Mechanism of Takifugu bimaculatus Skin Peptides in Alleviating Hyperglycemia in Rats with Type 2 Diabetic Mellitus Based on Microbiome and Metabolome Analyses.

In this study, we aimed to explore the hypoglycemic effects of a hydrolysate on Takifugu bimaculatus skin (TBSH). The effect of the dipeptidyl peptidase-IV (DPP-IV) inhibitory activities from different TBSH fractions was investigated on basic indexes, gut hormones, blood lipid indexes, viscera, and the gut microbiota and its metabolites in rats with type 2 diabetes mellitus (T2DM). The results showed that the <1 kDa peptide fraction from TBSH (TBP) exhibited a more potent DPP-IV inhibitory effect (IC50 = 0.45 ± 0.01 mg/mL). T2DM rats were induced with streptozocin, followed by the administration of TBP. The 200 mg/kg TBP mitigated weight loss, lowered fasting blood glucose levels, and increased insulin secretion by 20.47%, 25.23%, and 34.55%, respectively, rectified irregular hormonal fluctuations, lipid metabolism, and tissue injuries, and effectively remedied gut microbiota imbalance. In conclusion, TBP exerts a hypoglycemic effect in rats with T2DM. This study offers the potential to develop nutritional supplements to treat T2DM and further promote the high-value utilization of processing byproducts from T. bimaculatus. It will provide information for developing nutritional supplements to treat T2DM and further promote the high-value utilization of processing byproducts from T. bimaculatus.

Establishment and characterization of a gill cell line from Takifugu obscurus and transcriptome analysis of its gene expression profiles upon low temperature.

Takifugu obscurus is a farmed fish of great economic importance in China. The rapid development of T. obscurus aquaculture industry has been accompanied by disease and low-temperature stress, resulting in huge economic losses. Cell lines are used extensively in teleost physiology and pathology as the most cost-effective platform for in vitro research. A novel gill cell line of T. obscurus (named TOG) was first successfully established, and passed through 52 generations. The optimal conditions for TOG growth were 20 % FBS concentration and 24 °C, TOG could be grown in both hypotonic (150 mOsmol-kg-1) and hypertonic (600 mOsmol-kg-1) environments. TOG was determined to be derived from T. obscurus by sequencing the mitochondrial COI gene. Karyotype analysis revealed that the chromosome number of TOG was 44 (2n = 44). Transfection experiment showed that TOG was able to express foreign genes. Furthermore, several immune-related genes were significantly up-regulated in TOG after LPS and poly (I:C) stimulation, including tlr3, isg15, il1ß and il10. Additionally, transcriptome analysis of TOG under low-temperature stress (24 °C, 18 °C, 12 °C, 10 °C and 8 °C) found that differentially expressed genes (DEGs) were significantly clustered in several immunological and energy metabolic pathways, and cold stress could disrupt the immune barrier and reduce immunity by downregulating the immune-related pathways. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that bule module and turquoise module, which were closely correlated with low temperature and the degree of fish damage, were both predominantly found in PPAR, NOD-like receptor and Toll-like receptor signaling pathway. Hub genes were identified in these two modules, including mre11, clpb, dhx15, ddx18 and utp15. TOG cell line will become an effective experimental platform for genetic and immunological research, and our results would help us gain a deeper insight into the molecular mechanism of cold tolerance in teleost.

The potential regulatory role of the non-coding RNAs in regulating the exogenous estrogen-induced feminization in Takifugu rubripes gonad.

Estrogen plays a pivotal role in the early stage of sex differentiation in teleost. However, the underlying mechanisms of estrogen-induced feminization process are still needed for further clarification. Here, the comparative analysis of whole-transcriptome RNA sequencing was conducted between 17beta-Estradiol induced feminized XY (E-XY) gonads and control gonads (C) in Takifugu rubripes. A total of 57 miRNAs, 65 lncRNAs, and 4 circRNAs were found to be expressed at lower levels in control-XY (C-XY) than that in control-XX (C-XX), and were up-regulated in XY during E2-induced feminization process. The expression levels of 24 miRNAs, and 55 lncRNAs were higher in C-XY than that in C-XX, and were down-regulated in E2-treated XY. Furthermore, a correlation analysis was performed between miRNA-seq and mRNA-seq data. In C-XX/C-XY, 114 differential expression (DE) miRNAs were predicted to target to 904 differential expression genes (DEGs), while in C-XY/E-XY, 226 DEmiRNAs were predicted to target to 2,048 DEGs. In C-XX/C-XY, and C-XY/E-XY, KEGG pathway enrichment analysis showed that those targeted genes were mainly enriched in MAPK signaling, calcium signaling, steroid hormone biosynthesis and ovarian steroidogenesis pathway. Additionally, the competitive endogenous RNA (ceRNA) regulatory network was constructed by 24 miRNAs, 21 lncRNAs, 4 circRNAs and 5 key sex-related genes. These findings suggested that the expression of critical genes in sex differentiation were altered in E2-treated XY T. rubripes may via the lncRNA-miRNA-mRNA regulation network to facilitate the differentiation and maintenance of ovaries. Our results provide a new insight into the comprehensive understanding of the effects of estrogen signaling pathways on sex differentiation in teleost gonads.

The impact of tetrodotoxin (TTX) on the gut microbiome in juvenile tiger pufferfish, Takifugu rubripes.

Tetrodotoxin (TTX) is a potent neurotoxin that accumulates in Takifugu rubripes, commonly known as pufferfish, through the ingestion of TTX-bearing organisms as part of their food chain. Although researchers believe that pufferfish use TTX to relieve stress, data are not currently available on how TTX affects the gut microbiota of pufferfish. To address this gap, our study aimed to investigate whether administering TTX to fish could alter their gut microbiota and overall health under various salinity conditions, including 30.0 ppt, 8.5 ppt, and 1.7 ppt salinity, which represent full-strength, isosmotic, and low-salinity stress, respectively. We analyzed the effect of TTX ingestion on the community structure, core microbiome, and metabolic capabilities of the gut microbiome using high-throughput sequencing technologies. The predominant bacterial taxa within the gut microbiome were Firmicutes (21-85%), Campilobacterota (2.8-67%), Spirochaetota (0.5-14%), and Proteobacteria (0.7-9.8%), with Mycoplasma, uncultured Arcobacteraceae, Brevinema, Vibrio, Rubritalea, and uncultured Pirellulaceae as core genera. Our findings indicated that the impact of TTX on high-abundance genera at 30.0 ppt and 8.5 ppt salinity levels was negligible, indicating their stability and resilience to TTX ingestion. However, at 1.7 ppt, TTX-fed fish showed a significant increase in uncultured Arcobacteraceae. Furthermore, our analysis of TTX-fed fish revealed taxonomic alterations in low-abundance taxa, which altered the predicted functions of the gut microbiota at all salinity levels. These results suggest that TTX administration could cause subtle effects on the metabolic functions of gut microbial communities. Overall, our study provides insights into the complex relationship between a TTX-accumulating animal, T. rubripes, and its gut microbiota.

Investigation of copper-induced intestinal damage and proteome alterations in Takifugu rubripes: Potential health risks and environmental toxicology detection.

Copper is one of the predominant water pollutants. Excessive exposure to copper can cause harm to animal health, affecting the central nervous system and causing blood abnormalities. Cuproptosis is a novel form of cell death that differs from previous programmed cell death methods. However, the impact of copper on the intestines remains unclear. Therefore, we investigated the effects of different concentrations of copper exposure on the intestinal proteome of Takifugu rubripes (T. rubripes). Relevant biomarkers were used to detect cuproptosis. We revealed the crosstalk relationship between cuproptosis and self-rescue at different concentrations, and discussed the feasibility of using potential cuproptosis indicators as anti-infection factors. We observed intestinal damage in the three copper exposure groups, especially in T. rubripes treated with 100 and 500 µg/L copper, with shedding and breakage of intestinal villus and fuzzy and loose structure of intestinal mucosa. The presence of copper stress not only causes cuproptosis but also oxidative damage caused by reactive oxygen species (ROS). The results of quantitative proteomics by TMT showed that compared to the 50 and 100 µg/L copper exposure groups, the expression of glutaminase, pyruvate kinase, and skin mucus lectin in the 500 µg/L group was significantly increased. The positive mediators COX5A and CTNNB1, as well as the negative mediators CD4 and FDXR, were found to be differentially expressed. Using the protein expression trends of cuproptosis indicator factors FDX1 and DLAT to indicate the concentration of copper ions in the environment. In addition, we found a new effect of promoting ferroptosis: providing additional copper ions can activate the phenomenon of ferroptosis. Our results expand our understanding of the potential health risks of copper in T. rubripes. At the same time, it is of great significance for the process of copper poisoning and the development of new environmental toxicology detection reagents.

Teleost-specific TLR23 in Takifugu rubripes recruits MyD88 to trigger ERK pathway and promotes antibacterial defense.

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes, found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes, bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost.

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.

Distribution of pufferfish saxitoxin- and tetrodotoxin-binding protein homolog and tetrodotoxin in the brain and pituitary of juvenile tiger puffer Takifugu rubripes.

Pufferfish saxitoxin- and tetrodotoxin (TTX)-binding protein (PSTBP) is considered to transfer TTX between tissues. The immunohistochemical distribution of PSTBP-homolog (PSTBPh) and TTX in the brain and pituitary of hatchery-reared juvenile tiger puffer Takifugu rubripes was investigated. PSTBPh was observed mainly in the pars intermedia of the pituitary. TTX was only detected in a TTX-fed fish in the neurohypophysis of the pituitary and in several other brain regions. The relationship between PSTBPh and TTX is discussed.

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 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.

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.

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.