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.

Functional role of TrIL-1ß in Takifugu rubripes defense against Cryptocaryon irritans infection.

The Japanese puffer, Takifugu rubripes, is a commercially important fish species in China that is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. We previously found that interleukin-1ß (IL-1ß), an important cytokine with a potential role in resistance against pathogens, was one of the most significantly differentially up-regulated proteins in the gills and spleen of T. rubripes infected by the protozoan parasite Cryptocaryon irritans. In this study, we assessed the potential function of T. rubripes IL-1ß (TrIL-1ß) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1ß protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1ß may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1ß promoted the expression of endogenous IL-1ß, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1ß group had less tissue damage compared with control groups of fish. IL-1ß-small interfering RNA and IL-1ß overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1ß regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1ß also promoted the differential expression of IgM, showing that it was involved in humoral immunity of T. rubripes. The cumulative mortality experiment show that TrIL-1ß could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1ß. They also suggested that recombinant TrIL-1ß could be used as an adjuvant in a subunit vaccine against C. irritans infection, which is of profound importance for the prevention and control of parasitic diseases in T. rubripes.

Gluconeogenesis during development of the grass puffer (Takifugu niphobles).

During the development of teleost fish, the sole nutrient source is the egg yolk. The yolk consists mostly of proteins and lipids, with only trace amounts of carbohydrates such as glycogen and glucose. However, past evidence in some fishes showed transient increase in glucose during development, which may have supported the development of the embryos. Recently, we found in zebrafish that the yolk syncytial layer (YSL), an extraembryonic tissue surrounding the yolk, undergoes gluconeogenesis. However, in other teleost species, the knowledge on such gluconeogenic functions during early development is lacking. In this study, we used a marine fish, the grass puffer (Takifugu niphobles) and assessed possible gluconeogenic functions of their YSL, to understand the difference or shared features of gluconeogenesis between these species. A liquid chromatography (LC) / mass spectrometry (MS) analysis revealed that glucose and glycogen content significantly increased in the grass puffer during development. Subsequent real-time PCR results showed that most of the genes involved in gluconeogenesis increased in segmentation stages and/or during hatching. Among these genes, many were expressed in the YSL and liver, as shown by in situ hybridization analysis. In addition, glycogen immunostaining revealed that this carbohydrate source was accumulated in many tissues at segmentation stage but exclusively in the liver in hatched individuals. Taken together, these results suggest that developing grass puffer undergoes gluconeogenesis and glycogen synthesis during development, and that gluconeogenic activity is shared in YSL of zebrafish and grass puffer.

Intraperitoneal administration of arginine vasotocin (AVT) induces anorexigenic and anxiogenic actions via the brain V1a receptor-signaling pathway in the tiger puffer, Takifugu rubripes.

Arginine vasotocin (AVT) is produced mainly in the hypothalamus and as a neurohypophyseal hormone peripherally regulates water-mineral balance in sub-mammals. In addition, AVT-containing neurons innervate several areas of the brain, and AVT also acts centrally as both an anorexigenic and anxiogenic factor in goldfish. However, it is unclear whether these central effects operate in fish in general. In the present study, therefore, we investigated AVT-like immunoreactivity in the brain of the tiger puffer, a cultured fish with a high market value in Japan and also a representative marine teleost species, focusing particularly on whether AVT affects food intake and psychomotor activity. AVT-like immunoreactivity was distributed higher in the ventral region of the telencephalon, the hypothalamus and midbrain. Intraperitoneal (IP) administration of AVT at 100 pmol g-1 body weight (BW) increased the immunoreactivity of phosphorylated ribosomal proteinS6 (RPS6), a neuronal activation marker, in the telencephalon and diencephalon, decreased food consumption and enhanced thigmotaxis. AVT-induced anorexigenic and anxiogenic actions were blocked by IP co-injection of a V1a receptor (V1aR) antagonist, Manning compound (MC) at 300 pmol g-1 BW. These results suggest that AVT acts as an anorexigenic and anxiogenic factor via the V1aR-signaling pathway in the tiger puffer brain.

Japanese Planocerid Flatworms: Difference in Composition of Tetrodotoxin and Its Analogs and the Effects of Ingestion by Toxin-Bearing Fishes in the Ryukyu Islands, Japan.

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography-mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

DNA Methylation Analysis Reveals Potential Mechanism in Takifugu rubripes Against Cryptocaryon irritans Infection.

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection.

Copper exposure causes alteration in the intestinal microbiota and metabolites in Takifugu rubripes.

Copper is an environmental pollutant, and copper in aquatic environments mainly comes from soil and water. It enters the environment through atmospheric deposition, sewage discharge, and industrial production, and enters aquatic organisms, causing toxicity. Takifugu rubripes (T. rubripes) is a marine fish with high economic value. Due to the toxic effects of heavy metals on aquatic organisms such as fish, it can affect the gut community and metabolites of fish. The gut is an important channel for fish to communicate with the outside world and a necessary pathway for the metabolism of nutrients and toxic substances in the fish body. Studies have shown that due to changes in global water emissions and the high sensitivity of aquatic organisms to the environment, copper may pose greater potential hazards to aquatic organisms. Copper poses a greater risk to aquatic species than other heavy metals and metal/metal like pollutants (such as cadmium, lead, mercury, arsenic, etc.) . In order to elucidate the effects of copper exposure on the gut of T. rubripes. In this study, we exposed T. rubripes to 0, 50, 100, or 500 µg/L of copper for three days, the effects of copper exposure on the gut microbiota structure and metabolites of the T. rubripes were investigated using 16 S rRNA gene and metabolomics techniques. The research results indicate that with the increase copper concentration, the intestinal tissue of T. rubripes undergoes significant damage. 16 S rRNA sequencing results show that copper exposure alters the structure and metabolites of intestinal microbiota. Copper exposure of 100 and 500 µg/L inhibited the colonization of the bacterial gut, disrupted the intestinal barrier, and made the fish susceptible to the pathogens. Liquid chromatography-mass spectrometry analysis showed that copper regulated the production of metabolites such as L-histidine, arachidonic acid, and L-glutamic acid, which are related to energy and immunity. Microbiome-metabolome correlation analysis showed that Subdoligranulum, Family_XIII_AD3011_group, and Clostridium_sensu_stricto_1 were the key bacteria for copper ion intervention, and they might up-regulate the levels of metabolites such as indole-3-acetic acid, 3-indoleacrylic acid, and 5-hydroxyindole in the tryptophan metabolism pathway. In summary, our research has demonstrated that copper exposure can cause pathological changes in the intestinal tissue of the T. rubripes. High concentrations of copper ions can affect the colonization of the T. rubripes microbiota in the intestine, damage the fish's immune system, and alter the structure and metabolites of the intestinal microbiota, this can lead to intestinal metabolic dysfunction. providing a reference for the evaluation of the biological toxicity effects of heavy metal elements in the marine environment. This study provides a reference for evaluating the biological toxicity effects of heavy metal elements in marine environments.

Antioxidant response to ZnO nanoparticles in juvenile Takifugu obscurus: protective effects of salinity.

The extensive utilization of Zinc Oxide nanoparticles (ZnO NPs) has garnered significant attention due to their detrimental impacts on ecosystem. Unfortunately, ecotoxicity of ZnO NPs in coastal waters with fluctuating salinity has been disregarded. This study mainly discussed the toxic effects of ZnO NPs on species inhabiting the transition zones between freshwater and brackish water, who are of great ecological and economic importance among fish. To serve as the model organism, Takifugu obscurus, a juvenile euryhaline fish, was exposed to different ZnO NPs concentrations (0-200 mg/L) and salinity levels (0 and 15 ppt). The results showed that a moderate increase in salinity (15 ppt) could alleviate the toxic effect of ZnO NPs, as evidenced by improved survival rates. The integrated biomarker response index on oxidative stress also revealed that the toxicity of ZnO NPs was higher in freshwater compared to brackish water. These outcomes can be attributed to higher salinity (15 ppt) reducing the bioavailability of ZnO NPs by facilitating their aggregation and inhibiting the release of metal ions. It is noteworthy that elevated salinity was found to alleviate ZnO NPs toxicity by means of osmotic adjustment via the activation of Na+/K+-ATPase activity. This study demonstrates the salinity-dependent effect of ZnO NPs on T. obscurus, suggesting the possibility for euryhaline fish like T. obscurus to adapt their habitat towards more saline environments, under constant exposure to ZnO NPs.

Isolation and Purification of Protamine from the Cultured Takifugu flavidus and Its Physicochemical Properties.

Protamine is a cationic peptide derived from fish sperm and has several important functional properties: antibacterial properties, acting as a carrier for injectable insulin and as a heparin antagonist, combatting fatigue, etc. Thus, it has been widely used in medicinal applications and food products. Cultured Takifugu flavidus is a type of pufferfish with a delicious taste that is popular in China, and its production is increasing significantly. Therefore, protamine was extracted via acid extraction from the sperm of Takifugu flavidus and further isolated and purified via sephadex gel chromatography, ion exchange chromatography, and desalination chromatography. Furthermore, the physicochemical properties of protamine were investigated. The results showed that the sperm of the cultured T. flavidus were non-toxic, and the extracted and purified protamine had high contents of arginine (36.90%) and lysine (27.02%), respectively. The secondary structure of protamine was mainly ß-folded and irregularly curled. Additionally, protamine exhibited high thermal stability with a denaturation temperature of 176 °C. This study would provide a theoretical basis for the structural analysis, bioactivity, and resource development of pufferfish protamine and help to promote the development of the pufferfish industry.

Exposure of farmed fish to petroleum hydrocarbon pollution and the recovery process: A simulation experiment with tiger puffer Takifugu rubripes.

Petroleum hydrocarbon (PH) pollution threatens both wild and farmed marine fish. How this pollution affects the nutrient metabolism in fish and whether this effect can be recovered have not been well-known. The present study aimed to evaluate these effects with a feeding trial on tiger puffer, an important farmed species in Asia. In a 6-week feeding trial conducted in indoor flow-through water, fish were fed a control diet (C) or diets supplemented with diesel oil (0.02 % and 0.2 % of dry matter, named LD and HD, respectively). Following this feeding trial was a 4-week recovery period, during which all fish were fed a same normal commercial feed. At the end of the 6-week feeding trial, dietary PH significantly decreased the fish growth and lipid content. The PH significantly accumulated in fish tissues, in particular the liver, and caused damages in all tissues examined in terms of histology, anti-oxidation status, and serum biochemical changes. Dietary PH also changed the volatile flavor compound profile in the muscle. The hepatic transcriptome assay showed that the HD diet tended to inhibit the DNA replication, cell cycle and lipid synthesis, but to stimulate the transcription of genes related to liver protection/repair and lipid catabolism. The 4-week recovery period to some extent mitigated the damage caused by PH. After the recovery period, the inter-group differences in some parameters disappeared. However, the differences in lipid content, anti-oxidase activity, liver PH concentration, and histological structure still existed. In addition, differences in cellular chemical homeostasis and cytokine-cytokine receptor interaction at the transcriptional level can still be observed, indicated by the hepatic transcriptome assay. In conclusion, 6 weeks of dietary PH exposure significantly impaired the growth performance and health status of farmed tiger puffer, and a short-term recovery period (4 weeks) was not sufficient to completely mitigate this impairment.

Identification and characterization of an L-type lectin from obscure puffer Takifugu obscurus in response to bacterial infection.

L-type lectins (LTLs) contain a carbohydrate recognition domain homologous to leguminous lectins, and have functions in selective protein trafficking, sorting and targeting in the secretory pathway of animals. In this study, a novel LTL, designated as ToERGIC-53, was cloned and identified from obscure puffer Takifugu obscurus. The open reading frame of ToERGIC-53 contained 1554 nucleotides encoding 517 amino acid residues. The deduced ToERGIC-53 protein consisted of a signal peptide, a leguminous lectin domain (LTLD), a coiled-coil region, and a transmembrane region. Quantitative real-time PCR showed that ToERGIC-53 was expressed in all examined tissues, with the highest expression level in the liver. The expression of ToERGIC-53 was significantly upregulated after infection with Vibrio harveyi and Staphylococcus aureus. Recombinant ToERGIC-53-LTLD (rToERGIC-53-LTLD) protein could not only agglutinate and bind to one Gram-positive bacterium (S. aureus) and three Gram-negative bacteria (V. harveyi, V. parahaemolyticus and Aeromonas hydrophila), but also bind to glycoconjugates on the surface of bacteria such as lipopolysaccharide, peptidoglycan, mannose and galactose. In addition, rToERGIC-53-LTLD inhibited the growth of bacteria in vitro. All these results suggested that ToERGIC-53 might be a pattern recognition receptor involved in antibacterial immune response of T. obscurus.

Identification of lncRNA-based regulatory mechanisms of Takifugu rubripes growth traits in fast and slow-growing family lines.

Family selection is an important method in fish aquaculture because growth is the most important economic trait. Fast-and slow-growing families of tiger puffer fish (Takifugu rubripes) have been established through family selection. The development of teleost fish is primarily controlled by the growth hormone (GH)-insulin-like growth factor 1 (IGF-1) axis that includes the hypothalamus-pituitary-liver. In this study, the molecular mechanisms underlying T. rubripes growth were analyzed by comparing transcriptomes from fast- and slow-growing families. The expressions of 214 lncRNAs were upregulated, and those of 226 were downregulated in the brain tissues of the fast-growing T. rubripes family compared to those of the slow-growing family. Differentially expressed lncRNAs centrally regulate mitogen-activated protein kinase (MAPK) and forkhead box O (FoxO) signaling pathways. Based on the results of lncRNA-gene network construction, we found that lncRNA3133.13, lncRNA23169.1, lncRNA23145.1, and lncRNA23141.3 regulated all four genes (igf1, mdm2, flt3, and cwf19l1). In addition, lncRNA7184.10 may be a negative regulator of rasgrp2 and a positive regulator of gadd45ga, foxo3b, and dusp5. These target genes are associated with the growth and development of organisms through the PI3K/AKT and MAPK/ERK pathways. Overall, transcriptomic analyses of fast- and slow-growing families of T. rubripes provided insights into the molecular mechanisms of teleost fish growth rates. Further, these analyses provide evidence for key genes related to growth regulation and the lncRNA expression regulatory network that will provide a framework for improving puffer fish germplasm resources.

Role of TRAF6 from obscure puffer (Takifugu obscurus) in immune response against Edwardsiella tarda infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a vital molecule of inflammatory signaling pathways in innate immune response against pathogens. To elucidate its role in defense against Edwardsiella tarda infection in teleost fish, TRAF6 homologue was identified from obscure puffer (Takifugu obscurus) and functionally analyzed in this study. The obscure puffer TRAF6 (ToTRAF6) is a protein of 565 amino acids containing conserved RING domain, zinc finger-TRAF and MATH_TRAF6 domain. ToTRAF6 mRNA distributed in various healthy tissues of obscure puffer and was upregulated in the immune related tissues after E. tarda infection. ToTRAF6 protein was localized in the cytoplasm and aggregate as dots around the nuclei in FHM cells. The overexpression of ToTRAF6 in FHM cells decreased the quantity of E. tarda and induced the significant upregulation of downstream MAPK signaling pathway genes. These data suggest that ToTRAF6 is a key molecule of MAPK signaling pathway in defense against E. tarda infection.

5, 6, 11-trideoxy tetrodotoxin attracts tiger puffer Takifugu rubripes.

Tetrodotoxin (TTX) is a potent neurotoxin that binds to voltage-gated sodium channels and blocks the passage of sodium ions. TTX is widely distributed in both terrestrial and marine organisms, and the toxic puffers are believed to accumulate TTX through the food chain. Although pufferfish was previously thought to be attracted by TTX, recent finding from electroolfactogram (EOG) studies have indicated that the olfactory epithelium of T. alboplumbeus responded to 5, 6, 11-trideoxyTTX (TDT), but not to TTX itself. In this study, we examined behavioral experiments for Takifugu rubripes to distinguish between TTX and TDT under static and flow-through conditions. Our data clearly suggested that T. rubripes juveniles were attracted to TDT, not TTX. Moreover, we determined that the minimum effective dose of TDT to attract the puffer was 1-2 nmol of TDT under static conditions and 50-60 nmol of TDT under flow-through conditions. Following the experiments under static conditions, numerous bite marks by the pufferfish were found solely on the agarose gel infused with TDT. Based on these finding, we hypothesize that the pufferfish are attracted to TDT derived from prey, leading them effectively become toxic.

Comparison of specificity and sensitivity of diagnostic methods for Enteromyxum leei and Enteromyxum fugu detected from cultured tiger puffer, Takifugu rubripes in Korea.

Enteromyxum leei and Enteromyxum fugu, which are myxosporean parasites, were first found in cultured tiger puffer Takifugu rubripes in Korea. We collected four tiger puffers that showed severe emaciation signs for our experiments. DNA sequencing was confirmed that the tiger puffers were coinfected with E. leei and E. fugu. Furthermore, similar amounts of E. leei and E. fugu were confirmed using real-time PCR in the intestine. To the best of our knowledge, there have been no reports of E. fugu infection in the olive flounder Paralichthys olivaceus. However, the diagnosis of inflowing water, discharged water and olive flounder samples using highly sensitive diagnostic methods confirmed the presence of E. fugu in water and fish samples from olive flounder farms near the tiger puffer farm. Therefore, the present study aimed to develop highly sensitive diagnostic methods such as real-time and two-step PCR for early diagnosis and follow-up of the emaciation disease and multiplex PCR for rapid diagnosis. The multiplex PCR method exhibited the same sensitivity as the one-step PCR method developed in this study, demonstrating its efficacy for rapid diagnosis. Therefore, the suggested methods can be utilized for the early diagnosis and rapid diagnosis of emaciation diseases and reduction of economic losses through rapid disease control.

Preference of juvenile tiger puffer for light spectrum and tank colours based on different body size and breeding background.

As important environmental factors, the light spectra and tank colours have not received sufficient attention. Most fishes have the ability to perceive environment, distinguish colours, and exhibit preferences or aversions towards different environments, which can provide a reference for the design of their rearing environment. Tiger puffer (Takifugu rubripes) is an important mariculture species in China and East Asia, but its preference for illumination spectra and tank colours is unclear. This study focuses on the preferences of juvenile tiger puffers for different spectra and tank background colours in different rearing backgrounds and body sizes. The experiments were conducted in a preference testing device, and the behavioural videos were recorded and analysed using a motion behaviour tracking system (EthoVision XT 12). The results show that the puffers showed preference for short-wavelength lights ((i.e., cyan, green, etc.), avoidance of long-wavelength light (i.e., red) and less stay time in the full light spectrum and dark. For tank colours, the puffers showed a preference for light background colours (i.e., white), and avoidance of deep background colours (i.e., dark, red, etc.). Fish body sizes and original breeding environment could significantly affect the selective preference of juvenile puffer (P < 0.05). Large puffers preferred green tank colour than small ones, while small ones preferred grey and red. The puffers reared in green light and grey tank for 3 months preferred green light spectrum and green tank colour compared with those reared in full spectrum and grey tank, while the fish reared in full spectrum preferred grey tank colour and area without light. It was also found that the movement rate of juvenile puffers was affected by the light spectra and tank colours and was positively correlated with light wavelength (P < 0.05). Therefore, for tiger puffer breeding, short-wavelength light spectrums (cyan, green, etc.) and light-coloured tank backgrounds (white) are recommended. Long-wavelength Light-emitting diodes and dark tank colours should be avoided in breeding. This study would provide a reference basis for fish light spectra and background colour preference studies, as well as for the improvement of breeding welfare and production efficiency of juvenile tiger puffer.

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.

Construction of a high-density genetic linkage map and QTL mapping of growth and cold tolerance traits in Takifugu fasciatus.

Takifugu fasciatus is an aquaculture species with high economic value. In recent years, problems such as environmental pollution and inbreeding have caused a serious decline in T. fasciatus germplasm resources. In this study, a high-density genetic linkage map was constructed by whole-genome resequencing. The map consists of 4891 bin markers distributed across 22 linkage groups (LGs), with a total genetic coverage of 2381.353 cM and a mean density of 0.535 cM. Quantitative trait locus (QTL) localization analysis showed that a total of 19 QTLs associated with growth traits of T. fasciatus in the genome-wide significance threshold range, distributed on 11 LGs. In addition, 11 QTLs associated with cold tolerance traits were identified, each scattered on a different LG. Furthermore, we used QTL localization analysis to screen out three candidate genes (IGF1, IGF2, ADGRB) related to growth in T. fasciatus. Meanwhile, we screened three candidate genes (HSP90, HSP70, and HMGB1) related to T. fasciatus cold tolerance. Our study can provide a theoretical basis for the selection and breeding of cold-tolerant or fast-growing T. fasciatus.

l-fucoside localization in the gills of the genus Takifugu and its possible implication in the parasitism of Heterobothrium okamotoi (Monogenea: Diclidophoridae).

BACKGROUND: The monogenean parasite Heterobothrium okamotoi only parasitizes the gills of Takifugu rubripes. In this study, we hypothesized that the carbohydrates contribute to high host specificity of H. okamotoi. METHODS: T. rubripes, T. niphobles, T. snyderi, and T. pardalis were used for UEA I staining of the gills and an in vivo challenge test against H. okamotoi. To examine the effect of l-fucose, an in vitro detachment test was conducted using the host's gills. Additionally, fucosylated proteins were isolated from the membrane proteins of T. niphobles gills. RESULTS: The location of l-fucoside and the infection dynamics in four species were correlated to some extent; H. okamotoi detached relatively quickly from T. niphobles possessing l-fucoside both on the surface of the gills and in certain types of cells, including mucus cells, but detached slowly from T. snyderi possessing l-fucoside in only certain types of cells, including mucus cells. Under the conditions examined, H. okamotoi exhibited minimal detachment from T. rubripes and T. pardalis, and l-fucoside was not detected. The significantly higher detachment rate of H. okamotoi from the host's gills incubated in l-fucose-containing medium compared with the controls suggests that l-fucose in the non-host gills induced detachment of H. okamotoi. Four fucosylated proteins, including mucin5AC-like, were identified as potential factors for the detachment of H. okamotoi. CONCLUSIONS: Fucosylated proteins covering the surface of non-host gills might contribute to H. okamotoi detachment. GENERAL SIGNIFICANCE: This research shows the possible involvement of oligosaccharides in the host specificity of monogenean parasites.

Three novel L-type lectins from obscure puffer Takifugu obscurus promote antimicrobial immune response.

L-type lectins (LTLs) have leguminous lectin domains that bind to high-mannose-type oligosaccharides. LTLs are involved in glycoprotein secretory pathways and associated with many immune responses. In the present research, three LTL homologs from obscure puffer Takifugu obscurus, designated as ToVIP36-1, ToVIP36-2, and ToVIP36-3, were first cloned and identified. The open reading frames of ToVIP36-1, ToVIP36-2, and ToVIP36-3 were 1068, 1002, and 1086 bp in length, respectively, and encode polypeptides with 355, 333, and 361 amino acids, respectively. Key conserved residues and functional domains, including lectin_leg-like domain (LTLD), transmembrane region, and C-terminal trafficking signal KRFY, were identified in all ToVIP36s. Quantitative real-time PCR analysis showed that the three ToVIP36s were widely expressed in six examined tissues and had relatively high expression levels in the liver and intestine. The expression levels of ToVIP36s were remarkably altered in the liver and kidney after induction by Vibrio harveyi and Staphylococcus aureus. Subsequently, the recombinant LTLDs of ToVIP36s (rToVIP36-LTLDs) were prepared by prokaryotic expression. Three rToVIP36-LTLD proteins agglutinated with S. aureus, V. harveyi, Vibrio parahaemolyticus, and Aeromonas hydrophila in a calcium-dependent manner. In the absence of calcium, rToVIP36-LTLD proteins bound to the bacteria by binding to lipopolysaccharides, peptidoglycans, d-mannose, and d-galactose and inhibited the growth of S. aureus and V. harveyi. Our results indicated that ToVIP36s function as pattern-recognition receptors in T. obscurus immunity, providing insights into the role of LTLs in the antibacterial immunity of fishes.