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Background T. fasciatus ( Takifugu fasciatus ) faces the same problem as most warm water fish: the water temperature falls far below the optimal growth temperature in winter, causing a massive death of T. fasciatus and large economic losses. Understanding of the cold-tolerance mechanisms of this species is still limited. Integrated application of multi-omics research can provide a wealth of information to help us improve our understanding of low-temperature tolerance in fish. Results To gain a comprehensive and unbiased molecular understanding of cold-tolerance in T. fasciatus , we characterized mRNA-seq and metabolomics of T. fasciatus livers using Illumina HiSeq 2500 and UHPLC-Q-TOF MS. We identified 2544 up-regulated and 2622 down-regulated genes in the liver of T. fasciatus . A total of 40 differential metabolites were identified, including 9 down-regulated and 31 up-regulated metabolites. In combination with previous studies on proteomics, we have established an mRNA-protein-metabolite interaction network. There are 17 DEMs (differentially-expressed metabolites) and 14 DEGs-DEPs (differentially co-expressed genes and proteins) in the interaction network that are mainly involved in fatty acids metabolism, membrane transport, signal transduction, and DNA damage and defense. We then validated a number of genes in the interaction network by qRT-PCR. Additionally, a number of SNPs (single nucleotide polymorphisms) were revealed through the transcriptome data. These results provide key information for further understanding of the molecular mechanisms of T. fasciatus under cold stress. Conclusion The data generated by integrated application of multi-omics can facilitate our understanding of the molecular mechanisms of fish response to low temperature stress. We have not only identified potential genes and SNPs involved in cold tolerance, but also show that some nutrient metabolites may be added to the diet to help the overwintering of T. fasciatus .

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.

Tetrodotoxin (TTX) is a potent neurotoxin that accumulates in Takifugu rubripes , commonly known as pufferfish, through the ingestion of TTX-bearing organisms as part of their food chain. Although researchers believe that pufferfish use TTX to relieve stress, data are not currently available on how TTX affects the gut microbiota of pufferfish. To address this gap, our study aimed to investigate whether administering TTX to fish could alter their gut microbiota and overall health under various salinity conditions, including 30.0 ppt, 8.5 ppt, and 1.7 ppt salinity, which represent full-strength, isosmotic, and low-salinity stress, respectively. We analyzed the effect of TTX ingestion on the community structure, core microbiome, and metabolic capabilities of the gut microbiome using high-throughput sequencing technologies. The predominant bacterial taxa within the gut microbiome were Firmicutes (21–85%), Campilobacterota (2.8–67%), Spirochaetota (0.5–14%), and Proteobacteria (0.7–9.8%), with Mycoplasma , uncultured Arcobacteraceae , Brevinema , Vibrio , Rubritalea, and uncultured Pirellulaceae as core genera. Our findings indicated that the impact of TTX on high-abundance genera at 30.0 ppt and 8.5 ppt salinity levels was negligible, indicating their stability and resilience to TTX ingestion. However, at 1.7 ppt, TTX-fed fish showed a significant increase in uncultured Arcobacteraceae. Furthermore, our analysis of TTX-fed fish revealed taxonomic alterations in low-abundance taxa, which altered the predicted functions of the gut microbiota at all salinity levels. These results suggest that TTX administration could cause subtle effects on the metabolic functions of gut microbial communities. Overall, our study provides insights into the complex relationship between a TTX-accumulating animal, T. rubripes , and its gut microbiota .

The potent toxicity of tetrodotoxin (TTX) has long constrained sustainable growth in pufferfish aquaculture. Takifugu bimaculatus, an economically important species farmed along the coast of Fujian, China, remains poorly understood regarding how it transports and accumulates this potent neurotoxin. To address this gap, we combined transcriptomic and proteomic analyses to characterize the molecular responses of T. bimaculatus to TTX exposure. After oral administration, TTX primarily accumulated in the liver, ovaries, and skin. Multi-omics profiling revealed 163 differentially expressed genes (DEGs) and 88 differentially expressed proteins (DEPs) in liver tissue, together with 239 DEGs and 179 DEPs in ovarian tissue. KEGG pathway analysis suggests that the liver maintains homeostasis by regulating ion concentrations and restructuring lipid raft architectures, alongside coordinated carrier protein activity. This likely supports active TTX uptake and directed transport toward the ovaries and skin, followed by metabolic clearance. By contrast, ovarian tissues appear to establish a stable, long-term reservoir through cytoskeletal remodeling, enhanced interactions with the extracellular matrix, and activated endocytic pathways. Together, these findings offer insights into how T. bimaculatus accumulates and transports TTX, laying groundwork for identifying key transporter genes, clarifying TTX metabolic pathways, and developing practical food safety controls.

Tetrodotoxin (TTX) is a potent marine neurotoxin found in pufferfish, causing severe poisoning or death if consumed improperly. Studies have indicated that intestinal symbiotic microbiota are associated with the production and accumulation of TTX in pufferfish. However, the specific symbiotic microorganisms involved in these processes and their respective functions remain unclear. This study explored differences in intestinal microbiota related to the TTX content between toxic and non-toxic tiger puffer Takifugu rubripes. We found that the dominant phyla exhibiting significant abundance differences between the two groups were Proteobacteria and Bacteroidota, with the core bacterial genera being Rikenella, Vibrio, Photobacterium, and Bacteroides. Moreover, the genera Marinimicrobium, Idomarina, Galbibacter, and Brumimicrobium were reported for the first time to be potentially associated with TTX bioaccumulation in T. rubripes. In addition, an integrated analysis with our previous study indicated that the “ABC transporters” pathway may play significant roles in the production and transport of TTX in both symbiotic microorganisms and T. rubripes. This study preliminarily investigated the intestinal symbiotic bacteria associated with the accumulation and metabolism of TTX in T. rubripes, as well as screening potential microbial biomarkers for assessing the safety of pufferfish.

Taurine (TAU) plays important roles in the metabolism of bile acids, cholesterol and lipids. However, little relevant information has been available in fish where TAU has been identified as a conditionally essential nutrient. The present study aimed to investigate the effects of dietary TAU on the metabolism of bile acids, cholesterol and lipids in tiger puffer, which is both an important aquaculture species and a good research model, having a unique lipid storage pattern. An 8-week feeding trial was conducted in a flow-through seawater system. Three experimental diets differed only in TAU level, that is, 1·7, 8·2 and 14·0 mg/kg. TAU supplementation increased the total bile acid content in liver but decreased the content in serum. TAU supplementation also increased the contents of total cholesterol and HDL-cholesterol in both liver and serum. The hepatic bile acid profile mainly includes taurocholic acid (94·48 %), taurochenodeoxycholic acid (4·17 %) and taurodeoxycholic acid (1·35 %), and the contents of all these conjugated bile acids were increased by dietary TAU. The hepatic lipidomics analysis showed that TAU tended to decrease the abundance of individual phospholipids and increase those of some individual TAG and ceramides. The hepatic mRNA expression study showed that TAU stimulated the biosynthesis of both bile acids and cholesterol, possibly via regulation of farnesoid X receptor and HDL metabolism. TAU also stimulated the hepatic expression of lipogenic genes. In conclusion, dietary TAU stimulated the hepatic biosynthesis of both bile acids and cholesterol and tended to regulate lipid metabolism in multiple ways.

Tetrodotoxin (TTX), known as pufferfish toxin, is a potent neurotoxin blocking sodium channels in muscle and nerve tissues. TTX has been detected in various taxa other than pufferfish, including marine polyclad flatworms, suggesting that pufferfish toxin accumulates in fish bodies via food webs. The composition of TTX and its analogs in the flatworm Planocera multitentaculata was identical to those in wild grass puffer Takifugu alboplumbeus. Previously, Planocera sp. from Okinawa Island, Japan, were reported to possess high level of TTX, but no information was available on TTX analogs in this species. Here we identified TTX and analogs in the planocerid flatworm using high-resolution liquid chromatography–mass spectrometry, and compared the composition of TTX and analogs with those of another toxic and non-toxic planocerid species. We show that the composition of TTX and several analogs, such as 5,6,11-trideoxyTTX, dideoxyTTXs, deoxyTTXs, and 11-norTTX-6(S)-ol, of Planocera sp. was identical to those of toxic species, but not to its non-toxic counterpart. The difference in the toxin composition was reflected in the phylogenetic relationship based on the mitochondrial genome sequence. A toxification experiment using predatory fish and egg plates of P. multitentaculata demonstrated that the composition of TTX and analogs in wild T. alboplumbeus juveniles was reproduced in artificially toxified pufferfish. Additionally, feeding on the flatworm egg plates enhanced the signal intensities of all TTX compounds in Chelonodon patoca and that of deoxyTTXs in Yongeichthys criniger.

Pufferfish of the genus Takifugu possess tetrodotoxin (TTX), known as “pufferfish toxin” and it is believed that pufferfish eggs and newly hatched larvae utilize TTX as a defensive substance against predators. However, the mechanism for the placement of TTX to specific cells on the larval body surface during the developmental process remains unknown. In this study, we clarify the distribution and characteristics of TTX-rich cells. We performed whole-mount immunohistochemistry (IHC) using anti-TTX monoclonal antibody on larvae of two pufferfish species, Takifugu rubripes and Takifugu alboplumbeus, just after hatching. This allowed observation of the TTX location and compared it with those of wheat germ agglutinin (WGA)-positive (periodic acid-Schiff (PAS)-positive) cells for mucous cells and IHC using anti-Na+/K+-ATPase (NKA) monoclonal antibody for ionocytes. As a result, uniformly scattered localization of TTX-rich cells was commonly observed in the epidermis of the larvae of the two Takifugu species. TTX-rich cells were WGA-negative (PAS-negative) and structurally distinct from NKA-positive cells, suggesting that TTX-rich cells are unreported small cells unique to pufferfish skin, but not mucous cells nor ionocytes.

Pufferfish are considered a culinary delicacy but require careful preparation to avoid ingestion of the highly toxic tetrodotoxin (TTX), which accumulates in certain tissues. In this study, the tissue distribution of peroxiredoxin-1 from Takifugu bimaculatus was investigated. The peroxiredoxin-1 protein was obtained by in vitro recombinant expression and purification. The recombinant protein had a strong ability to scavenge hydroxyl radicals, protect superhelical DNA plasmids from oxidative damage, and protect L929 cells from H2O2 toxicity through in vitro antioxidant activity. In addition, we verified its ability to bind to tetrodotoxin using surface plasmon resonance techniques. Further, recombinant proteins were found to facilitate the entry of tetrodotoxin into cells. Through these analyses, we identified, for the first time, peroxiredoxin-1 protein from Takifugu bimaculatus as a potential novel tetrodotoxin-binding protein. Our findings provide a basis for further exploration of the application of peroxiredoxin-1 protein and the molecular mechanisms of tetrodotoxin enrichment in pufferfish.

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 .