A 16-day rearing trial was performed to investigate the influence of two supplemental levels (5% and 10%) of six dietary fat sources (linseed oil, peanut oil, coconut oil, soybean oil, lard oil and fish oil) on the growth, development and nutrient composition of black solider fly larvae. Our results demonstrated that the pre-pupa rate of larvae was linearly influenced by dietary C18:0, C18:3n-3 and C18:2n-6 content (pre-pupa rate = 0.927 × C18:0 content + 0.301 × C18:3n-3 content-0.258 × C18:2n-6 content p < 0.001)), while final body weight was linearly influenced by that of C16:0 (final body weight = 0.758 × C16:0 content, p = 0.004). Larval nutrient composition was significantly affected by dietary fat sources and levels, with crude protein, fat and ash content of larvae varying between 52.0 and 57.5, 15.0 and 23.8, and 5.6 and 7.2% dry matter. A higher level of C12:0 (17.4–28.5%), C14:0 (3.9–8.0%) and C16:1n-9 (1.3–4.3%) was determined in larvae fed the diets containing little of them. In comparison, C16:0, C18:1n-9, C18:2n-6 and C18:3n-3 proportions in larvae were linearly related with those in diets, with the slope of the linear equations varying from 0.39 to 0.60. It can be concluded that sufficient C16:0, C18:0 and C18:3n-3 supply is beneficial for larvae growth. Larvae could produce and retain C12:0, C14:0, and C16:1n-9 in vivo, but C16:0, C18:1n-9, C18:2n-6 and C18:3n-3 could only be partly incorporated from diets and the process may be enhanced by a higher amount of dietary fat. Based on the above observation, an accurately calculated amount of black soldier fly larvae could be formulated into aquafeed as the main source of saturated fatty acids and partial source of mono-unsaturated and poly-unsaturated fatty acids to save fish oil.

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n−6 to 18:3n−6, 18:2n−6 to 20:2n−6, and 18:3n−3 to 20:3n−3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

The rabbitfish Siganus canaliculatus is the first marine teleost shown to be able to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors catalyzed by two fatty acyl desaturases (fad) including Δ4 Fad and Δ6/Δ5 Fad as well as two elongases (Elovl4 and Elovl5). Previously, hepatocyte nuclear factor 4α (Hnf4α) was demonstrated to be predominant in the transcriptional regulation of two fads. To clarify the regulatory mechanisms involved in rabbitfish lipogenesis, the present study focused on the regulatory role of Hnf4α to elovl5 expression and LC-PUFA biosynthesis. Bioinformatics analysis predicted two potential Hnf4α elements in elovl5 promoter, one binding site was confirmed to interact with Hnf4α by gel shift assays. Moreover, overexpression of hnf4α caused a remarkable increase both in elovl5 promoter activity and mRNA contents, while knock-down of hnf4α in S. canaliculatus hepatocyte line (SCHL) resulted in a significant decrease of elovl5 gene expression. Meanwhile, hnf4α overexpression enhanced LC-PUFA biosynthesis in SCHL cell, and intraperitoneal injection to rabbitfish juveniles with Hnf4α agonists (Alverine and Benfluorex) increased the expression of hnf4α, elvol5 and Δ4 fad, coupled with an increased proportion of total LC-PUFA in liver. The results demonstrated that Hnf4α is involved in LC-PUFA biosynthesis by up-regulating the transcription of the elovl5 gene in rabbitfish, which is the first report of Hnf4α as a transcription factor of the elovl5 gene in vertebrates.

This study evaluated the effect of Lentinus edodes fermentation (LEF) on digestive enzyme activity, antioxidant capacity and morphology of the liver and intestine in largemouth bass (Micropterus salmoides) fed high plant protein diets (HPPD). LEF was supplemented in HPPD with 0 g kg⁻¹ (LEF0), 10 g kg⁻¹ (LEF1), 20 g kg⁻¹ (LEF2), 30 g kg⁻¹ (LEF3), 40 g kg⁻¹ (LEF4), 50 g kg⁻¹ (LEF5), respectively, and then the six diets were fed to largemouth bass with a body weight of 28.8 ± 0.05 g for eight weeks. Juvenile fish were randomized into 6 groups and each group had 4 replicates with 40 fish. Dietary LEF supplementation alleviated the liver inflammatory reaction of largemouth bass caused by HPPD and improved liver morphology. Goblet cells multiplied and the gut muscle layer thickened after LEF supplementation. The LEF significantly increased amylase activity in the liver and intestine of largemouth bass in individual experimental groups. The LEF could increase the activity of catalase in the liver and intestine of largemouth bass (p < 0.05). The content of malondialdehyde was significantly lower than that in the control group (p < 0.05). Dietary LEF supplementation had no significant effect on the intestinal flora of largemouth bass. These findings imply that LEF supplementation can reduce liver inflammation, enhance intestinal tissue morphology, and eventually benefit largemouth bass health.

Omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), particularly docosahexaenoic acids (22:6n-3, DHA), have positive effects on multiple biologic and pathologic processes. Fish are the major dietary source of n-3 LC-PUFA for humans. Growing evidence supports acyl-coenzyme A (acyl-CoA) synthetase 6 (acsl6) being involved in cellular DHA uptake and lipogenesis in mammals, while its molecular function and regulatory mechanism remain unknown in fish. The present study focused on investigating the molecular characterization and transcription regulation of the acsl6 gene in the freshwater teleost common carp (Cyprinus carpio). First, the full length of acsl6 cDNA contained a coding region of 2148 bp for 715 amino acids, which possessed all characteristic features of the acyl-CoA synthetase (ACSL) family. Its mRNA expression was the highest in the brain, followed by in the heart, liver, kidney, muscle, and eyes, but little expression was detected in the ovary and gills. Additionally, a candidate acsl6 promoter region of 2058 bp was cloned, and the sequence from −758 bp to −198 bp was determined as core a promoter by equal progressive deletion and electrophoretic mobility shift assay. The binding sites for important transcription factors (TFs), including stimulatory protein 1 (SP1), CCAAT enhancer-binding protein (C/EBPα), sterol-regulatory element binding protein 1c (SREBP1c), peroxisome proliferator activated receptor α (PPARα), and PPARγ were identified in the core promoter by site-directed mutation and functional assays. Furthermore, the intraperitoneal injection of PPARγ agonists (balaglitazone) increased the expression of acsl6 mRNA, coupling with an increased proportion of DHA in the muscle, while opposite results were obtained in the injection of the SREBP1c antagonist (betulin). However, the expression of acsl6 and DHA content in muscle were largely unchanged by PPARα agonist (fenofibrate) treatment. These results indicated that acsl6 may play an important role for the muscular DHA uptake and deposition in common carp, and PPARγ and SREBP-1c are the potential TFs involved in the transcriptional regulation of acsl6 gene. To our knowledge, this is the first report of the characterization of acsl6 gene and its promoter in teleosts.