Takifugu Rubripes Research Articles

Characterization of CLCN5 and Expression Profile under Low-Salinity Stress in Takifugu rubripes

Characterization of CLCN5 and Expression Profile under Low-Salinity Stress in Takifugu rubripes

Effects of the 5-HT1A receptor agonist 8-OH-DPAT on aggressive behavior in juvenile pufferfish, Takifugu rubripes.

Severe aggressive behavior of juvenile pufferfish affects economic efficiency and fish welfare in aquaculture. 5-HT plays an important role in regulating the aggressive behavior of fish in aquaculture environment. This study examined the effects of different concentrations (0, 0.25, 0.5, 1 mg/kg) of 8-OH-DPAT, a selective 5-HT1A receptor agonist, on the aggressive behavior of juvenile pufferfish. Forty-five minutes after drug injection, the aggressive behavior of juvenile fish was recorded for 20 min, including the latency to the first attack and the frequency of aggressive behaviors. The results showed no significant differences in the latency to the first attack of juvenile fish among treatment groups. During the first 10 min of the observation period, there was no significant difference in the total aggressive acts and locomotor activity among treatment groups. Total aggressive acts and locomotor activity were the least in the 1 mg/kg 8-OH-DPAT-treated during the 20 min observation period. Both aggressive behavior and locomotor activity were negatively correlated with 8-OH-DPAT treatment overall, respectively. The above results suggested that the serotonergic system activation had suppressive effects on aggressive behavior and locomotor activity in juvenile pufferfish.

From IgZ to IgT: A Call for a Common Nomenclature for Immunoglobulin Heavy Chain Genes of Ray-Finned Fish

From IgZ to IgT: A Call for a Common Nomenclature for Immunoglobulin Heavy Chain Genes of Ray-Finned Fish

A Transcriptomic Analysis of Gonads from the Low-Temperature-Induced Masculinization of Takifugu rubripes.

Simple SummaryOur study analyzed the differentiation of transcriptomes in normal and sex-reverse Takifugu rubripes, and screened out 13 differentially expressed genes related to sex differentiation. This is the first report on the gonadal transcriptome of pseudo-males in Takifugu rubripes. Our results provide an important contribution to the molecular mechanism of masculinization in a cultured fish subject to low-temperature treatment.The phenotypic sex of fish is usually plastic. Low-temperature treatment induces the masculinization of Takifugu rubripes, resulting in pseudo-males (PM) with the physiological sex of a male (M) and genetic sex of a female (F). For a comparison of gonadal transcriptomes, we collected gonads from three groups of T. rubripes (F, M, and PM) for high-throughput transcriptome sequencing. The results provided 467,640,218 raw reads (70.15 Gb) and a total of 436,151,088 clean reads (65.43 Gb), with an average length of 150 bp. Only 79 differentially expressed genes (DEGs) were identified between F and PM, whereas 12,041 and 11,528 DEGs were identified between F and M, and PM and M, respectively. According to the functional annotation of DEGs, 13 DEGs related to gonadal development were screened (LOC101066759, dgat1, limk1, fbxl3, col6a3, fgfr3, dusp22b, svil, abhd17b, srgap3, tmem88b, bud4, and mustn10) which might participate in formating PM. A quantitative PCR of the DEGs confirmed the reliability of the RNA-seq. Our results provide an important contribution to the genome sequence resources for T. rubripes and insight into the molecular mechanism of masculinization in a cultured fish subject to low-temperature treatment.

Profile of gene expression changes during estrodiol-17\u03b2-induced feminization in the Takifugu rubripes brain

BackgroundAs the critical tissue of the central nervous system, the brain has been found to be involved in gonad development. Previous studies have suggested that gonadal fate may be affected by the brain. Identifying brain-specific molecular changes that occur during estrodiol-17β (E2) -induced feminization is crucial to our understanding of the molecular control of sex differentiation by the brains of fish.ResultsIn this study, the differential transcriptomic responses of the Takifugu rubripes larvae brain were compared after E2 treatment for 55 days. Our results showed that 514 genes were differentially expressed between E2-treated-XX (E-XX) and Control-XX (C-XX) T. rubripes, while 362 genes were differentially expressed between E2-treated-XY (E-XY) and Control-XY (C-XY). For example, the expression of cyp19a1b, gnrh1 and pgr was significantly up-regulated, while st, sl, tshβ, prl and pit-1, which belong to the growth hormone/prolactin family, were significantly down-regulated after E2 treatment, in both sexes. The arntl1, bhlbe, nr1d2, per1b, per3, cry1, cipc and ciart genes, which are involved in the circadian rhythm, were also found to be altered. Differentially expressed genes (DEGs), which were identified between E-XX and C-XX, were significantly enriched in neuroactive ligand-receptor interaction, arachidonic acid metabolism, cytokine-cytokine receptor interaction and the calcium signaling pathway. The DEGs that were identified between E-XY and C-XY were significantly enriched in tyrosine metabolism, phenylalanine metabolism, arachidonic acid metabolism and linoleic acid metabolism.ConclusionA number of genes and pathways were identified in the brain of E2-treated T. rubripes larvae by RNA-seq. It provided the opportunity for further study on the possible involvement of networks in the brain-pituitary-gonadal axis in sex differentiation in T. rubripes.

Effects of different feeding frequencies on the growth, plasma biochemical parameters, stress status, and gastric evacuation of juvenile tiger puffer fish (Takifugu rubripes)

Tiger puffer fish (Takifugu rubripes)is one of the most economically valuable aquaculture species in Asia. However, farming tiger puffer fish at the juvenile stage has been greatly restrained by cannibalization caused by inappropriate feeding regimes, resulting in very high mortality and great economic loss, particularly in intensive recirculating aquaculture systems. Therefore, the feeding strategy should adapt to fish appetite, nutrition requirements and welfare in intensive aquaculture. A 60-day feeding experiment was conducted to evaluate the effects of feeding frequency on the overall health of juvenile tiger puffer fish. Fish were randomly assigned to four groups with different feeding frequencies using the same ration size per feeding. P1: two daily meals; P2: four daily meals; P3: six daily meals; P4: continuous diurnal feeding using a belt feeder. Fish in the P2 group exhibited optimal values for growth, including final weight, weight gain rate, specific growth rate, feed efficiency, and survival (P < 0.05), followed by those in the P3 group. The crude lipid content increased significantly with increasing feeding frequency (P < 0.05), indicating that high frequency feeding could accumulate higher body fat content. The apparent digestibility coefficients of dry matter, protein, and lipid were significantly lower for fish in the P4 group than for those in the other groups (P < 0.05). The total cholesterol, triglyceride, and glucose levels increased significantly with increasing feeding frequency (P < 0.05), indicating that high frequency feeding leads to excessive accumulation of dietary carbohydrates and lipids in juvenile tiger puffer fish. Fish in the P1 and P4 groups exhibited relatively higher antioxidant enzyme activities (P < 0.05), which suggested that low and high feeding frequencies cause oxidative stress in juvenile tiger puffer fish. A comparison of non-linear model parameters revealed that an exponential model best described the gastric evacuation rate of juvenile tiger puffer fish, with approximately half the meal passed within 3 h, which further confirmed that feeding intervals in four times/day may more closely align with the digestive physiological characteristics of juvenile tiger puffer fish. Overall, these results indicate that the best growth performance and physiological indicators can be achieved when fed at 4 times/day.

Responses to graded levels of leucine and branched-chain amino acid imbalance in tiger puffer Takifugu rubripes

In fish, it has been debatable whether branched-chain amino acid (BCAA) imbalance will show antagonistic effects on the growth and the concentrations of plasma or tissue BCAAs. The objective of this study were to investigate the effects of incremental levels of dietary leucine on the growth (trial 1) and potential antagonistic effects between BCAAs (trial 2) in tiger puffer. This study was composed of two separate trials. In trial 1, six experimental diets were prepared by the basal diet containing incremental levels of leucine (19.3, 24.9, 28.6, 32.6, 36.3 and 40.7 g kg−1 of dry diet). In trial 2, five experimental diets were formulated to contain a proper amount of leucine (29.0 g kg−1 of dry diet), a deficiency of leucine (12.6 g kg−1 of dry diet), an excess of leucine (55.9 g kg−1 of dry diet), an excess of isoleucine (43.2 g kg−1 of dry diet) and an excess of valine (49.6 g kg−1 of dry diet). All diets in two trials were fed to fish in triplicate net cages and polyethylene tanks, respectively. In trial 1, growth increase relative to initial body weight was 206.2–227.5%, while growth performance and body condition indices were not significantly affected by different levels of dietary leucine and did not show significantly linear or quadratic relationships with incremental levels of dietary leucine. In trial 2, growth performance also did not exhibit significant differences in fish fed imbalanced BCAAs diets. Compared with a proper amount of leucine group, the activities of branched-chain α-keto acid dehydrogenase enzyme complex (BCKDH) and BCKDH kinase increased in the leucine or valine excessive diet. The expressions of amino acid transporters (B0AT1 and y+LAT2) and peptide transporter 1 (PepT1) were down-regulated by excessive dietary leucine, isoleucine or valine. Free valine concentration in plasma and muscle increased in the leucine deficient diet compared to a proper amount and an excess of leucine in diets. In conclusion, the growth of tiger puffer was not affected by the diets containing 12.6–55.9 g kg−1 leucine. An excess of dietary leucine, isoleucine or valine possibly resulted in antagonism between BCAAs based on the activities of enzymes related to BCAA catabolism and the expressions of amino acid transporters, although the growth was not significantly depressed by one excessive BCAA when the other two BCAAs met the requirements for tiger puffer. In addition, plasma and muscle valine levels indicated that the antagonism of valine to leucine were observed in the leucine deficient diet, but not in the leucine excessive diet.

Genetic parameter estimates for three antioxidant factors in cultured Takifugu rubripes

Genetic parameters of three antioxidant factors, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), were evaluated in liver samples from 840 Takifugu rubripes individuals from 28 full-sib families. Heritability values of SOD, CAT, and GPX were 0.17, 0.18, and 0.14, respectively, and the full-sib family effect values for these antioxidant factors were 0.46, 0.47, and 0.49, respectively. The ranges of phenotypic and genetic correlations among the three immune factors were 0.748–0.848 and 0.726–0.806, respectively. Considering the low heritability and high full-sib family effect of the three antioxidant indexes, the use of both genome-wide selection and clustered regularly interspaced short palindromic repeats (CRISPR) is promising for genetically improving the three antioxidant indexes in cultured fish. In addition, given positive phenotypic and genetic correlations among the three antioxidant enzymes SOD, CAT and GPX, the antioxidant competence of Takifugu rubripes can be improved by genetically improving these three antioxidant traits via multi-trait integrated breeding technology or indirect selection.

Effects of food quantity on aggression and monoamine levels of juvenile pufferfish (Takifugu rubripes).

Aggressive behavior is important for animals to obtain limited resources. Understanding fish behavior and physiological response is of great significance to evaluate aquaculture production and fish welfare. Food is an important trigger of aggressive behavior in juvenile fish under high-density aquaculture conditions. The aim of this study was to investigate the aggressive behavior and monoamine levels of juvenile pufferfish (mean body mass of 6.29 ± 0.33 g) under normal feeding and restricted feeding. Our main results included the following: (1) The mortality and fin damage were higher and aggression was more intense of juvenile pufferfish at the 1% ration than those of the 3% ration; (2) during feeding, the velocity, body contact, and activity at the 1% ration were significantly higher than that of the 3% ration; (3) the concentrations of brain 5-hydroxyindoleacetic acid (5-HIAA) and monoamine oxidase A (MAOA) at the 1% ration were significantly lower, and dopamine (DA) concentrations were significantly higher. These results suggest that juvenile pufferfish shows serious aggressive behavior at the low ration, which may be related to the decrease of 5-HIAA and MAOA concentrations, and the increase of DA concentrations.

Calreticulin functions in antimicrobial immunity of obscure puffer Takifugu obscurus

Calreticulin (Crt) is a highly conserved and multi-functional protein with lectin-like properties and important immunological activities. In this study, a Crt homolog, namely, ToCrt, was cloned and characterized from the obscure puffer Takifugu obscurus with an open reading frame of 1278 bp encoding a putative protein of 425 amino acids. The deduced amino acid sequence of ToCrt consisted of three conserved structural domains: N-domain, P-domain, and C-terminal domain. In the phylogenetic tree, ToCrt formed a separate cluster with three Crts from other pufferfish species (Takifugu rubripes, Takifugu flavidus, and Takifugu bimaculatus). The mRNA transcript of ToCrt was ubiquitously expressed in all the examined tissues in a decreasing order: liver, spleen, kidney, gills, intestine, and heart. After Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila stimulations, the levels of ToCrt mRNA in the kidney and spleen were significantly upregulated compared with that in the control group. The recombinant calreticulin domain of ToCrt (rToCrt) could bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and polysaccharides from bacterial cell walls such as lipopolysaccharide and peptidoglycan. Meanwhile, rToCrt could agglutinate different kinds of microorganisms and exhibit antimicrobial activity. These results suggested that T. obscurus ToCrt could serve as an antimicrobial effector in the host immune response against invading microorganisms.

Effects of illumination intensities on growth, digestive and metabolic enzyme activities and antioxidant capacities of juvenile Takifugu rubripes

In this study, the impacts of various illumination intensities on growth, digestion and metabolism as well as antioxidant enzyme dynamics of Takifugu rubripes (0.03 ± 0.01 g) were investigated. Activities of alkaline phosphatase (AKP), acid phosphatase (ACP), pepsin (PPS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) were measured. Juveniles were reared under four degrees of illumination intensities: 50 mW/m2, 250 mW/m2, 500 mW/m2 and 750 mW/m2, at a constant photoperiod (12D:12 L). Samples were collected at days 7, 14, 21, 28, and 35. After 35 days, the 750 mW/m2 light intensity was found to have significantly increased the final body weight, which was 1.1 ± 0.005 g. On day 35, PPS activities of T. rubripes juveniles in the 750 mW/m2 sample group were significantly low when compared to those of the 250 mW/m2 group while AKP activities in the 750 mW/m2 group were significantly higher than those of the 50 and 500 mW/m2 groups (p < 0.05). Low light intensity was unsuitable for the initial stages of T. rubripes. The 50 mW/m2 light intensity had a negative effect on weight gain of juveniles during the trial period. Moreover, at the end of the experiment, metabolic activities (ALT and LDH) in the 50 mW/m2 group were significantly low than those of other groups (p > 0.05). Even though they were relatively lower, there were no significant differences in SOD activities in the 250 mW/m2 group relative to the 500 and 750 mW/m2 groups. On day 35, high light intensity (750 mW/m2) resulted in stress responses that were characterized by T-AOC activities, and activity values were significantly lower than those of other groups (p < 0.05). These findings imply that different illumination intensities impact the early growth and physiology stages of T. rubripes, with the 250–500 mW/m2 light intensity being the most suitable.

Genomic prediction for testes weight of the tiger pufferfish, Takifugu rubripes, using medium to low density SNPs

Aquaculture production is expected to increase with the help of genomic selection (GS). The possibility of performing GS using only a small number of SNPs has been examined in order to reduce genotyping costs; however, the practicality of this approach is still unclear. Here, we tested whether the effects of reducing the number of SNPs impaired the prediction accuracy of GS for standard length, body weight, and testes weight in the tiger pufferfish (Takifugu rubripes). High values for predictive ability (0.563–0.606) were obtained with 4000 SNPs for all traits under a genomic best linear unbiased predictor (GBLUP) model. These values were still within an acceptable range with 1200 SNPs (0.554–0.588). However, predictive abilities and prediction accuracies deteriorated using less than 1200 SNPs largely due to the reduced power in accurately estimating the genetic relationship among individuals; family structure could still be resolved with as few as 400 SNPs. This suggests that the SNPs informative for estimation of genetic relatedness among individuals differ from those for inference of family structure, and that non-random SNP selection based on the effects on family structure (e.g., site-FST, principal components, or random forest) is unlikely to increase the prediction accuracy for these traits.

A new calnexin modulates antibacterial immune response in obscure puffer Takifugu obscurus

Calnexin (Cnx) is a membrane-bound lectin chaperone of the endoplasmic reticulum. In this study, a novel Cnx homologue from the obscure puffer Takifugu obscurus was characterized, tentatively named ToCnx. The cDNA of ToCnx was 1803 bp, and it contained an open reading frame encoding a polypeptide of 600 amino acid residues with a calculated molecular weight of 67.5 kDa. Multiple alignment of the deduced amino acid sequences of ToCnx and other related fish Cnxs revealed that ToCnx had typical characteristics of fish Cnxs. Sequence comparison and phylogenetic tree analysis showed that ToCnx had the closest relationship with Cnxs from Takifugu flavidus and Takifugu rubripes. ToCnx transcripts were detected in all the tissues examined, and they were mainly expressed in the liver, kidney, and intestine. Upon Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila infection, ToCnx transcripts were all significantly upregulated in the kidneys. The recombinant calreticulin domain of ToCnx (rToCnx) was prepared by prokaryotic expression. In the absence of calcium, rToCnx was able to bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and two bacterial saccharides, such as lipopolysaccharide and peptidoglycan. In the presence of calcium, rToCnx could agglutinate all the detected microorganisms. In addition, rToCnx possessed the effect of inhibiting the growth of three microbe strains. These observations suggested that ToCnx is an important participant in host immune defense against bacteria.

Differences in DNA methylation between slow and fast muscle in Takifugu rubripes.

Fish skeletal muscle is comprised of fast muscle (FM) and slow muscle (SM), which constitutes 60% of total the body mass. Fish skeletal muscle can affect fish swimming activity, which is important for aquaculture due to its growth-potentiating effects. DNA methylation can influence gene expression level. We previously identified multiple differentially expressed genes (DEGs) between FM and SM in Takifugu rubripes. However, it is unknown if the expression levels of these DEGs are influenced by DNA methylation. In the present study, we used DNA methylation sequencing to study the DNA methylation profiles of FM and SM in T. rubripes. SM had higher overall methylation levels than FM. A total of 8479 differentially methylated genes (DMGs) and 3407 DMGs containing differentially methylated regions (DMRs) in the promoter regions between FM and SM were identified. After enrichment analysis, we found functionally relevant DMGs between FM and SM, including Kapca, Plcd3a, Plcd1, Pi3k, Tsp4b and Pgfrb in the hedgehog signaling pathway and phosphatidylinositol (PI)-related pathways. Due to the different methylation levels of these genes between FM and SM, the expression levels of Kapca, Plcd3a, Plcd1, Pi3k, and Tsp4b were higher in FM and Pgfrb was higher in SM. There were differences in the hedgehog signaling pathway and PI-related pathways between FM and SM. In SM, the cytokine-cytokine receptor interaction promoted focal adhesion, while ECM-receptor interactions promoted focal adhesion in FM. These results provide information regarding the difference between FM and SM in T. rubripes.

Functional characterization of a novel tumor necrosis factor gene (TNF-New) in rock bream (Oplegnathus fasciatus)

The novel tumor necrosis factor (TNF-New or TNFN) gene has been identified only in teleost such as zebrafish, medaka (Oryzias latipes), fugu (Takifugu rubripes), and rainbow trout (Oncorhynchus mykiss). In this study, a putative TNFN gene in rock bream (named RB-TNFN) was cloned and its functional expression in the immune system was analyzed. Although it was previously reported to share a high degree of homology with mammalian lymphotoxin (LT)-β, in silico analysis revealed that RB-TNFN differed slightly from mammalian LT-β in its genomic structure, phylogenetic relationship, and predicted protein tertiary structure, whereas the genomic location of TNFN (immediately behind TNF-α) was the same as that of LT-β. In healthy rock bream, RB-TNFN gene expression was the highest in the liver and the lowest in the head kidney. In vitro, it was significantly upregulated in head kidney cells following polyinosinic:polycytidylic acid, concanavalin A, phytohemagglutinin, or calcium ionophore (CI) stimulation and in spleen cells by lipopolysaccharide (LPS), CI, and rock bream iridovirus (RBIV). In vivo, it was upregulated in the spleen, liver, and gut on day 1 and in the blood on day 3 following LPS injection, and in the blood, head kidney, and liver following RBIV vaccination. Post-RBIV infection, the vaccinated group showed a significantly higher TNFN gene expression in the head kidney and blood than the unvaccinated group. Treatment with recombinant TNFN protein (RB-rTNFN) resulted in significantly upregulated interleukin-1β expression in the head kidney, spleen, blood, liver, and peritoneal cells. It also enhanced IL-8 gene expression in the head kidney, blood, and peritoneal cells, and interferon γ gene expression in the gut and gills on day 1. TNFN and cyclo-oxygenase-2 gene expression was upregulated in peritoneal cells on day 3. Flow cytometry analysis revealed a significant increase in the peritoneal lymphocyte population after the intraperitoneal (i.p.) injection of RB-rTNFN. These results suggest that RB-TNFN mediated innate and adaptive immunity in rock bream.

Molecular cloning, characterization and expression patterns of HSP70 and HSP60 in the pufferfish (Takifugu rubripes)

Takifugu rubripes is a commercially important fish in the Chinese aquaculture industry due to its excellent meat quality. In eukaryotic cells, heat shock proteins (HSPs) play a crucial role in maintaining intracellular protein homeostasis. They play significant roles in response to adverse environmental stresses in aquatic animals. In this study, we used bioinformatics to comprehensively compare two different full-length cDNA sequences at the molecular level in the HSP family of T. rubripes. We performed various physicochemical studies such as race cloning and RT-qPCR, and found that TrHSP60 (575 amino acids) has a larger polypeptide, followed by TrHSP70–13 (441 amino acids). Homology and phylogenetic studies showed that these sequences have the greatest homology to Takifugu bimaculatus. The mRNA of the two genes were constitutively expressed in all tested tissues of untreated T. rubripes, including brain, muscle, kidney, liver, spleen, gill, and intestine, with highest expression levels in the intestine. Three experimental groups were established in this study, the H1 group (20 °C), H2 group (24 °C) and H3 group (28 °C), in order to analyze the expressed levels of TrHSP70 in T. rubripes. To understand the role of HSPs in temperature stress in T. rubripes, we analyzed the expression levels of HSPs in T. rubripes at different temperatures. The results showed that the mRNA expression of the HSP gene were influenced by temperature stress. Gene expression of TrHSP70 and TrHSP60 were mainly up-regulated at 12 h and 48 h in the H1 group, respectively. Meanwhile, gene expression of TrHSP70 was highest at 48 h in the H3 group and TrHSP60 reached was lowest at 24 h in the H3 group. The results showed that the two genes were constitutively expressed genes, which play an important role in maintaining normal physiological function and stress responses. The results show that TrHSP70 and TrHSP60 are constitutively expressed genes, which plays important role in maintaining normal physiological function and coping with stress.

Impact of cooking on the sensory perception and volatile compounds of Takifugu rubripes

Takifugu rubripes is well-known for its unique flavour but can also develop a putrid off-note. To eliminate off-note and promote desirable flavour, four cooking processes (boiling, steaming, microwave-heating and roasting) were explored to determine their effects on cooked T. rubripes. The temperature and water dynamics, physico-chemical properties were analysed and correlated with sensory qualities. The changes of centre temperature dynamics during cooking decreased the water mobility and led to varied sensory properties. Six out of ten orthonasal aroma attributes and four out of five mouthfeel attributes were significantly different among samples (p < 0.05). Based on partial least squares regression analysis, orthonasal aroma attributes “roasted” and “earthy/putrid fish” highly correlated with the volatile compounds generated from Maillard reaction and lipid oxidation, respectively; meanwhile mouthfeel attributes of chewy/fibre and tender/juicy were highly associated with water loss and moisture, respectively. This study provides insights for optimising cooking conditions to create desirable fish flavour.

Characterization and expression analysis of the complement component 8β from pufferfish (Takifugu rubripes)

Complement 8β (C8β) is a critical component in the complement system, which mediates bacterial lysis and acts as an essential molecule for membrane attack complex (MAC) formation. This study cloned a partial C8β gene by transcriptome sequencing and RACE approaches from pufferfish Takifugu rubripes (designated as Tr-C8β). Tr-C8β had a 3′ untranslated region (UTR) of 112 bp, a putative open reading frame (ORF) of 1761 bp encoding a polypeptide of 586 amino acid residues, with an estimated molecular mass of 65.26 kDa and a predicted isoelectric point (pI) of 7.86. For adult T. rubripes, spatial distribution expression indicated Tr-C8β expressed in all examined tissues with predominant expression in the liver, moderate in muscle, and weak in the brain. Moreover, the expression of Tr-C8β in the liver was significantly upregulated and peaked at 24 h after lipopolysaccharide (LPS) stimulation. For the larval T. rubripes, Tr-C8β mRNA expression increased dramatically during the heartbeat phase and peaked in the hatching prophase, then decreased significantly from 3 to 5 days post hatch (dph), and bottomed at 9 dph. Taken together, all these results suggested that Tr-C8β is an immune factor that might be involved in anti-bacteria response and development in pufferfish.

Microbiota Composition and Quality Changes of Tiger Puffer (Takifugu rubripes) Fillets during 4°C Refrigerated and Ice Storage

ABSTRACT This study was conducted to evaluate the microbiota composition and quality changes of the tiger puffer (Takifugu rubripes) during 4°C refrigerated storage and ice storage for 14 days. The results showed that the sensorial shelf life of refrigerator-stored and ice-stored tiger puffer was 6 and 12 days, respectively. Macroccus and Kocuria were dominant microbiota in fresh fish fillets. Pseudomonas became dominant in refrigerated fillets on day 6, while its dominance was largely weakened in the ice-stored group. At the end of storage, Pseudomonas and Carnobacterium became dominant microbiota in the refrigerated group, while Carnobacterium was inhibited in the ice-stored condition. Furthermore, total viable counts, color, texture, sensory evaluation, K-value and related values, and biogenic amines of puffer fillets in the two groups showed that the quality gradually deteriorated during the storage, and puffer fillets in the ice-stored group decayed slower than that in the refrigerator-stored group. In conclusion, ice storage inhibited the growth of Pseudomonas and Carnobacterium. It also can inhibit the increase of K-value and related values, which was beneficial to the maintenance of fish fillet quality and thereby extended the shelf life of the tiger puffer when compared with refrigerated storage.

Screening of reference genes in tiger puffer (Takifugu rubripes) across tissues and under different nutritional conditions.

The present study was aimed at screening suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in tiger puffer (Takifugu rubripes), an important aquaculture species in Asia and also a good model species for lipid research. Specifically, this reference gene screening was targeted at standardization of gene expression in different tissues (liver, muscle, brain, intestine, heart, eye, skin, and spleen) or under different nutritional conditions (starvation and different dietary lipid levels). Eight candidate reference genes (ribosomal protein L19 and L13 (RPL19 and RPL13), elongation factor-1 alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase1 (HPRT1), beta-2-Microglobulin (B2M), 18S ribosomal RNA (18SrRNA), and beta actin (ACTB)) were evaluated with four algorithms (geNorm, NormFinder, BestKeeper, and comparative ΔCt method). The results showed that different algorithms generated inconsistent results. Based on these findings, RPL19, EF1α, 18SrRNA, and RPL13 were relatively stable in different tissues of tiger puffer. During starvation conditions, ACTB/RPL19 was the best reference gene combination. Under different dietary lipid levels, ACTB/RPL13 was the most suitable reference gene combination. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in tiger puffer.