Gamma-Aminobutyric Acid (GABA) Avoids Deterioration of Transport Water Quality, Regulates Plasma Biochemical Indices, Energy Metabolism, and Antioxidant Capacity of Tawny Puffer (Takifugui flavidus) under Transport Stress.

Live fish transportation is crucial for managing aquaculture but can pose health risks to fish due to stressors encountered during transportation. Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that plays a crucial role in the central nervous system and is considered to exhibit anti-stress effects. This study aims to investigate the effects of GABA on the transport water quality, plasma biochemical indices, energy metabolism, and antioxidant capacity of tawny puffer (Takifugu flavidus) under transport stress. Tawny puffer were pretreated by immersing in aquariums containing GABA (final concentrations at 0, 5, 50, and 150 mg/L) seawater for 3 days; then, simulated transport was conducted using oxygen-filled polyethylene bags containing the same concentration of GABA seawater as the pretreatment period. Water samples, plasma, and liver were collected after 0, 6, and 12 h of transport. The results revealed that with the prolongation of transportation time, the control group's water quality deteriorated, stress-related plasma biochemical indices increased, glycolytic substrate contents decreased, glycolytic enzyme activities and product contents increased, and aerobic metabolic enzyme activities exhibited initial increases followed by declines, ATPase activities decreased, antioxidant enzyme activities decreased, and the lipid peroxidation marker contents increased. It is noteworthy that GABA treatment could avoid water quality deterioration during transportation, inhibit an elevation in stress-related biochemical indicators, regulate energy metabolism, and reduce oxidative damage in tawny puffer, especially at 50 and 150 mg/L concentrations. In summary, GABA treatment can effectively alleviate the transport stress of tawny puffer.

Anti-Melanogenic Effects of Takifugu flavidus Muscle Hydrolysate in B16F10 Melanoma Cells and Zebrafish.

Abnormal melanogenesis can lead to hyperpigmentation. Tyrosinase (TYR), a key rate-limiting enzyme in melanin production, is an important therapeutic target for these disorders. We investigated the TYR inhibitory activity of hydrolysates extracted from the muscle tissue of Takifugu flavidus (TFMH). We used computer-aided virtual screening to identify a novel peptide that potently inhibited melanin synthesis, simulated its binding mode to TYR, and evaluated functional efficacy in vitro and in vivo. TFMH inhibited the diphenolase activities of mTYR, reducing TYR substrate binding activity and effectively inhibiting melanin synthesis. TFMH indirectly reduced cAMP response element-binding protein phosphorylation in vitro by downregulating melanocortin 1 receptor expression, thereby inhibiting expression of the microphthalmia-associated transcription factor, further decreasing TYR, tyrosinase related protein 1, and dopachrome tautomerase expression and ultimately impeding melanin synthesis. In zebrafish, TFMH significantly reduced black spot formation. TFMH (200 µg/mL) decreased zebrafish TYR activity by 43% and melanin content by 52%. Molecular dynamics simulations over 100 ns revealed that the FGFRSP (T-6) peptide stably binds mushroom TYR via hydrogen bonds and ionic interactions. T-6 (400 µmol/L) reduced melanin content in B16F10 melanoma cells by 71% and TYR activity by 79%. In zebrafish, T-6 (200 µmol/L) inhibited melanin production by 64%. TFMH and T-6 exhibit good potential for the development of natural skin-whitening cosmetic products.

Affinity Purification and Molecular Characterization of Angiotensin-Converting Enzyme (ACE)-Inhibitory Peptides from Takifugu flavidus.

An affinity chromatography filler of CNBr-activated Sepharose 4B-immobilized ACE was used to purify ACE-inhibitory peptides from Takifugu flavidus protein hydrolysate (<1 kDa). Twenty-four peptides with an average local confidence score (ALC) ≥ 80% from bounded components (eluted by 1 M NaCl) were identified by LC-MS/MS. Among them, a novel peptide, TLRFALHGME, with ACE-inhibitory activity (IC50 = 93.5 µmol·L-1) was selected. Molecular docking revealed that TLRFALHGME may interact with the active site of ACE through H-bond, hydrophobic, and electrostatic interactions. The total binding energy (ΔGbinding) of TLRFALHGME was estimated to be -82.7382 kJ·mol-1 by MD simulations, indicating the favorable binding of peptides with ACE. Furthermore, the binding affinity of TLRFALHGME to ACE was determined by surface plasmon resonance (SPR) with a Kd of 80.9 µmol, indicating that there was a direct molecular interaction between them. TLRFALHGME has great potential for the treatment of hypertension.

Transcriptome analysis revealed gene expression feminization of testis after exogenous tetrodotoxin administration in pufferfish Takifugu flavidus.

Tetrodotoxin (TTX) is a deadly neurotoxin and usually accumulates in large amounts in the ovaries but is non-toxic or low toxic in the testis of pufferfish. The molecular mechanism underlying sexual dimorphism accumulation of TTX in ovary and testis, and the relationship between TTX accumulation with sex related genes expression remain largely unknown. The present study investigated the effects of exogenous TTX treatment on Takifugu flavidus. The results demonstrated that exogenous TTX administration significantly incresed level of TTX concentration in kidney, cholecyst, skin, liver, heart, muscle, ovary and testis of the treatment group (TG) than that of the control group (CG). Transcriptome sequencing and analysis were performed to study differential expression profiles of mRNA and piRNA after TTX administration of the ovary and testis. The results showed that compared with female control group (FCG) and male control group (MCG), TTX administration resulted in 80 and 23 piRNAs, 126 and 223 genes up and down regulated expression in female TTX-treated group (FTG), meanwhile, 286 and 223 piRNAs, 2 and 443 genes up and down regulated expression in male TTX-treated group (MTG). The female dominant genes cyp19a1, gdf9 and foxl2 were found to be up-regulated in MTG. The cyp19a1, whose corresponding target piRNA uniq_554482 was identified as down-regulated in the MTG, indicating the gene expression feminization in testis after exogenous TTX administration. The KEGG enrichment analysis revealed that differentially expressed genes (DEGs) and piRNAs (DEpiRNAs) in MTG vs MCG group were more enriched in metabolism pathways, indicating that the testis produced more metabolic pathways in response to exogenous TTX, which might be a reason for the sexual dimorphism of TTX distribution in gonads. In addition, TdT-mediated dUTP-biotin nick end labeling staining showed that significant apoptosis was detected in the MTG testis, and the role of the cell apoptotic pathways was further confirmed. Overall, our research revealed that the response of the ovary and testis to TTX administration was largely different, the ovary is more tolerant whereas the testis is more sensitive to TTX. These data will deepen our understanding on the accumulation of TTX sexual dimorphism in Takifugu.

A rational tool for the umami evaluation of peptides based on multi-techniques.

Umami peptides have become of key interest in the development of flavoring agents. However, the lack of known umami peptides further prevents the understanding of the umami mechanism. The famous pufferfish (Takifugu flavidus) is a great resource for novel umami peptides, and we further analyze the umami characteristics of peptides based on multi-evaluation. In this study, five novel umami peptides, DF9, TK18, AK11, IK10, and GT12 were found; DF9 having the highest umami intensity, followed by AK11. Moreover, biosensor results showed DF9 with the lowest Ka value of 6.85 × 10-13 mol/L, followed by AK11. These data are mostly in agreement with sensory evaluation and fully reveal the umami mechanism of peptides. Quantum chemical and molecular docking demonstrated active site D in peptides bound with T1R1 receptor. Our results open up new strategies to estimate the taste characteristics of umami peptides and provide rational tools for screening umami peptides in food.

A Novel Angiotensin-I-Converting Enzyme (ACE) Inhibitory Peptide from Takifugu flavidus.

Alcalase, neutral protease, and pepsin were used to hydrolyze the skin of Takifugu flavidus. The T. flavidus hydrolysates (TFHs) with the maximum degree of hydrolysis (DH) and angiotensin-I-converting enzyme (ACE)-inhibitory activity were selected and then ultra-filtered to obtain fractions with components of different molecular weights (MWs) (<1, 1-3, 3-10, 10-50, and >50 kDa). The components with MWs < 1 kDa showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.58 mg/mL. Purification and identification using semi-preparative liquid chromatography, Sephadex G-15 gel chromatography, RP-HPLC, and LC-MS/MS yielded one new potential ACE-inhibitory peptide, PPLLFAAL (non-competitive suppression mode; IC50 of 28 µmmol·L-1). Molecular docking and molecular dynamics simulations indicated that the peptides should bind well to ACE and interact with amino acid residues and the zinc ion at the ACE active site. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that PPLLFAAL could significantly decrease the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHRs after intravenous administration. These results suggested that PPLLFAAL may have potential applications in functional foods or pharmaceuticals as an antihypertensive agent.

Multi-Omics Analysis of Gene and Protein Candidates Possibly Related to Tetrodotoxin Accumulation in the Skin of Takifugu flavidus.

Pufferfish is increasingly regarded by many as a delicacy. However, the tetrodotoxin (TTX) that accumulates in its body can be lethal upon consumption by humans. TTX is known to mainly accumulate in pufferfish skin, but the accumulation mechanisms are poorly understood. In this study, we aimed to explore the possible mechanism of TTX accumulation in the skin of the pufferfish Takifugu flavidus following treatment with TTX. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we detected 37.3% of toxin accumulated in the skin at the end of the rearing period (168 h). Transcriptome and proteome analyses revealed the mechanism and pathways of TTX accumulation in the skin of T. flavidus in detail. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes analyses strongly suggest that cardiac muscle contraction and adrenergic signaling in cardiomyocyte pathways play an important role in TTX accumulation. Moreover, some upregulated and downregulated genes, which were determined via RNA-Seq, were verified with qPCR analysis. This study is the first to use multi-omics profiling data to identify novel regulatory network mechanisms of TTX accumulation in the skin of pufferfish.

Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus.

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein-toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

Comparing the metabolic profiles of raw and cooked pufferfish (Takifugu flavidus) meat by NMR assessment.

The difference of metabolite profiles between raw and cooked pufferfish (Takifugu flavidus) meat was explored by 1H NMR technique and multivariate statistical methods. The orthogonal projection to latent structure-discriminant analysis (OPLS-DA) results showed an obvious separation between two samples. There were 24 dominating metabolites in the pufferfish muscle extraction, of which 11 metabolites changed significantly (p < 0.05), including 9 amino acids (alanine, leucine, methionine, tyrosine, glutamate, glycine, arginine, lysine, taurine), 1 organic acid (lactate) and 1 alkaloid (betaine). Moreover, the Student's t-test was performed to identify the different levels of metabolites. Sensory intensity experiments showed that there was a significant difference in the umami taste between raw and cooked pufferfish meat (p < 0.05). The content of glutamate, aspartate and 5'-IMP in the cooked meat increased, making the umami taste more intense. This study provided essential information about the metabolites explaining the taste difference between raw and cooked pufferfish meat.

Electrodialysis Extraction of Pufferfish Skin (Takifugu flavidus): A Promising Source of Collagen.

Collagen is widely used in drugs, biomaterials, foods, and cosmetics. By-products of the fishing industry are rich sources of collagen, which can be used as an alternative to collagen traditionally harvested from land mammals. However, commercial applications of fish-based collagen are limited by the low efficiency, low productivity, and low sustainability of the extraction process. This study applied a new technique (electrodialysis) for the extraction of Takifugu flavidus skin collagen. We found electrodialysis to have better economic and environmental outcomes than traditional dialysis as it significantly reduced the purification time and wastewater (~95%) while maintaining high extraction yield (67.3 ± 1.3 g/100 g dry weight, p < 0.05). SDS-PAGE, amino acid composition analysis, and spectrophotometric characterization indicated that electrodialysis treatment retained the physicochemical properties of T. flavidus collagen. Heavy metals and tetrodotoxin analyses indicated the safety of T. flavidus collagen. Notably, the collagen had similar thermal stability to calf skin collagen, with the maximum transition temperature and denaturation temperature of 41.8 ± 0.35 and 28.4 ± 2.5 °C, respectively. All evidence suggests that electrodialysis is a promising technique for extracting collagen in the fishing industry and that T. flavidus skin collagen could serve as an alternative source of collagen to meet the increasing demand from consumers.

Mitochondrial DNA sequence of the hybrid of Takifugu flavidus (♀) × Takifugu rubripes (♂).

The complete mitochondrial genome of the hybrid of Takifugu flavidus (♀) × Takifugu rubripes (♂) was sequenced and characterized. The total length of the mitogenome is 16,443 bp. It contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 2 non-coding regions: origin of light-strand replication (OL) and control region (D-loop). The overall nucleotide composition of the heavy strand is 30.05% A, 25.79% T, 15.07% G and 29.09% C, with an A + T content of 55.84%. Except for ND6 gene and eight tRNA genes, other genes are encoded on the heavy strand. The complete mitogenome of the hybrid of T. flavidus (♀) × T. rubripes (♂) should contribute to further studies on mitochondrial inheritance mechanism.

The complete mitochondrial genome sequence of Takifugu flavidus (Tetraodontiformes: Tetrodontidae).

The complete mitochondrial genome of Takifugu flavidus (Tetraodontiformes: Tetrodontidae) was obtained in this study. The mitogenome is 16,449 bp in size and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 2 non-coding regions: origin of light-strand replication (OL) and control region (D-loop). The overall nucleotide composition of the heavy strand was 29.88% A, 25.81% T, 15.28% G and 29.03% C, with a slight AT bias of 55.69%. Except for ND6 gene and eight tRNA genes, other genes are encoded on the heavy strand. The mitochondrial genome data of T. flavidus should contribute to phylogenetic analysis and studies on genetic structure, as well as molecular phylogeny and species identification of Tetrodontidae.

Draft sequencing and analysis of the genome of pufferfish Takifugu flavidus.

The pufferfish Takifugu flavidus is an important economic species due to its outstanding flavour and high market value. It has been regarded as an excellent model of genetic study for decades as well. In the present study, three mate-pair libraries of T. flavidus genome were sequenced by the SOLiD 4 next-generation sequencing platform, and the draft genome was constructed with the short reads using an assisted assembly strategy. The draft consists of 50,947 scaffolds with an N50 value of 305.7 kb, and the average GC content was 45.2%. The combined length of repetitive sequences was 26.5 Mb, which accounted for 6.87% of the genome, indicating that the compactness of T. flavidus genome was approximative with that of T. rubripes genome. A total of 1,253 non-coding RNA genes and 30,285 protein-encoding genes were assigned to the genome. There were 132,775 and 394 presumptive genes playing roles in the colour pattern variation, the relatively slow growth and the lipid metabolism, respectively. Among them, genes involved in the microtubule-dependent transport system, angiogenesis, decapentaplegic pathway and lipid mobilization were significantly expanded in the T. flavidus genome. This draft genome provides a valuable resource for understanding and improving both fundamental and applied research with pufferfish in the future.