miR-24 is involved in vertebrate LC-PUFA biosynthesis as demonstrated in marine teleost Siganus canaliculatus

Recently, microRNAs (miRNAs) have emerged as crucial regulators of lipid metabolism. However, the miRNA-mediated regulatory mechanism on long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis in vertebrates remains largely unknown. Here, we address a potentially important role of miRNA-24 (miR-24) in the regulation of LC-PUFA biosynthesis in rabbitfish Siganus canaliculatus. miR-24 showed significantly higher abundance in liver of rabbitfish reared in brackish water than in seawater for fish fed vegetable oil diets and in S. canaliculatus hepatocyte line (SCHL) cells incubated with alpha-linolenic acid (ALA) than the control group. Similar expression patterns were also observed on the expression of sterol regulatory element-binding protein-1 (srebp1) and LC-PUFA biosynthesis related genes. While opposite results were observed on the expression of insulin-induced gene 1 (insig1), an endoplasmic reticulum membrane protein blocking Srebp1 proteolytic activation. Luciferase reporter assays revealed rabbitfish insig1 as a target of miR-24. Knockdown of miR-24 in SCHL cells resulted in increased Insig1 protein, and subsequently reduced mature Srebp1 protein and expression of genes required for LC-PUFA biosynthesis, and these effects could be attenuated after additional insig1 knockdown. Opposite results were observed with overexpression of miR-24. Moreover, increasing endogenous insig1 by knockdown of miR-24 inhibited Srebp1 processing and consequently suppressed LC-PUFA biosynthesis in rabbitfish hepatocytes. These results indicate a potentially critical role for miR-24 in regulating LC-PUFA biosynthesis through the Insig1/Srebp1 pathway by targeting insig1. This is the first report of miR-24 involved in LC-PUFA biosynthesis and thus may provide knowledge on the regulatory mechanisms of LC-PUFA biosynthesis in vertebrates

Long-chain polyunsaturated fatty acid biosynthesis in the euryhaline herbivorous teleost Scatophagus argus: Functional characterization, tissue expression and nutritional regulation of two fatty acyl elongases

Both the spotted scat Scatophagus argus and rabbitfish Siganus canaliculatus belong to the few cultured herbivorous marine teleost, however, their fatty acyl desaturase (Fad) system involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is different. The S. argus has a △6 Fad, while the rabbitfish has △4 and △6/△5 Fads, which were the first report in vertebrate and marine teleost, respectively. In order to compare the characteristics of elongases of very long-chain fatty acids (Elovl) between them, two Elovl cDNAs were cloned from S. argus in the present study. One has 885bp of open read fragment (ORF) encoding a protein with 294 amino acid (aa) showing Elovl5 activity functionally characterized by heterologous expression in yeast, which was primarily active for the elongation of C18 and C20 PUFAs. The other has 915bp of ORF coding for a 305 aa protein showing Elovl4 activity, which was more efficient in the elongation of C20 and C22 PUFAs. Tissue distribution analyses by RT-PCR showed that elovl5 was highly expressed in the liver compared to other tissues determined, whereas elovl4 transcripts were only detected in the eye. The expression of elovl5 and elovl4 were significantly affected by dietary fatty acid composition, with highest expression of mRNA in the liver and eye of fish fed a diet with an 18:3n−3/18:2n−6 ratio of 1.7:1. These results indicated that the S. argus has a similar Elovl system in the LC-PUFA biosynthetic pathway to that of rabbitfish although their Fad system was different, suggesting that the diversification of fish LC-PUFA biosynthesis specificities is more associated with its Fad system. These new insights expand our knowledge and understanding of the molecular basis and regulation of LC-PUFA biosynthesis in fish

miR-17 is involved in the regulation of LC-PUFA biosynthesis in vertebrates: Effects on liver expression of a fatty acyl desaturase in the marine teleost Siganus canaliculatus

Biosynthesis in vertebrates of long-chain polyunsaturated fatty acids (LC-PUFA) such as arachidonic (ARA; 20:4n-6), eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids requires the catalysis by fatty acyl desaturases (Fads). A vertebrate Fad with Δ4 activity catalyzing the direct conversion of 22:5n-3 to DHA was discovered in the marine teleost rabbitfish Siganus canaliculatus. Recent studies in vertebrates have shown that miRNAs may participate in the regulation of lipid metabolism at post-transcription level. However, their roles in LC-PUFA biosynthesis were not known. In the present study, in silico analysis predicts that the rabbitfish Δ4 Fad may be a target of miR-17 and thus we cloned miR-17, which is located at the forepart of the miR-17-92 cluster. Dual luciferase reporter assays demonstrated that miR-17 targeted the 3'UTR of Δ4 Fad directly. Furthermore, the expression level of miR-17 displayed an inverse pattern with that of Δ4 Fad mRNA in gill, liver and eyes, and also the Δ4 Fad protein quantity in rabbitfish liver. Incubation of rabbitfish primary hepatocytes with linoleic acid (LA; 18:2n-6), α-linolenic acid (LNA; 18:3n-3), EPA or DHA showed differential effects on miR-17, Δ4 Fad and Δ6/Δ5 Fad expression. LNA promoted the expression of miR-17 and Δ6/Δ5 Fad, but suppressed the expression of Δ4 Fad. In contrast, LA and EPA decreased the expression of miR-17 and Δ6/Δ5 Fad, but had no effect on Δ4 Fad. However, all the above were down-regulated by DHA. These data indicate that miR-17 was involved in the regulation of LC-PUFA biosynthesis in rabbitfish liver by targeting Δ4 Fad

Cloning, Functional Characterization and Nutritional Regulation of Delta 6 Fatty Acyl Desaturase in the Herbivorous Euryhaline Teleost Scatophagus Argus

Marine fish are generally unable or have low ability for the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, with some notable exceptions including the herbivorous marine teleost Siganus canaliculatus in which such a capability was recently demonstrated. To determine whether this is a unique feature of S. canaliculatus or whether it is common to the herbivorous marine teleosts, LC-PUFA biosynthetic pathways were investigated in the herbivorous euryhaline Scatophagus argus. A putative desaturase gene was cloned and functionally characterized, and tissue expression and nutritional regulation were investigated. The full-length cDNA was 1972 bp, containing a 1338 bp open-reading frame encoding a polypeptide of 445 amino acids, which possessed all the characteristic features of fatty acyl desaturase (Fad). Functional characterization by heterologous expression in yeast showed the protein product of the cDNA efficiently converted 18:3n-3 and 18:2n-6 to 18:4n-3 and 18:3n-6, respectively, indicating D6 desaturation activity. Quantitative real-time PCR showed that highest D6 fad mRNA expression was detected in liver followed by brain, with lower expression in other tissues including intestine, eye, muscle, adipose, heart kidney and gill, and lowest expression in stomach and spleen. The expression of D6 fad was significantly affected by dietary lipid and, especially, fatty acid composition, with highest expression of mRNA in liver of fish fed a diet with a ratio of 18:3n-3/18:2n-6 of 1.72:1. The results indicated that S. argus may have a different LC-PUFA biosynthetic system from S. canaliculatus despite possessing similar habitats and feeding habits suggesting that LC-PUFA biosynthesis may not be common to all marine herbivorous teleosts

Dietary DHA Enhanced the Textural Firmness of Common Carp (<i>Cyprinus carpio</i> L.) Fed Plant-Derived Diets through Restraining FoxO1 Pathways

Omega-3 fatty acids have a positive effect on the muscle textural firmness of fish, while the intrinsic mechanism is poorly understood. To investigate the potential mechanism of textural modification caused by dietary docosahexaenoic acid ( DHA) in common carp (Cyprinus carpio L.), three plant-derived diets with varying DHA levels (0%, 0.5%, 1%, D1–D3) were prepared to feed juveniles (initial weight 15.27 ± 0.77 g) for 8 weeks, and the muscular texture, fibers density, and transcriptome were analyzed. The results showed that the growth performance, muscular DHA content, fibers density, and texture of the fish fed diets D2 and D3 were significantly ameliorated compared with the fish fed diet D1. The muscular transcriptome profiles indicated that the up-regulated genes of fish fed dietary DHA mainly in response to muscle proliferation, as well as the FoxO pathway, were significantly enriched in the D2 and D3 groups. Consistent with this, the Quantitative Real-Time PCR (qRT-PCR ) assays indicated that the expression of myogenic regulatory factors (myog, myod, mrf4, mrf5) was up-regulated in the high-DHA groups. Additionally, the expression of foxo1 (inhibitor of myofiber development) mRNA was down-regulated, while its negative regulatory pathway (MAPK and PI3K) was activated in the D2 and D3 groups. The results suggested that the DHA supplementation is beneficial to modifying the muscular textural firmness of common carp fed plant-derived diets, which could be attributed to the inhibition of FoxO1 pathways

Characteristics of LC-PUFA biosynthesis in marine herbivorous teleost Siganus canaliculatus under different ambient salinities

The present study aimed to characterize the influence of salinity on the biosynthesis of long-chain polyunsaturated fatty acid (LC-PUFA) in rabbitfishSiganus canaliculatus. An eight-week feeding trial was performed in rabbitfish juveniles with diets containing fish oil (FO) or a blend of vegetable oils (perilla and Canola oils, VO) at two salinities, 32 and 10ppt. The whole-body fatty acid mass balance (FAMB) method was used to evaluate thein vivoLC-PUFA biosynthetic activities, and the hepatic mRNA levels of &Delta;4 and &Delta;6/&Delta;5 fatty acyl desaturases (Fad) and elongase of very long-chain fatty acids (Elovl5) genes were determined by real-time quantitative PCR. The results showed that theex novoproduction of LC-PUFA in fish receiving the VO diet was significantly higher than fish fed the FO diet at both salinities. Furthermore, LC-PUFA production at 10ppt salinity was significantly higher than that at 32ppt salinity in the VO dietary groups, whereas no effect of salinity was found in the FO dietary groups. Consistent with this, the calculated apparentin vivodesaturation and elongation activities were also higher in VO and low-salinity treatments. In addition, higher levels of&Delta;4and&Delta;6/&Delta;5 fadsmRNA expression were obtained at low salinity, which was consistent with the calculated enzyme activities. In contrast, the expression ofelovl5was lower than that offads,and the levels were not consistent with the elongase activity. The results suggest that ambient salinity may affect the activity of the LC-PUFA biosynthetic pathway in rabbitfish through regulating fatty acyl desaturase and elongase activities, partly through a transcriptional control mechanism in the case of desaturases

Effects of Dietary Animal Protein Source Composition on the Growth Performance, Intestinal Health, and Protein Metabolism of Largemouth Bass (Micropterus salmoides)

Terrestrial animal protein sources contain less antinutritional factors, high protein content, and functional factors, which are beneficial to fish health. Among them, poultry byproduct meal (containing 65%-73% protein rich in vitamins) and porcine meat meal (containing 45%-60% protein and high contents of proline and glycine) are the most widely used meals in aquatic compound feeds, and are important fish meal replacement sources. As a carnivorous fish species, largemouth bass (Micropterus salmoides) is highly dependent on dietary fish meal, and the level of fish meal added in its commercial feeds is up to 50%. However, the rising price of fish meal increases the farming cost of M. salmoides. Therefore, it is necessary to identify a suitable alternative protein source to reduce the amount of dietary fish meal and the feed cost. Therefore, seven compound feeds (D1-D7) were prepared in this study. The added ratios of fish meal/poultry byproduct meal/porcine meat meal were as follows: 45.0/22.6/0, 37.1/22.6/8.0, 28.8/22.6/16.0, 45.0/14.5/8.0, 45.0/5.3/16.0, 41.6/18.0/8.0, and 37.0/13.8/16.0. Juvenile M. salmoides (initial body weight ~55 g) were fed the above diets for 60 days with five replicates in each group. The effects of the animal protein source combination on the growth performance, tissue biochemical indices, muscle texture characteristics, liver protein metabolism, and intestinal inflammatory factor-related gene expression were evaluated. The water temperature during the feeding trial was 27.4-32.3 ℃ and the ammonia nitrogen concentration was 0.1-0.2 mg/L. After the feeding experiment, three fish were randomly selected from each cage to collect the serum, liver, intestinal tract, muscle, and other samples, which were then stored at -80 ℃. In addition, three fish were randomly selected from each cage to determine their morphological indices. At the same time, two fish were selected from each cage to determine the muscle texture characteristics and the whole fish proximate composition. Physiological and biochemical indices of serum and liver tissues, albumin, urea nitrogen (BUN), total amino acid (T-AA), alanine aminotransferase (ALT), aspartate aminotransferase, total protein (TP), and blood ammonia (SA) levels), were determined using commercial kits, and the texture characteristics of muscle were determined by using a texture analyzer. The moisture, crude fat, crude protein, and ash contents of whole fish and muscle were determined by atmospheric drying, Soxhlet extraction, Kjeldahl nitrogen determination, and Muffle furnace incineration, respectively. Real-time quantitative PCR was used to determine the expression levels of genes related to liver protein metabolism and the intestinal inflammatory response. All test data were expressed as the mean±standard error, and multiple comparisons were made by the Tukey test, with P 0.05). The whole-body crude protein content in the D3 group was significantly higher than that in the D1 group, and the crude lipid level in the D3 group was significantly lower than that in the D6 group (P 0.05). The serum T-AA content of fish in the D3 group was significantly higher than that in the D1 and D4 groups (P 0.05). In terms of muscle quality, the muscle hardness, adhesion, and mastication in the D3 group were significantly lower than those in the D4 and D6 groups, respectively (P 0.05). In addition, the mRNA expression levels of intestinal il-10 and liver tor, s6k1, akt, and pi3k in the D3 group were upregulated, and were significantly higher than those in the D7 group (P < 0.05). The mRNA expression levels of il-1β and il-6 in the intestines and 4ebp-1 in the liver of the D3 group were significantly lower than those of the D1 group (P < 0.05). These results indicated that combined use of 28.8% fish meal, 16.0% porcine meat meal, and 22.6% poultry byproduct meal had the best growth promotion effect on M. salmoides, and was able to improve liver protein synthesis and maintain intestinal health. The results of this study provided technical support for reducing the dependence of M. salmoides compound feed on fish meal

Evaluation of High Efficiency and Low Fish Meal Diets for Golden Pompano (Trachinotus ovatus) in Deep-Sea Cage Culture

Trachinotus ovatus, commonly known as golden pompano, is a euryhaline warm water carnivorous fish. It has the characteristics of fast growth, simple feeding, delicious meat, strong stress resistance, and high survival rate. It can accept compound feed throughout its growth. It is popular among fish breeders and consumers because of its moderate specifications and affordable price. With an annual output of 240 000 tons, it has become one of the most important marine fish breeding species in the southern coastal areas of China. As a marine carnivorous fish, it has specific requirements relating to the levels and sources of dietary protein and fat, and a strong dependence on fish meal and fish oil, which are limited resources with high prices, which also determines its high feed cost. However, compared with other rare sea fish, its price is low and the profit margin of breeding is low (2–4 CNY/kg), thus, easily leading to the loss of breeding enterprises and individual businesses. Therefore, it is necessary to develop efficient and low-cost compound diets and reduce the supplemental level of fish meal oil in diets to solve the bottleneck problem of golden pompano fish breeding. Previous studies have shown that T. ovatus subjected to a high efficiency and low fish meal diet exhibited excellent growth and health in pond cage culture. To further evaluate the application effect of this feed in deep-sea cage culture, an experimental feed (crude protein 47.66%, crude fat 7.98%) based on the formula feed of a low fish meal diet was produced by a feed company with a large-scale production process (feed production using large machinery and mass production in a feed mill with an hourly output that can reach more than 10 t using equipment such as oil sprayer machines, where the fat source is added by spraying). A commercial feed from a well-known brand was used as the control diet (crude protein 47.75%, crude fat 9.63%). Large-sized golden pompano (mean body weight ~262 g) were provided by Yangjiang Haina Fisheries Limited and kept for 2 weeks at the deep-sea cage breeding base in Dasuo Island, Yangjiang (12–20 m depth, about 15 km offshore) to adapt to the test environment. During the temporary feeding period, a well-known commodity was used for feed. Overall, 150 000 healthy large-sized golden pompano with neat specifications (initial body weight ~260 g) were selected and randomly assigned to six deep-sea cages (HDPE C60 floating cages, circumference 60 m, 25 000 fish per cage). Each feed was provided in three parallel cages for 33 days (April 29 to May 31, 2021). During breeding, full food was provided twice a day (07:00 and 17:00). During the experiment, the seawater temperature was 20.00~29.00 ℃. Dissolved oxygen was 6.30~7.80 mg/L. The results showed that the growth performance of fish was not statistically different between the two groups (P > 0.05). However, compared with the control group, the weight gain rate and specific growth rate of fish-fed experimental diets increased by 14.43 % and 8.19 %, respectively, and the average daily weight gain increased by 0.68 g. In terms of muscle nutrition and texture characteristics, the muscle lipid contents of the fish-fed experimental diets were significantly higher than those of fish-fed control diets (P 0.05). Compared with the control group, the serum protein, triglyceride, total cholesterol, and low-density lipoprotein contents, as well as the activity of aspartate aminotransferase, of fish fed the experimental diet were significantly decreased (P 0.05). In addition, the feed cost per 1 kg of fish receiving the experimental diet was 18.80% lower than that of fish receiving the control diets, and its culture benefit was increased by 62.12%. The results showed that the experimental diet (high efficiency and low fish meal diet) not only promoted growth, but also improved the muscle fat level and serum lipid metabolism of the fish. These results indicate that the high efficiency and low fish meal diet can be applied in the culture of golden pompano within deep-sea cages. In this study, a high efficiency and low fish meal diet for T. ovatus was developed by using amino acid balance technology and fatty acid precision nutrition technology in deep-sea cage large-scale culture. Through the analysis of growth performance, serum biochemical parameters, liver lipid metabolism, and antioxidant properties, it was found that the growth promoting effect of test material was comparable to that of commercial material, and could improve the muscle quality and liver health of golden pompano. Use of the experimental diet could also reduce the cost of breeding, improve the economic benefits, and result in high economic value. The results indicate that the experimental high efficiency and low fish meal diet for T. ovatus has a good application effect and excellent market development prospects, and also has important practical guiding significance for the large-scale production and application of high efficiency low fish meal compound feed, solving the problem of aquaculture bottleneck and facilitating deep-sea golden pompano culture

Regulation of long-chain polyunsaturated fatty acid biosynthesis in teleost fish

Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA, C20-24), including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), are involved in numerous biological processes and have a range of health benefits. Fish have long been considered as the main source of n-3 LC-PUFA in human diets. However, the capacity for endogenous biosynthesis of LC-PUFA from C18 PUFA varies in fish species based on the presence, expression and activity of key enzymes including fatty acyl desaturases (Fads) and elongation of very long-chain fatty acids (Elovl) proteins. In this article, we review progress on the identified Fads and Elovl, as well as the regulatory mechanisms of LC-PUFA biosynthesis both at transcriptional and post-transcriptional levels in teleosts. The most comprehensive advances have been obtained in rabbitfish Siganus canaliculatus, a marine teleost demonstrated to have the entire pathway for LC-PUFA biosynthesis, including the roles of transcription factors hepatocyte nuclear factor 4α (Hnf4α), liver X receptor alpha (Lxrα), sterol regulatory element-binding protein 1 (Srebp-1), peroxisome proliferator-activated receptor gamma (Pparγ) and stimulatory protein 1 (Sp1), as well as post-transcriptional regulation by individual microRNA (miRNA) or clusters. This research has, for the first time, demonstrated the involvement of Hnf4α, Pparγ and miRNA in the regulation of LC-PUFA biosynthesis in vertebrates. The present review provides readers with a relatively comprehensive overview of the progress made into understanding LC-PUFA biosynthetic systems in teleosts, and some insights into improving endogenous LC-PUFA biosynthesis capacity aimed at reducing the dependence of aquafeeds on fish oil while maintaining or increasing flesh LC-PUFA content and the nutritional quality of farmed fish.This work was financially supported by the National Key R&D Program of China (2018YFD0900400) and National Natural Science Foundation of China (31873040, 31110103913, 30972266, 30671629, 31702357), and partly funded through the project IMPROMEGA of the Ministry of Science, Innovation and Universities, Spanish Government (grant no. RTI2018-095119-B-I00, MCIU/AEI/FEDER, UE).Peer reviewe

Effects of dietary n-3 LC-PUFA levels on the growth, immunity, and lipid metabolism of freshwater carnivorous teleost largemouth bass (Micropterus salmoides) juveniles

The n-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA) play important roles in the normal growth and metabolism of fish . In order to explore the appropriate requiremnts of n-3 LC-PUFA in juvenile largemouth bass Micropterus salmoides, a freshwater carnivorous teleost, four isoproteic (50 %) and isolipidic (11 %) diets were formulated. The control diet used fish oil (FO) as the sole lipid source, and the other three diets used mixed vegetable oil as the main lipid source and supplemented with 0.50 %, 0.80 % and 1.10 % n-3 LC-PUFA, respectively. After the juvenile largemouth bass (initial weight 12.11 ± 0.01 g) fed with the four diets for 75 days, the growth, serum and heptic biochemical indices, as well as hepatic lipid metabolism were investigated. The results showed that the growth performance of 0.50 %-1.10 % groups were high that of control group; comparing to the 1.10 % group, high weight gain, specific growth rate and daily gain, as well as muscle hardness, chewiness and gumminess were detected in the 0.80 % group (P < 0.05). With the increase of dietary n-3 LC-PUFA levels, the serum biochemical indices (total cholesterol, triglyceride, and aspartate aminotransferase activity) and hepatic triglyceride were significantly decreased, while the serum acid phosphatase and alkaline phosphatase, as well as hepatic and muscle n-3 LC-PUFA contents were significantly increased (P < 0.05). Compared with the 0.50 % group, high hepatic mRNA expression levels of anti-inflammatory genes (il-10, tgf-β) were observed in the 0.80 % group (P < 0.05); and the relatively high transcript levels of genes related to lipid transport and catabolism (hsl, pparα, fatp and mtp) and low transcript levels of genes related to lipid anabolism (accα and pparγ) were detected in the 0.80 % and 1.10 % groups, as well as high mRNA levels of accα and pparγ were measured in the 0.80 % group (P < 0.05). The results indicated that 0.80 % dietary n-3 LC-PUFA level is suitable for largemouth bass concerning the growth performance, muscle quality and hepatic lipid metabolism. When subjected the weight gain to the second-order polynomial regression analysis, the maximum value was achieved at 0.76 % n-3 LC-PUFA level, which provide a basis for the development of formula feed for largemouth bass