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Sexual maturity in tiger pufferfish ( ) consumes substantial metabolic energy, constraining somatic growth and limiting meat yield. Artificial triploid induction (sterility) may redirect energy toward somatic growth. Cold-shock induced triploids were compared with diploid controls using muscle and liver tissues, and the phenotype, histology, nutritional composition, hormone levels, as well as transcriptome profiles were assessed. After 5 months, triploids attained significantly greater body length and body weight, with larger muscle fiber diameter but lower fiber density. The triploids yielded lower ash content and higher amounts of total (TAA), essential (EAA), non-essential (NEAA), and delicious amino acids (DAA), as well as higher total fatty acids (TFA), total polyunsaturated fatty acids (PUFA), and omega 3 polyunsaturated fatty acids (n-3 PUFA). Growth-related hormones were elevated, including growth hormone (GH), testosterone (T), triiodothyronine (T ), and thyroxine (T ). Muscle transcriptome sequencing identified 231 differentially expressed genes, predominantly enriched in pathways related to cell growth regulation, metabolic processes, and energy conversion. These results provide reference data for breeding programs.

Background Takifugu rubripes is an economically valuable fish species in Asia. The implementation of all-male culture for T. rubripes is highly anticipated in aquaculture. Aromatase inhibitor (AI, letrozole) treatment was found to be an efficient method to induced masculinization in T. rubripes , as reported in our previous study. Here, to further explore the underlying regulation mechanism of AI-induced masculinization, a whole-transcriptome analysis comparing was conducted between AI-induced masculinized XX (AI-XX) gonads and control (Con) gonads in T. rubripes . Results In Con-XX/Con-XY comparison, 1,172 differential expression (DE) mRNAs, 129 DEmiRNAs, 210 DElncRNAs, and 4 DEcircRNAs were identified. In the Con-XX/AI-XX comparison, 1,329 DEmRNAs, 174 DEmiRNAs, 6 DEcircRNAs and 280 DElncRNAs were found. Con-XX/Con-XY and Con-XX/AI-XX comparisons shared 690 DEmRNAs, 50 DEmiRNAs, 3 DEcircRNAs, and 105 DElncRNAs. The analyses of protein-protein interaction (PPI) and competitive endogenous RNA (ceRNA) network identified interactions among these shared DERNAs. Kcnh2b , trim27 , cnnm2b , reln , cckbra , pkd1l2 , steap4 , gsg1l , hamp , and foxg1c were predicted as the top ten of hub genes. miRNAs included miRNA-27 family and miRNA-489 family showed targeting relationship with hub genes. GO and KEGG functional enrichment analysis showed that the targeted genes were mainly enriched in GO:0065008 regulation of biological quality and TGF-beta signaling pathway. qPCR validation confirmed the differential expression of selected mRNAs, and ncRNAs. Conclusions This research comprehensively reveals the potential regulatory effects of ncRNAs on cellular motility, fate regulation, and hormonal regulation during gonadal masculinization in T. rubripes . It may provide significant insights into the regulation mechanisms underlying sex reversal in fish.

To identify candidate genes and pathways involved in testicular development in Takifugu rubripes , a comparative transcription analysis was conducted across the various developmental stages of the testis (stages II to V). A total of 9520 differentially expressed genes (DEGs) were identified among the different stages, and they were significantly clustered into six clusters ( P  < 0.05). One thousand four hundred eleven DEGs such as gndf , wnt1 , and cyp17b1 were found to be decreased from stage II to V. In contrast, 994 DEGs such as fn1 , ift81 , and cdc25a were found to be increased from stage II to V. Six thousand three hundred eighteen DEGs (e.g., dmrt1 , sdk2 , and chrna1 ) were identified as being expressed at similar levels at stages II and III. However, they were subsequently found to be decreased from stage III to IV. Four hundred one DEGs exhibited a significant upregulation trend from stage II to III. These genes were expressed at similar levels in stages III, IV, and V, including chrnd , wnt4a , and cyp7a1 . The highest expression levels of 200 DEGs (e.g., ccnb2 , cdk1 , and sycp2 ) were observed in stage IV, while 196 DEGs (e.g., chmp1b , hsd17b3 , and zp3 ) exhibited the highest expression level in stage III. Those DEGs were mainly enriched in the pathways (e.g., neuroactive ligand-receptor interaction, cell adhesion molecules, and calcium signaling pathways) associated with testicular development. Quantitative polymerase chain reaction of eight randomly selected genes validated the RNA sequencing results. This study may provide new insights into the molecular regulatory mechanisms governing testicular development and spermatogenesis in T. rubripes .

Background The regulatory roles of non-coding RNAs (ncRNAs) during sex differentiation in teleosts have received widespread attention recently. Mifepristone (RU486, a progesterone antagonist), which acts as an endocrine disruptor, can affect reproduction and sex differentiation in teleosts. Results The expression of ncRNAs in the gonads of tiger puffer ( Takifugu rubripes ) during RU486 (500 µg/g diet) induced masculinization process was examined by RNA-sequencing. A total of 4,381 long non-coding RNAs (lncRNAs), 309 circular RNAs (circRNAs), and 1,020 microRNAs (miRNAs) were identified. The expression of 41 differentially expressed (DE) lncRNAs and 20 DE miRNAs, which showed sexual dimorphic expression patterns in genetic female gonads in the control group (C-XX) vs. genetic male gonads in the control group (C-XY), were altered in genetic female gonads in the RU486 treated group (RU-XX). The genes targeted by DE ncRNAs were mainly enriched in sex-related pathways, such as calcium signaling, ovarian steroidogenesis, and cortisol synthesis and secretion. The results of co-expression and competing endogenous RNA (ceRNA) network analysis indicated that miRNAs (e.g., miR-205-z and fru-miR-122) and lncRNAs (including XR_003890915.1 and XR_003885862.1) may have pivotal roles, and lncRNAs (including XR_003890295.1, MSTRG.11750.1, and XR_003888827.1) may act as miRNA sponges, involved in the competition between miRNAs and sex-related genes during tiger puffer masculinization process. Dual luciferase reporter assay results identified that ovarian steroidogenesis related gene hsd17b1 is a downstream target of fru-miR-122. The expression of 4 lncRNAs, 4 circRNAs, and 6 miRNAs were validated by qPCR, indicating the accuracy and dependability of RNA-Seq. Conclusions This study provided the evidence that ncRNAs may participate in RU486-induced masculinization in T. rubripes , and may enhance our understanding of the regulatory network of sex differentiation in fugu.

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 ˂ 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 ˂ 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.

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.

The allometric relationships between resting metabolism (VO2) and body mass (M), VO2 = aiMb, 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 (ai, 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 ai 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 ai. 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 ‘ai’ develop more anti-predator adaptation as a result of the decreased predatory risk.

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.

Closely related species have often evolved dramatic differences in body size. Takifugu rubripes (fugu) is a large marine pufferfish whose genome has been sequenced, whereas T. niphobles is the smallest species among Takifugu. We show that, unsurprisingly, the juvenile growth rate of T. rubripes is higher than that of T. niphobles in a laboratory setting. We produced F 2 progenies of their F 1 hybrids and found one quantitative trait locus (QTL) significantly associated with variation in juvenile body size. This QTL region (3.5 Mb) contains no known genes directly related to growth phenotype (such as IGF s ) except Fgf21, which inhibits growth hormone signaling in mouse. The QTL in Takifugu spp. is distinct from the region previously known to control body size variations in stickleback or tilapia. Our results suggest that in the fish tested herein, genomic regions underlying body size evolution might have different genetic origins. They also suggest that many diverse traits in Takifugu spp. are amenable to genetic mapping.

Myosin heavy chain (MYH) genes belong to a multigene family, and the regulated expression of each member determines the physiological and contractile muscle properties. Among these, MYH6 , MYH7 , and MYH14 occupy unique positions in the mammalian MYH gene family because of their specific expression in slow/cardiac muscles and the existence of intronic micro(mi) RNAs. MYH6 , MYH7 , and MYH14 encode miR-208a, miR-208b, and miR-499, respectively. These MYH encoded miRNAs are designated as myomiRs because of their muscle-specific expression and functions. In mammals, myomiRs and host MYHs form a transcription network involved in muscle fiber-type specification; thus, genomic positions and expression patterns of them are well conserved. However, our previous studies revealed divergent distribution and expression of MYH14 /miR-499 among teleosts, suggesting the unique evolution of myomiRs and host MYHs in teleosts. Here, we examined distribution and expression of myomiRs and host MYHs in various teleost species. The major cardiac MYH isoforms in teleosts are an intronless gene, atrial myosin heavy chain ( amhc ), and ventricular myosin heavy chain ( vmhc ) gene that encodes an intronic miRNA, miR-736. Phylogenetic analysis revealed that vmhc /miR-736 is a teleost-specific myomiR that differed from tetrapoda MYH6 / MYH7 /miR-208s. Teleost genomes also contain species-specific orthologs in addition to vmhc and amhc , indicating complex gene duplication and gene loss events during teleost evolution. In medaka and torafugu, miR-499 was highly expressed in slow/cardiac muscles whereas the expression of miR-736 was quite low and not muscle specific. These results suggest functional diversification of myomiRs in teleost with the diversification of host MYHs .