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.

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.

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.

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.

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.

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.

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.

Identification of two SNPs in myostatin (MSTN) gene of Takifugu rubripes and their association with growth traits.

The myostatin (MSTN) is a member of transforming growth factor-ß superfamily which inhibits muscle growth. In this study, the genomic DNA sequence of MSTN gene was cloned from Takifugu rubripes (T. rubripes). Two polymorphisms of the MSTN gene were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods in 296 T. rubripes. One A748G locates in exon 2 and the other, C1197T, in intron 2. Analysis showed that the A748G mutation caused an amino acid change from Thr to Ala (Ala166Glu). These two SNPs showed a low degree of linkage disequilibrium and four haplotypes were identified. The most frequent haplotype was AC, which occurred at a frequency of 44.3%. Association analyses between these two SNPs and growth traits showed that the individuals with genotype CT and TT of the mutation C1197T had significantly higher body mass, body length and body height than those with genotype CC (P < 0.05). These results show that MSTN gene can be utilized as a candidate gene for molecular marker-assisted breeding of T. rubripes.

Effects of dietary amylose/amylopectin ratio on growth performance, feed utilization, digestive enzymes, and postprandial metabolic responses in juvenile obscure puffer Takifugu obscurus.

The effect of dietary amylose/amylopectin (AM/AP) ratio on growth, feed utilization, digestive enzyme activities, plasma parameters, and postprandial blood glucose responses was evaluated in juvenile obscure puffer, Takifugu obscurus. Five isonitrogenous (430 g kg(-1) crude protein) and isolipidic (90 g kg(-1) crude lipid) diets containing an equal starch level (250 g kg(-1) starch) with different AM/AP ratio diets of 0/25, 3/22, 6/19, 9/16 and 12/13 were formulated. Each experimental diet was fed to triplicate groups (25 fish per tank), twice daily during a period of 60 days. After the growth trial, a postprandial blood response test was carried out. Fish fed diet 6/19 showed best growth, feed efficiency and protein efficiency ratio. Hepatosomatic index, plasma total cholesterol concentration, liver glycogen and lipid content, and gluconokinase, pyruvate kinase and fructose-1,6-bisphosphatase activities were lower in fish fed highest AM/AP diet (12/13) than in fish fed the low-amylose diets. Activities of liver and intestinal trypsin in fish fed diet 3/22 and diet 6/19 were higher than in fish fed diet 9/16 and diet 12/13. Activities of liver and intestinal amylase and intestinal lipase, and starch digestibility were negatively correlated with dietary AM/AP ratio. Fish fed diet 3/22 and diet 6/19 showed higher plasma total amino acid concentration than fish fed the other diets, while plasma urea nitrogen concentration and activities of alanine aminotransferase and aspartate aminotransferase showed the opposite trend. Equal values were found for viscerosomatic index and condition factor, whole body and muscle composition, plasma high-density and low-density lipoprotein cholesterol concentrations, and activities of lipase and hexokinase and glucose-6-phosphatase in liver. Postprandial plasma glucose and triglyceride peak value of fish fed diet 12/13 were lower than in fish fed the low-amylose diets, and the peak time of plasma glucose was later than in fish fed the other diets. Plasma glucose and triglyceride concentrations showed a significant difference at 2 and 4 h after a meal and varied between dietary treatments. According to regression analysis of weight gain against dietary AM/AP ratio, the optimum dietary AM/AP ratio for maximum growth of obscure puffer was 0.25. The present result indicates that dietary AM/AP ratio could affect growth performance and feed utilization, some plasma parameters, digestive enzyme as well as hepatic glucose metabolic enzyme activities in juvenile obscure puffer.

Correlation with larval body size of mRNA levels of growth hormone, growth hormone receptor I and insulin-like growth factor I in larval torafugu Takifugu rubripes.

The full-length of insulin-like growth factor (IGF) complementary (c)DNAs encoded by igf-I and igf-II from torafugu pufferfish Takifugu rubripes were cloned in the present study. The deduced amino acid sequences of the two genes showed c. 80% identity each with those of Igf-I and Igf-II from other teleosts, respectively. Two growth hormone (GH) receptors, ghr1 and ghr2, were also cloned in silico using the T. rubripes Fugu genome database. The transcripts of T. rubripes igf-I were detected in slow muscle, heart, skin, gill, liver and intestine but not in fast muscle, spleen and testis of adult fish, whereas those of igf-II were found in all tissues examined. Subsequently, the accumulated messenger (m)RNA levels of igf-I and igf-II were investigated in an F(2) population derived from a male of an apparent fast-growing T. rubripes strain and a wild female T. rubripes together with those of other growth-related genes encoding Gh, Ghr1 and Ghr2, and with those of prolactin (Prl) and leptin (Lep) previously reported. The accumulated mRNA levels of igf-I, gh and ghr1 were significantly correlated to growth rate at larval stages in the population, but not for those of igf-II, prl, ghr2 and lep. Although it is unclear whether or not this phenotype is directly related to the heredity of the fast-growing strain, the findings suggest that the expression of igf-I, gh and ghr1 is involved in the regulation of growth rate at larval stages in T. rubripes.

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.

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.

Ontogenetic phase shifts in metabolism: links to development and anti-predator adaptation.

The allometric relationships between resting metabolism (VO(2)) and body mass (M), VO(2) = a(i)M(b), are considered a fundamental law of nature. A distinction though needs to be made between the ontogeny (within a species) and phylogeny (among species) of metabolism. However, the nature and significance of the intraspecific allometry (ontogeny of metabolism) have not been established in fishes. In this study, we present experimental evidence that a puffer fish ranging 0.0008-3 g in wet body mass has four distinct allometric phases in which three stepwise increases in scaling constants (a(i), i = 1-4), i.e. ontogenetic phase shifts in metabolism, occur with growth during its early life stages at around 0.002, 0.01 and 0.1 g, keeping each scaling exponent constant in each phase (b = 0.795). Three stepwise increases in a(i) accompanied behavioural and morphological changes and three peaks of severe cannibalism, in which the majority of predation occurred on smaller fish that had a lower value of a(i). Though fishes are generally highly fecund, producing a large number of small eggs, their survivability is very low. These results suggest that individuals with the ability to rapidly grow and step up 'a(i)' develop more anti-predator adaptation as a result of the decreased predatory risk.

Comparative proteomics of kidney samples from puffer fish Takifugu rubripes exposed to excessive fluoride: an insight into molecular response to fluorosis.

To investigate comparative proteomics of the pufferfish kidney exposed to excessive fluoride, the authors randomly put 16 fish into the control and treated groups that were raised in softwater alone (F(-) = 0.4 mg/L) or with sodium fluoride of 35 mg/L for 3 days, respectively. Then proteins of the fish kidneys were profiled by two-dimensional electrophoresis, and the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) was applied to identify the spots on the gel with altered densities. On average, 547 and 516 protein spots were detected in the control and the treated groups, respectively. Among them, 32 protein spots showed significant alteration (p < 0.05) between the fluoride-treated and the control groups, and 22 differentially expressed protein spots were identified by MALDI TOF-TOF MS. Consistent with their previously annotated functions, these proteins appear to be involved in the biological functions associated with fluorosis. These results will greatly advance one's understanding of the effects of fluoride exposure on the physiological and biochemical functions of takifugu kidney as well as the toxicological mechanism of fluoride-causing fluorosis in both fish and humans.

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.

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.

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.

Estradiol-17beta treatment induces intersexual gonadal development in the pufferfish, Takifugu rubripes.

Estrogens are responsible for most characteristics of the female sex of a species, such as metabolic, behavioral, and morphological changes during reproduction. Artificial estradiol-17beta (E2) treatment Induces sex reversal in some fish. The Japanese pufferfish (Takifugu rubripes) has the most compact genome among vertebrates and great pottial for comparative genome analysis. In this paper, we describe the Influence of E2 treatment during gonadal development in the pufferfish. After hatching, fry were treated with no (control) or a 0.1, 1, 10, or 100 microg/g diet from 21 to 80 days after hatching (dah). Doublesex-mab3-related transcription factor (DMRT1) is Involved in testicular development. VASA is responsible for germ cell development, and CYP19A plays a role in E2 biosynthesis during ovarian development across animal phyla as well as in gonadal morphology after E2 treatment. DMRT1, VASA, and CYP19A were Investigated in the gonads of E2-treated pufferfish. Fish fed with the highest dose (E2 100 microg/g diet) developed Intersexual gonads in the testis; the majority of germ cells were oocytes, but some spermatocytes were detected. RT-PCR results showed the expression of VASA and CYP19A in all intersexual gonads and DMRT1 in some. Furthermore, abnormalities in the epithelium-tunica layer were detected, and gonadal somatic cells (e.g., granulosa cells, theca cells, or germinal epithelium) proliferated extensively in the intersexual gonad. These results suggest that E2 treatment Induces ovarian development in the bipotential gonads of genetic males by modification of gonadal somatic cells and E2 production, mediated by CYP19A.

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.