9cRA) or 13-cis RA (13cRA) at 6-9 dph. All RA isomers induced deformities in the lower jaw, caudal fin and vertebrae. In the lower jaw, growth retardation of the dentary was evident. In the vertebrae, the major abnormalities were hypertrophy of the centrum, central fusion, and an increase in the number of abdominal vertebrae. Caudal fin deformities included deformity of caudal bone complex and absence of the entire caudal fin. The absence of the hypural primordium at 12 dph was the first sign of abnormality in caudal fin development, and resulted in complete blocking of the caudal fin development. Among the RA isomers, atRA induced the most severe deformity in all skeletons examined. Retinoic acid receptor (RAR) expression was activated by atRA and 9cRA, and pitx2 expression was inhibited in the lower jaw by atRA and 9cRA. Vitamin D receptor (VDR) expression was specifically inhibited by atRA treatment, suggesting that RA inhibits the lower jaw growth by suppressing the expression of these genes. These results suggest that RA exerted toxic effects on the skeletal systems, mainly through the RAR pathway.