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.

Effects of the Thyroid Endocrine System on Gonadal Sex Ratios and Sex-Related Gene Expression in the Pufferfish Takifugu rubripes.

To examine the effect and mechanism of thyroid hormone on gonadal sex differentiation, Takifugu rubripes larvae were treated with goitrogen (methimazole, MET, 1000 g/g), and thyroxine (T4, 2nM) from 25 to 80 days after hatching (dah). Gonadal histology and sex ratios of fish were then determined at 80 dah. MET treatment induced masculinization, but T4 treatment did not induce feminization in T. rubripes larvae. Transcriptomic analysis of gonads at 80 dah was then conducted. Among the large number of differentially expressed genes between the groups, the expression of foxl2, cyp19a1a, and dmrt1 was altered. The expression of foxl2, cyp19a1a, dmrt1 and gsdf at 25, 40, 55 days after treatment (dat) was further analyzed by qPCR. MET treatment suppressed the expression of foxl2 and cyp19a1a, and induced the expression of dmrt1 in genetic females (p < 0.05). Additionally, T4 treatment induced an increase in the expression of cyp19a1a in genetic XY gonads only at 25 dat. However, the increase in cyp19a1a expression did not continue to 40 and 55 dat. This may explain why feminization of larvae was not found in the T4-treated group. Thus, the present study provides the first evidence that MET treatment causes masculinization in teleost fish. The effects of MET-induced masculinization in T. rubripes may act primarily via suppression of the expression of foxl2 and cyp19a1a, and stimulation of the expression of dmrt1. Moreover, the effects of higher concentrations of T4 or different concentrations of T3, on sex differentiation require further testing.

Genetic Dissection of a Precocious Phenotype in Male Tiger Pufferfish (Takifugu rubripes) using Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di).

The novel non-targeted PCR-based genotyping system, namely Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di), is characterized by the simplicity in library construction and robustness against DNA degradation and is expected to facilitate advancements in genetics, in both basic and applied sciences. In this study, we tested the utility of GRAS-Di for genetic analysis in a cultured population of the tiger pufferfish Takifugu rubripes. The genetic analyses included family structure analysis, genetic map construction, and quantitative trait locus (QTL) analysis for the male precocious phenotype using a population consisting of four full-sib families derived from a genetically precocious line. An average of 4.7 million raw reads were obtained from 198 fish. Trimmed reads were mapped onto a Fugu reference genome for genotyping, and 21,938 putative single-nucleotide polymorphisms (SNPs) were obtained. These 22 K SNPs accurately resolved the sibship and parent-offspring pairs. A fine-scale linkage map (total size: 1,949 cM; average interval: 1.75 cM) was constructed from 1,423 effective SNPs, for which the allele inheritance patterns were known. QTL analysis detected a significant locus for testes weight on Chr_14 and three suggestive loci on Chr_1, Chr_8, and Chr_19. The significant QTL was shared by body length and body weight. The effect of each QTL was small (phenotypic variation explained, PVE: 3.1-5.9%), suggesting that the precociousness seen in the cultured pufferfish is polygenic. Taken together, these results indicate that GRAS-Di is a practical genotyping tool for aquaculture species and applicable for molecular breeding programs, such as marker-assisted selection and genomic selection.

Light-induced and circadian expressions of melanopsin genes opn4xa and opn4xb in the eyes of juvenile grass puffer Takifugu alboplumbeus.

Animals regulate a variety of aspects of physiology according to environmental light conditions via nonvisual opsins such as melanopsin. In order to study photic regulation of fish physiology, expression changes of the genes for melanopsin (opn4xa and opn4xb) and effects of light on them were examined in juvenile grass puffer Takifugu alboplumbeus using quantitative real-time PCR. In the brain of juvenile fish, no significant diurnal nor circadian changes were observed in opn4x mRNA levels. On the other hand, in the eyes, the mRNA level of opn4xa showed a significant diurnal rhythm with a peak at Zeitgeber time (ZT) 4, while no apparent circadian changes were observed. The mRNA level of opn4xb in the eyes showed a diurnal change similar to that of opn4xa, while it showed a significant circadian change. Furthermore, continuous exposure to light during a subjective night significantly increased the mRNA levels of opn4xa in the eyes at ZT24, suggesting that light induces gene expression of opn4xa in the eyes and that the induction occurs only during the night-day transition period. These results suggest that Opn4xa and Opn4xb play differential roles in the eyes of juvenile grass puffer to mediate the physiological effects of environmental light information.

Expression profile of kalliklectin, a soluble-type mannose receptor, during embryogenesis and early larval development in fugu (Takifugu rubripes).

Kalliklectin is a unique fish-specific lectin, whose sequence is similar to the heavy chain of mammalian plasma kallikrein and coagulation factor XI. In this study, we aimed to evaluate dynamic expression profiles of the lectin gene, during early developmental stages, in fugu, Takifugu rubripes. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the kalliklectin gene was not expressed until 14 h post-fertilization (hpf), while the mRNA was detected after 30 hpf. In real-time quantitative PCR (qPCR), the gene was first expressed at 10.5 hpf; then, the expression level increased with a peak at 30 hpf and then gradually decreased. On the other hand, western blotting with specific antibody detected the lectin protein at all tested stages, including the unfertilized egg, which suggests that the lectin detected in the early stages was a maternal factor. Immunohistochemistry demonstrated that kalliklectin was localized at the basement membranes of the newly hatched larvae, while the lectin was widely detected in epidermal cells in larva at 5 dph. A 40-kDa lectin was partially purified from unfertilized eggs using mannose-affinity chromatography, and the lectin was determined as kalliklectin by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis, which indicated that the lectin is functional in the eggs. The egg lectin can bind to Gram-positive bacterial pathogens of fish, such as Lactococcus garvieae and Streptococcus iniae. We conclude that fugu kalliklectin might be an important immunocomponent, transferred from mother to offspring.

Transcriptomic analyses reveal novel genes with sexually dimorphic expression in Takifugu rubripes brain during gonadal sex differentiation.

BACKGROUND: Quantification of mRNAs in gonads and other tissues at the early critical development stage of sex differentiation may help to provide a global view of regulatory mechanisms underlying sex differentiation. We have recently reported the transcriptomic profiling of fugu gonad associated with sex differentiation. OBJECTIVES: This study attempted to identify the genes in the brain that are involved in gonadal differentiation and development. METHODS: In this study, a transcriptomic scan of potential candidate genes involved in sex differentiation was conducted in the brains of fugu larvae at 30 and 40 dah (morphological gonadal sex differentiation had not yet occurred). The dimorphic expression patterns of several candidate genes were verified using quantitative PCR. RESULTS: A total of 28.24 Gb of clean reads were obtained and 22,337 genes were identified in the brains of fugu larvae. These included 1008 novel genes that provide abundant data for functional analysis of sex differentiation. 229 genes were identified in the 30 dah larvae that were abundant in the XY brain and 21 that were abundant in the XX brain. In the 40 dah larvae, 325 genes were identified abundant in the XY brain and 174 were identified abundant in the XX brain. CONCLUSION: This is the first investigation into the transcriptome of the fugu larvae brain at the early sex differentiation stage. The results obtained here will enhance the understanding of molecular mechanisms that underly fugu sex differentiation.

Construction of a High-Density Genetic Linkage Map and QTL Mapping for Growth-Related Traits in Takifugu bimaculatus.

Takifugu bimaculatus is a euryhaline species, distributed ranging from the southern Yellow Sea to the South China Sea. Their tolerance to a wide range of salinity and temperature, coupled with a desirable firm texture, makes T. bimaculatus a strong candidate for Takifugu aquaculture in subtropics areas. Due to the increasing demand in markets and emerging of the Takifugu aquaculture industry, close attention has been paid to improvement on the T. bimaculatus production. In aquaculture, the great effort has been put into marker-assisted selective breeding, and efficient improvement was realized. However, few genetic resources on T. bimaculatus are provided so far. Aiming at understanding the genetic basis underlying important economic growth traits, facilitating genetic improvement and enriching the genetic resource in T. bimaculatus, we constructed the first genetic linkage map for T. bimaculatus via double digestion restriction-site association DNA sequencing and conducted quantitative traits locus (QTL) mapping for growth-related traits. The map comprised 1976 single nucleotide polymorphism markers distributed on 22 linkage groups (LG), with a total genetic distance of 2039.74 cM. Based on the linkage map, a chromosome-level assembly was constructed whereby we carried out comparative genomics analysis, verifying the high accuracy on contigs ordering of the linkage map. On the other hand, 18 QTLs associated with growth traits were detected on LG6, LG7, LG8, LG10, LG20, and LG21 with phenotypical variance ranging from 15.1 to 56.4%. Candidate genes participating in cartilage development, fat accumulation, and other growth-related regulation activities were identified from these QTLs, including col11a1, foxa2, and thrap3. The linkage map provided a solid foundation for chromosomes assembly and refinement. QTLs reported here unraveled the genomic architecture of some growth traits, which will advance the investigation of aquaculture breeding efforts in T. bimaculatus.

Exposure to nitrite alters thyroid hormone levels and morphology in Takifugu rubripes.

Nitrite (NO2-) can act as a toxic nitrogenous compound with the potential to disrupt endocrine systems in fish. The aim of the present study was to investigate the effects of nitrite on the thyroid endocrine system of Takifugu rubripes. Fish were exposed to 0, 0.5, 1, 3, and 6 mM nitrite concentrations. Blood was collected to assay the concentrations of thyroid-stimulating hormone (TSH), thyroxine (T4), triiodothyronine (T3), free thyroxine (FT4), free triiodothyronine (FT3), and 3,3,5'-triiodothyronine (rT3), as well as the activity of iodothyronine deiodinases (Dio1, Dio2, and Dio3,) after 0, 12, 24, 48, and 96 h of exposure to nitrite. The first branchial arch to the third branchial arch of T. rubripes were sampled and fixed, and thyroid morphology was observed. The results showed that exposure to nitrite significantly increased the concentrations of TSH, T3, FT3, and reduced the concentrations of T4, FT4, and rT3. The activity of Dio1 and Dio2 increased significantly, whereas Dio3 activity decreased significantly. Additionally, thyroid follicles degenerated and became blurred and most colloid material disappeared 96 h after exposure to high nitrite concentrations. Based on these results, high nitrite concentration exposure can disturb thyroid hormone homeostasis, alter thyroid follicle morphology, and result in thyroid endocrine toxicity.

Studies of the mechanism of fatty liver formation in Takifugu fasciatus following copper exposure.

Fatty liver is widely observed during Takifugu fasciatus production, but the mechanisms underlying fatty liver formation remain unknown. The present study was conducted to determine the potential effects of copper (Cu) on hepatic lipid deposition and metabolism in T. fasciatus after 21 days of exposure to Cu (levels: 0, 20 and 100 µg/L). Copper exposure decreased the weight gain rate (WG) in T. fasciatus, but increased the values of the viscerosomatic index (VSI) and hepatosomatic index (HSI) compared with the control. The time-dependent Cu accumulation in tissues increased as the Cu concentration increased. The order of Cu accumulation was liver > intestine > muscle. The lipid content, triglyceride (TG) content and lipoprotein lipase (LPL) activity increased after Cu exposure compared with the control. In addition, more lipid droplets and greater vacuolization were observed in the liver after exposure to 20 µg/L Cu than after 100 µg/L Cu. The expression of genes involved in lipogenesis (g6pd, 6pgd, lpl, fas and acc), lipolysis (hsl and cpt 1) and transcription (ppar α and ppar ©) was dependent on Cu. An analysis of the intestinal microbiome community showed that the highest values of the Chao 1 index, ACE, Shannon index and Simpson index were obtained in fish exposed to 20 µg/L Cu, whereas the lowest values were obtained after the 100 µg/L Cu treatment. The Principal Coordinates Analysis (PCoA) plots of the data revealed structural differences in the groups treated with Cu compared with the control group. At the phylum level, the intestinal microbiota in the Cu-treated and control fish were dominated by Proteobacteria and Bacteroidetes. The higher Firmicutes to Bacteroidetes ratio was observed in fish treated with 20 µg/L Cu compared with other groups, while the lowest ratio was observed in fish exposed to 100 µg/L Cu. Our study revealed the mechanisms by which Cu exposure altered (i) lipid deposition in the body and (ii) the intestinal microbiome, which may contribute to maintain the health status of T. fasciatus for the aquaculture.

Growth and survival of Takifugu rubripes larvae cultured under different light conditions.

We assessed the effects of light intensity and spectrum on the growth and survival of Takifugu rubripes larvae from 30 to 69 days after hatching. Five lighting regimes were applied using 0.5, 1.5, and 3.0 W m-2 full spectrum white (W0.5, W1.5, W3.0), 0.5 W m-2 yellow (Y0.5), and 0.5 W m-2 blue light (B0.5). At the end of the experiment, body length, wet weight, and specific growth rate from day 0 to day 39 were significantly greater in larvae reared under W3.0 than under B0.5 (P Ë‚ 0.05). No significant differences were observed among W0.5, W1.5, and W3.0, or among W0.5, Y0.5, and B0.5 (P > 0.05). Survival rate was significantly higher in larvae reared under W1.5 than W0.5 (P Ë‚ 0.05), but no significant differences were observed among W0.5, Y0.5, and B0.5 (P > 0.05). Additionally, light conditioning did not affect the total thickness of the retina. Although the ratio of the thickness of the retinal pigment epithelium layer/total thickness (TT) was significantly higher in larvae exposed to W3.0 compared with those exposed to other light conditions, and the thickness of the outer nuclear layer/TT was significantly lower in larvae exposed to W3.0 compared with those exposed to W0.5 (P < 0.05), no relationship was confirmed between the structure of the retina and the growth performance of the T. rubripes larvae. Expression patterns of two stress-related and seven growth-related genes were also compared with the biometric parameters investigated in the experimental groups. No significant differences in the aanat1a, crh, ss1, igf1, or igf2 expression were observed among the five treatments. Pomc expression was significantly lower in larvae exposed to W1.5 than the larvae exposed to W0.5, and it was significantly lower in larvae exposed to Y0.5 than in larvae exposed to W0.5 or B0.5 (P < 0.05). Significant differences were also found in the expression of gh, with the highest levels being observed under W3.0, while the lowest levels were observed in B0.5 (P < 0.05). Ghrh expression was significantly higher in W3.0 (P < 0.05). These results should be considered when designing rearing protocols for fugu larvae in aquaculture systems.

Dietary methionine increased the lipid accumulation in juvenile tiger puffer Takifugu rubripes.

Methionine (Met) is one of the most important amino acids in fish feed. The effects of dietary Met on lipid deposition in fish varied a lot among different studies. The present study was aimed at investigating the effects of dietary Met supplementation on the lipid accumulation in tiger puffer, which have a unique lipid storage pattern. Crystalline L-Met was supplemented to a low-fishmeal control diet to obtain two experimental diets with a low (1.1% of dry weight, L-MET) or high Met level (1.6% of dry weight, H-MET). A 67-day feeding trial was conducted with juvenile tiger puffer (average initial weight, 13.83 g). Each diet was fed to triplicate tanks (30 fish in each tank). The results showed that the total lipid contents in whole-body and liver significantly increased with increasing dietary Met levels. The hepatosomatic index, weight gain, and total bile acid content in serum showed similar patterns in response to dietary Met treatments, while the lipid content in muscle was not affected. The hepatic contents of 18-carbon fatty acids were elevated by dietary Met supplementation. The Hepatic mRNA expression of lipogenetic gene such as FAS, GPAT, PPARγ, ACLY, and SCD1 was down-regulated, while the gene expression of lipolytic genes ACOX1 and HSL, as well as that of ApoB100, were up-regulated by increasing dietary Met levels. The hepatic lipidomics of experimental fish was also analyzed. In conclusion, increasing dietary Met levels (0.61%, 1.10%, and 1.60%) increased the hepatic lipid accumulation in tiger puffer. The mechanisms involved warrant further studies.

Interactive effects of temperature and salinity on the survival, oxidative stress, and Na+/K+-ATPase activity of newly hatched obscure puffer (Takifugu obscurus) larvae.

Obscure puffer (Takifugu obscurus) is an anadromous fish widely distributed around the coastal and inland rivers in East Asia. T. obscurus often encounters fluctuations in temperature and salinity. This study aimed to investigate the effect of the interactions of temperature and salinity on survival and oxidative stress response of newly hatched T. obscurus larvae. A combination of three temperatures (19, 25, and 31 °C) and three salinities (0, 10, and 20 ppt) was applied for 96 h under laboratory conditions. The newly hatched larvae could not tolerate 31 °C for 96 h. No death was recorded at other temperatures during this experiment. Malondialdehyde concentrations increased significantly after 6 h of exposure to high salinity (10 and 20 ppt) and then decreased until the end of the experiment at each temperature. The highest superoxide dismutase activity was observed under the exposure to 20 ppt for 24 h at 31 °C. Na+/K+-ATPase activity significantly increased as salinity increased, especially at low temperatures. With the prolong of exposure time, the integrated biomarker response (IBR) values showed an increase until 48 h and then declined at 96 h in most treatments. The largest IBR value appeared when larvae were exposed to the highest temperature and salinity for 24 h. Our study indicated that high temperature with high salinity may negatively affect the early development of T. obscurus and their combined effects should be considered in the larvae culture.

Effects of replacing fish meal with soybean meal on growth performance, feed utilization and physiological status of juvenile obscure puffer, Takifugu obscurus.

An 8-week feeding trial was conducted to evaluate the growth performance, feed utilization and physiological status of obscure puffer, Takifugu obscurus (13.03 ±â€¯0.14 g) fed diets in which fish meal (FM) was replaced with various levels of dehulled and defatted soybean meal (SBM): 0% (SBM0), 15% (SBM15), 30% (SBM30), 45% (SBM45), 60% (SBM60) and 75% (SBM75). No significant differences were observed in weight gain and specific growth rate (SGR) of fish when FM replacement level was lower than 30%, and the broken-line model of SGR showed the maximum replacement level was 40%. Fish fed the SBM-containing diets had a lower red blood cell value compared to the control. The hemoglobin and methemoglobin values showed a declining tendency as dietary SBM level increased. Plasma triacylglycerol, cholesterol and low-density lipoprotein cholesterol levels also showed a decreasing trend that was associated with the reduced crude lipid content of whole body as dietary SBM level increased. The activities of alanine aminotransferase and aspartate aminotransferase in fish fed the SBM-containing diets were all higher than those fed the control diet while glutathione peroxidase and catalase activities were lower than the control group. Results indicated that up to 40% FM protein, based on the broken-line analysis of SGR, can be replaced with SBM in diet for obscure puffer juveniles with supplemental lysine, methionine and taurine.

Multiple transcription factors mediating the expressional regulation of myosin heavy chain gene involved in the indeterminate muscle growth of fish.

Torafugu myosin heavy chain gene, MYHM2528-1, is specifically expressed in neonatal slow and fast muscle fibers, suggesting its functional role in indeterminate muscle growth in fish. However, the transcriptional regulatory mechanisms of MYHM2528-1 involved in indeterminate muscle growth in fish remained unknown. We previously isolated a 2100 bp 5'- flanking sequence of torafugu MYHM2528-1 that showed sufficient promoter activity to allow specific gene expression in neonatal muscle fibers of zebrafish. Here, we examined the cis-regulatory mechanism of 2100 bp 5'-flanking region of torafugu MYHM2528-1 using deletion-mutation analysis in zebrafish embryo. We discovered that myoblast determining factor (MyoD) binding elements play a key role and participate in the transcriptional regulation of MYHM2528-1 expression in zebrafish embryos. We further discovered that paired box protein (Pax3) are required for promoting MYHM2528-1 expression and myocyte enhancer factor-2 (MEF2) binding sites participate in the transcriptional regulation of MYHM2528-1 expression in slow/fast skeletal muscles. Our study also confirmed that the nuclear factor of activated T-cell (NFAT) binding sites take part in the transcriptional regulation of MYHM2528-1 expression in slow and fast muscles fiber in relation to indeterminate muscle growth. These results obviously confirmed that multiple cis-elements in the 5'-flanking region of MYHM2528-1 function in the transcriptional regulation of its expression.

Identification of genes involved in gonadal sex differentiation and the dimorphic expression pattern in Takifugu rubripes gonad at the early stage of sex differentiation.

Quantifying the expression of mRNAs in the gonads at the critical stage of molecular sex differentiation stage might help to clarify the regulatory network during early sex differentiation and provide new information on the role of sex-related genes in gonadal function. In this study, transcriptomic analysis of sex-related genes expression profiles in fugu gonads at 60 and 90 days after hatching (dah) was conducted firstly, and a total of 112,504,991 clean reads, encompassing 28.35 Gb of sequences were retrieved. Twenty-three thousand eight hundred ten genes were found to be expressed in juvenile fugu gonads, and we mainly focused on the differentially expressed genes that have the potential to be involved in the gonadal sex differentiation. For 60-dah juveniles, we identified 1014 genes that were upregulated in the ovary and 1570 that were upregulated in the testis. For 90-dah juveniles, we identified 1287 genes that were upregulated in the ovary and 1500 that were upregulated in the testis. The dimorphic expression patterns of 15 genes in gonads at 30 and 40 dah were further investigate using qPCR. Cyp11b and star were expressed at higher levels in XY than in XX, while cyp11a1 and cyp19a1a were expressed at higher levels in XX than in XY at 30 dah. At 40 dah, the levels of gsdf, dmrt1, dmrt3, cyp11c1, star, and hsd3b expression were higher in XY, while the levels of foxl2, cyp19a1a, wnt9b, and foxD4 expression were higher in XX. Sox9, cyp11a1, cyp17a1, cyp17a2, and nr5a2 were expressed at similar levels in XX and XY at 40 dah. This is the first report of gonadal transcriptome of fugu at early sex differentiation stage, and our results provide an archive for further study on molecular mechanism underlying sex differentiation in this species.

Role of maternal tetrodotoxin in survival of larval pufferfish.

It is known that tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin and had been detected in various taxa. However, the exact function of the toxin in TTX-bearing organisms has remained unclear. In Takifugu pufferfish species, it has been suggested that TTX is utilized to protect larvae from predators but no experimental proof exists. In the present study, we used pufferfish Takifugu alboplumbeus larvae from wild and cultured parents to determine the effects of the maternal TTX on the survival of toxic and non-toxic pufferfish larvae, respectively. TTX contents in the larval pufferfish differed between the larvae derived from wild and cultured parents (1.23 ±â€¯0.20 ng/individual vs. undetectable levels, respectively). Immunohistochemical staining with anti-TTX monoclonal antibody demonstrated that the TTX-specific signals were primarily observed at the body surface of the larvae of wild parents, but not of cultured parents. Predation experiments demonstrated that the juveniles of Girella punctata and Chaenogobius gulosus, used as predator fish, ingested the pufferfish larvae derived from either type of parents, but instantly spat out those from wild parents only. These results indicate that larvae, which are at the most vulnerable stage in the life of pufferfish, are protected by maternal TTX.

The protective effects of taurine on oxidative stress, cytoplasmic free-Ca2+ and apoptosis of pufferfish (Takifugu obscurus) under low temperature stress.

The present study was aimed to investigate the low temperature toxicity and its protection by taurine in pufferfish. The experimental basal diets supplemented with taurine at the rates of 250 (control), 550, 850, 1140, 1430, 1740 mg kg-1 were fed to fish for 8 weeks. The results showed that fish fed diet with taurine had significantly improved weight gain and specific growth rate. After the feeding trial, the fish were then exposed to low temperature stress. The results showed that low temperature stress could induce reactive oxygen species (ROS) generation, disturb the cytoplasm Ca2+ homeostasis, and lead to oxidative stress and apoptosis. Compared with the control group, dietary taurine supplementation groups increased antioxidant enzyme genes such as manganese superoxide dismutase (Mn-SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT), heat shock proteins (HSP70) and complement C3 (C3) mRNA levels under low temperature stress. Meanwhile, dietary taurine supplementation groups reduced ROS generation, and stabilized the cytoplasm Ca2+ under low temperature stress. Furthermore, dietary taurine supplementation groups reduced apoptosis via decreasing caspase-3 activity. This is the first report to demonstrate the mechanisms of taurine against low temperature stress in fish.

Growth performance and protective effect of vitamin E on oxidative stress pufferfish (Takifugu obscurus) following by ammonia stress.

This study was conducted to determine the effects of vitamin E on growth performance, biochemical parameters, and antioxidant capacity of pufferfish (Takifugu obscurus) exposed to ammonia stress. The experimental basal diets supplemented with vitamin E at the rates of 2.31 (control), 21.84, 40.23, 83.64, 158.93, and 311.64 mg kg-1 dry weight were fed to fish for 60 days. After the feeding trial, the fish were exposed to 100 mg L-1 ammonia-nitrogen for 48 h. The results shown that the vitamin E group significantly improved weight gain, specific growth rate, and the expression levels of growth hormone receptors and insulin-like growth factor. Fish fed with the vitamin E-supplemented diets could increase plasma alkaline phosphatase activities and decrease plasma glutamicoxalacetic transaminase and glutamic-pyruvic transaminase activities. The relative expression levels of heat shock proteins (40.23-311.64 mg kg-1 vitamin E diet group), manganese superoxide dismutase (83.64-158.93 mg kg-1 vitamin E diet group), catalase (40.23-311.64 mg kg-1 vitamin E diet group), and glutathione reductase (40.23-311.64 mg kg-1 vitamin E diet group) were upregulated. On the other hand, the decreased level of reactive oxygen species (ROS) was observed in the 83.64-311.64 mg kg-1 vitamin E additive group. These results showed that vitamin E might have a potentially useful role as an effective antioxidant to improve resistance in pufferfish.

Molecular cloning and characterization of secretory carbonic anhydrase VI in pufferfish (Takifugu rubripes).

Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish (Takifugu rubripes). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%-77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%-37% identity with mammalian and fish CA II sequences. An NH2-terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue.

Production of Tiger Puffer Takifugu rubripes Offspring from Triploid Grass Puffer Takifugu niphobles Parents.

The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2-5 kg) much larger than average market size (0.6-1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.