Takifugu Obscurus Research Articles

Identification and functional characterization of three irf7 transcript variants in obscure puffer (Takifugu obscurus)

Interferon regulatory factor 7 (IRF7) is a key mediator in regulating the type Ι IFN response. Although irf7 has been identified in more than twenty fish species, alternative splicing has not been found in teleost irf7. Alternative splicing is an important mechanism expanding the transcriptomic and proteomic diversity, and has been found in several IRF family members. Here, three alternative splicing variants of irf7 were identified and characterized in obscure puffer. The first splicing transcript (Toirf7v1) was predicted to encode 428 amino acids with a DNA-binding domain (DBD), an interaction-associated domain (IAD) and a serine-rich domain (SRD). Toirf7v2 encoded 430 amino acids caused by the intron retention, and contained the whole conserved domains. Toirf7v3 encoded a truncated protein with 337 amino acids resulting from the alternative 5′ splice-site selection, and lacked part of IAD domain and the entire SRD domain. Functional studies demonstrated that all of the three isoforms could activate the expression of type I IFN and IFN-stimulated genes (ISGs). Nevertheless, the two variants (Toirf7v2 and Toirf7v3) exhibited much less ability to induce transcription of IFN and ISGs compared to the Toirf7v1. Our findings suggest that these splicing variants may have distinct roles in the regulation of immune response. These results will be beneficial to understand the functional characteristics of irf7 variants in fish.

Transcriptome changes of Takifugu obscurus liver after acute exposure to phenanthrene.

Phenanthrene (Phe) is a model compound in polycyclic aromatic hydrocarbon (PAH) research. Reportedly, Phe treatment induced oxidative stress and histological disorders to Takifugu obscurus liver. In this study, to further explore the molecular responses of T. obscurus liver to Phe exposure, transcriptome sequencing was applied to compare mRNA transcription profiles between Phe treatment and the control. Compared with the control, 1,581 and 1,428 genes were significantly upregulated and downregulated in Phe treatment, respectively. Further analysis revealed that Phe treatment mainly upregulated genes in Ras-MAPK and PI3K-akt signaling pathways, which represented insulin resistance and further activated the FOXO signaling pathway. The triacylglycerol biosynthesis was promoted but the gluconeogenesis process was inhibited in response to Phe treatment, demonstrating that Phe exposure disturbed the sugar and lipid metabolism. Moreover, Phe treatment upregulated the Apelin-APJ and ErbB signaling pathways, promoting angiogenesis in T. obscurus liver. Insulin resistance, promoted triacylglycerol biosynthesis, and angiogenesis might explain the molecular mechanisms underlying carcinogenic toxicity of Phe. Overall, this study provides new insights to understand the environmental risk of Phe to fishes.

Effects of slightly acidic electrolyzed water pretreatment combined with biopreservatives on the shelf life of refrigerated obscure pufferfish (Takifugu obscurus).

This study assessed the effects of ε-polylysine hydrochloride (ε-PLH) and rosemary extract (RE) combined with slightly acidic electrolyzed water (SAEW) on the shelf life of obscure pufferfish (Takifugu obscurus) during refrigerated storage at (4± 1 °C). The samples were first dipped in SAEW and then treated with sterilized water (CK) and other solutions (0.2% RE [RE], 0.3% ε-PLH [PLH], and 0.2% RE + 0.3% ε-PLH [RE+PLH], respectively). Afterwards, microbiological, physicochemical, water migration, and sensory attributes were periodically assessed. The results showed that compared with CK and RE groups, ε-PLH treatment could effectively inhibit microbial growth and delay the increase in total volatile base nitrogen (TVB-N). In addition, microbial indexes, TVB-N, thiobarbituric acid, and metmyoglobin value in the samples treated with RE+ε-PLH were lower than those with ε-PLH or RE alone during storage. Meanwhile, samples treated with RE+ε-PLH exhibited the best sensory properties and no effect on color parameters. Based on the results of microbiological analysis, the shelf life of obscure pufferfish treated with RE+ε-PLH was 14 days during refrigerated storage at (4± 1 °C). PRACTICAL APPLICATION: In our study, the combined application of ε-PLH, RE, and SAEW was effective to extend the shelf life of obscure pufferfish. Based on the results of total viable counts, the RE+PLH group had a shelf life of approximately 14 days. Accordingly, bio-preservatives combined with SAEW technology have a broad application potential in the storage of obscure pufferfish.

The regulation of fenofibrate on lipid metabolism in obscure puffer ( Takifugu obscurus ) is dependent on dietary lipid content

The regulation of fenofibrate on lipid metabolism in obscure puffer ( <i>Takifugu obscurus</i> ) is dependent on dietary lipid content

Response of gut microbiota to feed-borne bacteria depends on fish growth rate: a snapshot survey of farmed juvenile Takifugu obscurus.

Environmental bacteria have a great impact on fish gut microbiota, yet little is known as to where fish acquire their gut symbionts, and how gut microbiota response to the disturbance from environmental bacteria. Through the integrative analysis by community profiling and source tracking, we show that feed-associated bacteria can impose a strong disturbance upon the hindgut microbiota of cultured fugu. Consequently, marked alterations in the composition and function of gut microbiota in slow growth fugu were observed, implying a reduced stability upon bacterial disturbance from feed. Moreover, quantitative ecological analyses indicated that homogeneous selection and dispersal limitation largely contribute to the community stability and partial variations among hosts in the context of lower degree of disturbance. While the disturbance peaked, variable selection leads to an augmented interaction within gut microbiota, entailing community unstability and shift. Our findings emphasized the intricate linkage between feed and gut microbiota and highlighted the importance of resolving the feed source signal before the conclusion of comparative analysis of microbiota can be drawn. Our results provide a deeper insight into aquaculture of fugu and other economically important fishes and have further implications for an improved understanding of host-microbe interactions in the vertebrate gastrointestinal tract.

Effects of Incubation Method and Transfer Timing on the Hatching Rate of Fertilized Eggs of the River Puffer Takifugu obscurus

Effects of Incubation Method and Transfer Timing on the Hatching Rate of Fertilized Eggs of the River Puffer Takifugu obscurus

Genetic Differences in Natural and Cultured River Pufferfish Populations by PCR Analysis.

Genomic DNA (gDNA) extracted from two populations of natural and cultured river pufferfish (Takifugu obscurus) was amplified by polymerase chain reaction (PCR). The complexity of the fragments derived from the two locations varied dramatically. The genetic distances (GDs) between individuals numbered 15 and 12 in the cultured population was 0.053, which was the lowest acknowledged. The oligonucleotide primer OPC-11 identified 88 unique loci shared within each population reflecting the natural population. The OPC-05 primer identified 44 loci shared by the two populations. The average band-sharing (BS) values of individuals in the natural population (0.683±0.014) were lower than in those derived from the cultured population (0.759±0.009) (p<0.05). The shortest GD demonstrating a significant molecular difference was found between the cultured individuals # 15 and # 12 (GD=0.053). Individual # 02 of the natural population was most distantly related to cultured individual # 22 (GD=0.827). A cluster tree was built using the unweighted pair group method with arithmetic mean (UPGMA) Euclidean GD analysis based on a total of 578 various fragments derived from five primers in the two populations. Obvious markers identified in this study represent the genetic structure, species security, and proliferation of river pufferfish in the rivers of the Korean peninsula.

Chitosan-sodium alginate bioactive coatings containing ε-polylysine combined with high CO2 modified atmosphere packaging inhibit myofibril oxidation and degradation of farmed pufferfish (Takifugu obscurus) during cold storage

In this study, we explored the effectiveness of chitosan (CH) and sodium alginate (SA) bioactive coatings containing increasing concentrations of ε-polylysine (ε-PL) (0.1%, 0.2%, and 0.3%) combined with modified atmosphere packaging (MAP) in 60%CO2/5%O2/35%N2 to inhibit myofibril oxidation and degradation of farmed pufferfish during a cold storage period of 18 days. Our research revealed that ε-PL-enriched CH-SA coatings and high CO2 MAP noticeably delayed myofibril oxidation by inhibiting the formation of carbonyl groups and by keeping higher sulfhydryl content. We also observed that these treatments preserved myofibril Ca2+-ATPase activity, protected their organized secondary structure by retaining higher contents of α-helix and β-sheet, and also stabilised tertiary structure during cold storage. The transmission electron microscope (TEM) images showed that the distortion degree of Z-disk and M-line had become deeper and the gap between sarcomeres had expanded gradually when a comparison was carried out between fresh fish and fish at the end of the cold storage period of 18 days. However, the treated samples were still regular in appearance with distinct light I-bands, dark A-bands and Z-disk. Overall, we report that coatings containing ε-PL and high CO2 MAP treatments appear to protect the myofibrils of farmed pufferfish during cold storage.

Molecular cloning, characterization and expression analysis of major histocompatibility complex class I alpha gene of pufferfish (Takifugu obscurus).

The major histocompatibility complex (MHC) genes play a key role in immune response in vertebrates. In this study, an MHC I alpha homolog gene (PfMHC Ⅰα) from pufferfish (Takifugu obscurus) was identified and its subcellular localization and expression patterns of PfMHC Ⅰα after challenge in vivo and in vitro were analysed. The open reading frame of PfMHC Ⅰα was 1,089bp in length, encoding 362 aa. The immunofluorescence result revealed that PfMHC Ⅰα was presented on the membrane of lymphocytes. qRT-PCR analysis indicated that PfMHC Ⅰα was expressed in all examined tissues, with the highest expression in skin, followed by the expression in gills and whole blood. After challenge of Aeromonas hydrophila or polyinosinic: polycytidylic acid (Poly I:C) in vitro, the expression levels of PfMHC Ⅰα on pufferfish kidney lymphocytes were significantly up-regulated, with the highest expression level at 48hr post-challenge. After infection with A. hydrophila or Poly I:C in vivo, the expression levels of PfMHC Ⅰα in the skin, whole blood and kidneys were significantly up-regulated. Taken together, it is speculated that PfMHC Ⅰα associates with resistance to both intracellular and extracellular antigens and plays an important role in the host response against pathogen infection in pufferfish.

The factors influencing the flavor characteristics of frozen obscure pufferfish (Takifugu Obscurus) during storage: Ice crystals, endogenous proteolysis and oxidation

The effects of ice crystals, endogenous proteolysis and oxidization on flavor-related compounds of obscure pufferfish fillets during frozen storage were investigated in terms of biogenic amines, trimethylamine (TMA), free amino acids (FAA), nucleotides and electronic nose. These three factors were separately controlled by pretreatments of liquid nitrogen, iodoacetic acid and natural antioxidants, respectively. In general, the smaller ice crystals in fillets reduced accumulation of off-flavor compounds (such as hypoxanthine, TMA, lysine and putrescine), and increased umami-associated FAA and inosine monophosphate to a greater extend during storage when compared to other groups. According to the analysis of electronic nose and principal component analysis (PCA), the control pretreatments of endogenous proteolysis and oxidation were both beneficial to maintain the flavor quality of fillets, while the best preservation effect was observed in fillets frozen by liquid nitrogen, suggesting ice crystals played a more critical role causing the flavor loss of pufferfish during long-term frozen storage.

Effect of different extent of protein oxidation on the frozen storage stability of muscle protein in obscure pufferfish (Takifugu obscurus)

The objective of this study was to explore frozen storage stability of muscle protein of the obscure pufferfish using a semi-model system based on light exposure (LE), oxygen concentration (OC), and multiple freeze-thaw cycles (FTCs). The dorsal muscle of fifteen frozen fish was cut into small pieces (2 cm × 1.5 cm × 1 cm) and divided into 4 groups stored in differing conditions: one was stored in light with 0% oxygen (L-0), one in light with 20% oxygen (L-20), one in dark with 0% oxygen (D-0), and one in dark with 20% oxygen (D-20). All were subsequently subjected to five FTCs. Protein oxidation (PO) levels, protein structure, and water states were all assessed. The results showed that both LE and OC could significantly accelerate sulfhydryl and tryptophan loss, in the order of L-20 > L-0 and D-20 > D-0 during FTCs. Changes of protein structure were in line with the extent of PO indicated by surface hydrophobicity and UV absorbance. An increased PO also affected the water states, with higher PO resulting in immobilized water being more tightly trapped within the protein. However, thawing loss was shown to be independent of PO and the structural changes of myofibrillar protein.

Identification and functional characterization of three caspases in Takifugu obscurus in response to bacterial infection

Caspases are evolutionarily conserved proteases, which are inextricably linked with the apoptosis and immune system in mammals. However, the expression pattern and function of some caspases remain largely unknown in pufferfish. In this study, three different pufferfish caspases (caspase-2 (Pfcasp-2), caspase-3 (Pfcasp-3), and caspase-8 (Pfcasp-8)) were characterized, and their expression patterns and functions were determined following Aeromonas hydrophila infection. The open reading frames of Pfcasp-2, -3, and -8 are 1,320, 846, and 1455 bp, respectively. Analyses of sequence alignment and phylogenetic tree showed that casp-2, -3, and -8 share 52%-65%, 33%-40%, 63%-78% overall sequence identities with those of other vertebrates, respectively. 3D structures of Pfcasp-2, -3, and -8 enjoy conservation in core area together, while each owns a distinctive profile. Comparisons of deduced amino acid sequences indicated that Pfcaspases possessed the caspase domain and conserved active sites like ‘HG’ and ‘QACXG’ (X for R or G). qRT-PCR results revealed that Pfcasp-2, -3, and -8 were expressed constitutively in a wide range of organs, especially in immune-related organs including whole blood and kidney. In vitro, the expressions of the three caspases (Pfcasp-2, 3, and -8) and immune-related genes (IgM and IL-8) were significantly up-regulated in kidney leukocytes after A. Hydrophila challenge and inhibitors treatment. The expressions of Pfcasp-2 and Pfcasp-3 were successfully inhibited in the kidney leukocytes by Ac-DEVD-CHO (an inhibitor to caspase-3), but the expression of Pfcasp-8 was not affected. Cellular localization analysis showed that the distribution of Pfcasp-2, -3, and -8 was in cytoplasm. Further, overexpression of Pfcasp-2, -3, or -8 was found to cause DNA damage and apoptosis, suggesting that three caspases may be related to apoptosis and mediate different apoptosis pathways in pufferfish. Moreover, the expressions of these caspases were also up-regulated in whole blood and kidney after A. hydrophila challenge, indicating their possible involvement in the immune response against A. hydrophia stimulation. Taken together, the results of this study suggest that the caspase-2,-3, and -8 may play an important role in the apoptosis and immune response in pufferfish.

Immunohistochemical characterization and change in location of branchial ionocytes after transfer from freshwater to seawater in the euryhaline obscure puffer, Takifugu obscurus.

The obscurepuffer Takifugu obscurus is a euryhaline fish species suitable for studying the molecular mechanism of osmoregulation. The distributional changes of branchial ionocytes were detected following the transfer from freshwater (FW) to seawater (SW) based on two main ion transporters, Na+/K+-ATPase (NKA) and Na+/K+/ 2Cl- cotransporter 1 (NKCC1). The mRNA and protein expression levels of NKA and NKCC1 in the gills all increased rapidly in the first four days after transfer to SW. Double immunofluorescence staining showed that NKCC1 and NKA were colocalized in the branchial ionocytes and the immunoreaction of NKCC1 was stronger after transfer. Moreover, following transfer to SW, the number of lamellar ionocytes in the gills is reduced and the number of filament ionocytes is increased significantly. Taken together, these findings indicated that SW transfer of obscure puffer promotes the changes of distribution, function and size of branchial ionocytes.

Cloning and characterization of carnitine palmitoyltransferase Iα (CPT1α) from obscure puffer (Takifugu obscurus), and its gene expression in response to different lipid sources

In this study, CPT Іα cDNA of obscure puffer (Takifugu obscurus) was cloned and characterized, and its expression in response to replacement of dietary fish oil with vegetable oil was investigated following a feeding trial. The sequence of obscure puffer CPT Іα cDNA contains a 5’ UTR of 113 bp, a 3’ UTR of 294 bp and an open reading frame of 2316 bp encoding a polypeptide of 771 amino acids with an estimated molecular mass of 88.5 kDa and a predicted isoelectric point of 8.81. Moreover, bioinformatics analysis of CPT Іα sequences indicated that this gene shared 65 %–98 % identity with CPT Іs from other piscine species of which the most closely related one was tiger puffer. And the results of qRT-PCR revealed that CPT Іα gene was ubiquitously expressed in all tested tissues and the highest expression level was found in liver, followed by heart and blood. The lowest expression level of CPT Іα was observed in spleen and eye. Replacing dietary fish oil with 50 % vegetable oil (soybean oil, corn oil and rapeseed oil) or different proportion of soybean oil (0 %, 25 %, 50 % and 75 %) has no significant effect on CPT Іα gene expression in liver of obscure puffer. These results suggest that CPT Іα contains highly-conserved domains and fish oil replacement by vegetable oil has no remarkable effect on hepatic CPT Іα expression. Our results provide the vital data for the further research on the mechanism of lipid catabolism, as the pivotal role of CPT Іα in lipid metabolism.

Effects of 9,10-phenanthrenequione on antioxidant indices and metabolite profiles in Takifugu obscurus plasma.

Derived from polycyclic aromatic hydrocarbons (PAHs), oxygenated-PAHs (oxy-PAHs) may pose hazards to aquatic organisms, which remain largely unknown. Takifugu obscurus is an important anadromous fish species of high economic and ecological values. In the present study, T. obscurus was acutely exposed to 44.29 µg l−1 9,10-phenanthrenequione (9,10-PQ) for 96 h. Changes of antioxidant indices and metabolite profiles in plasma were compared between 9,10-PQ treatment and the control. The results showed that 9,10-PQ treatment significantly increased malondialdehyde (MDA) content during 6 to 96 h, increased superoxide dismutase (SOD) and catalase (CAT) activities at 6 h, but decreased them at 96 h. These results indicated that 9,10-PQ induced oxidative stress to fish. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis revealed that four metabolic pathways were influenced in response to treatment with 9,10-PQ, including glycerophospholipid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, purine metabolism and sulfur metabolism. These pathways are associated with antioxidant mechanisms, biosynthesis of neurotransmitters and innate immune functions. Thus, the as-obtained results confirmed that 9,10-PQ induced oxidative stress and raised concerns of neurotoxicity and immunotoxicity to fish. Overall, the present study posed a high environmental risk of oxy-PAHs to aquatic ecosystems.

Texture and Quality Assessment of Ready-to-eat Farmed Obscure Puffer Fish (Takifugu obscurus) Fillet by Evaluating Bacterial and Myofibrillar Degradation and Biochemical Changes during Refrigerated Storage

ABSTRACT Obscure puffer fish (Takifugu obscurus) flesh, often eaten as raw fish fillet, is susceptible to softening. To investigate the mechanisms for the softening of flesh and changes in firmness, the proliferation of total microbial flora, Enterobacteriaceae, Pseudomonas spp., and lactic acid bacteria (LAB), total volatile basic nitrogen (TVBN), trimethylamine (TMA), and pH were periodically assayed during storage at 0, 4, and 8ºC. Myofibril extraction and separation of puffer fish flesh was carried out and the length of the separated myofibrils was measured periodically using an optical microscope during the entire storage time. Transmission electron microscopy was also used to inspect the changes in myofibrillar characteristics of the fish flesh at the beginning and end of the storage period. The results indicated that lower chilled temperature can contribute to reduced proliferation of microbial flora and less variation in TVBN, TMA, and pH and further maintain the integrity of myofibrils and retain firmness of puffer fish flesh during storage.

Transcriptome analysis of Takifugu obscurus liver in response to acute retene exposure

Retene (1-methyl-7-isopropyl-phenanthrene, RET) is an alkyl polycyclic aromatic hydrocarbon (PAH) with environmental risk to aquatic animals. Takifugu obscurus is a migratory fish species with high economic and ecological value. To assess the toxic effects of RET on molecular metabolism, juvenile T. obscurus in this study were acutely exposed to 44.30 µg/L of RET for four days. The transcriptome profiles of livers were compared between RET treatment group and the control, and the results revealed that 1,897 genes were significantly differentially expressed (DEGs) after exposure to RET, which enriched 17 KEGG pathways. Among these, glycerolipid metabolism, glycerophospholipid metabolism, insulin signaling pathway, and FOXO signaling pathways were significantly activated. Further exploration indicated that RET exposure disrupted glucose metabolism, stimulated insulin metabolism, and activated cell proliferation genes. Overall, these findings help explain the molecular mechanisms underlying RET toxicity, and may offer evidence to support T. obscurus protection.

Transcriptome changes of Takifugu obscurus liver after acute exposure to the oxygenated-PAH 9,10-phenanthrenequione.

Contamination with polycyclic aromatic hydrocarbons (PAHs) causes noticeable ecological problems in aquatic ecosystems. 9,10-Phenanthrenequione (9,10-PQ) is an oxidized PAH and is highly toxic to aquatic animals. However, the effects of 9,10-PQ on the molecular metabolism of fish remain largely unknown. In this study, Takifugu obscurus juveniles were acutely exposed to 44.30 µg/L 9,10-PQ for 3 days. The transcriptome profile changes in their livers were compared between the 9,10-PQ treatment group and the control using T. rubripes as the reference genome. The results identified 22,414 genes in our transcriptome. Among them, 767 genes were differentially expressed after exposure to 9,10-PQ, which enriched 16 KEGG pathways. Among them, the glycolysis, phagosome, and FOXO signaling pathways were significantly activated in 9,10-PQ treatment compared with the control. These data indicate that 9,10-PQ increased the glycolysis capacity to produce more energy for resistance and harmed immune function. Moreover, several genes related to tumorigenesis were significantly upregulated in response to 9,10-PQ, displaying the carcinogenic toxicity of 9,10-PQ to T. obscurus. Genes in steroid biosynthesis pathways were downregulated in the 9,10-PQ treatment group, suggesting interference with the endocrine system. Overall, these findings provide information to help evaluate the environmental risks that oxygenated-PAHs present to T. obscurus.

Adaptive evolution of low-salinity tolerance and hypoosmotic regulation in a euryhaline teleost, Takifugu obscurus

The mechanism of osmoregulation is crucial for maintaining growth, development, and life activities in teleosts. Takifugu obscurus, the only euryhaline species in the genus Takifugu, is a proper model organism for studying the mechanism of low-salt tolerance and hypoosmotic regulation. In this study, whole-genome sequencing data were obtained from 90 pufferfish representing five species within this genus, T. rubripes, T. obscurus, T. flavidus, T. niphobles, and T. bimaculatus. Using a phylogeny, PCA, and population structure analyses, we observed similar amounts of population genetic differentiation among species. The five species are closely related to each other and have differentiated within a relatively short period, while T. bimaculatus and T. flavidus shared the most similar genetic backgrounds. We further identified hundreds of genes under selection related to hypoosmotic regulation between T. obscurus and other Takifugu species, including 16 representative genes involving ion transporters (atp1a3, atp2a2, atp2a3, slc13a1, slc5a8, slc12a2, slc12a4, slc26a2, scn1b, and kcna2/3/10), genes involved in hormone regulation (fyn, prlr, and grb2), and a gene associated with water absorption (aqp3). Our findings provide preliminary insight into the mechanism of osmoregulation and will facilitate follow-up validation of candidate genes related to osmoregulation in T. obscurus.

Effects of chitosan and sodium alginate active coatings containing ε-polysine on qualities of cultured pufferfish (Takifugu obscurus) during cold storage

The present study evaluated the effects of chitosan (CH) and sodium alginate (SA) bioactive coatings containing 0.1, 0.2 and 0.3% ε-polylysine (ε-PL) combined with high CO2 modified atmosphere packaging conditions (MAP, 60% CO2/5% O2/35% N2) on the quality of cultured pufferfish (Takifugu obscurus) at 4 °C for 18 days. Results showed that CH-SA coatings containing ε-PL and high CO2 MAP treatments could significantly lower total viable count (TVC), Pseudomonas spp., H2S-producing bacteria and lactic acid bacteria counts (LAB), and reduce the productions of off-flavor compounds including total volatile basic nitrogen (TVB-N), trimethylamine (TMA) and ATP-related compounds. Volatile compounds were identified and quantified by SPME gas chromatography–mass spectrometer (SPME-GC/MS) for pufferfish during cold storage and the bioactive coating treated pufferfish significantly lowered the relative content of fishy flavor compounds, such as 1-octen-3-ol, hexanal, (E)-2-octenal, octanal and 2,3-butanedione. Further, CH-SA coatings containing ε-PL and high CO2 MAP treatments could more efficiently retard the water migration by magnetic resonance imaging (MRI) results, and maintained the quality of pufferfish during storage at 4 °C according to organoleptic evaluation results.