Cells and Fugu Response to Capsid of BFNNV Genotype.

The nervous necrosis virus (NNV) of the BFNNV genotype is the causative agent of viral encephalopathy and retinopathy (VER) in cold water fishes. Similar to the RGNNV genotype, BFNNV is also considered a highly destructive virus. In the present study, the RNA2 of the BFNNV genotype was modified and expressed in the EPC cell line. The subcellular localization results showed that the capsid and N-terminal (1-414) were located in the nucleus, while the C-terminal (415-1014) of the capsid was located in the cytoplasm. Meanwhile, cell mortality obviously increased after expression of the capsid in EPC. EPC cells were transfected with pEGFP-CP and sampled at 12 h, 24 h and 48 h for transcriptome sequencing. There are 254, 2997 and 229 up-regulated genes and 387, 1611, and 649 down-regulated genes post-transfection, respectively. The ubiquitin-activating enzyme and ubiquitin-conjugating enzyme were up-regulated in the DEGs, indicating that cell death evoked by capsid transfection may be related to ubiquitination. The qPCR results showed that heat stock protein 70 (HSP70) is extremely up-regulated after expression of BFNNV capsid in EPC, and N-terminal is the key region to evoke the high expression. For further study, the immunoregulation of the capsid in fish pcDNA-3.1-CP was constructed and injected into the Takifugu rubripes muscle. pcDNA-3.1-CP can be detected in gills, muscle and head kidney, and lasted for more than 70 d post-injection. The transcripts of IgM and interferon inducible gene Mx were up-regulated after being immunized in different tissues, and immune factors, such as IFN-γ and C3, were also up-regulated in serum, while C4 was down-regulated one week after injection. It was suggested that pcDNA-3.1-CP can be a potential DNA vaccine in stimulating the immune system of T. rubripes; however, NNV challenge needs to be conducted in the following experiments.

Levels of Tetrodotoxins in Spawning Pufferfish, Takifugu alboplumbeus.

Tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin thought to be used as a biological defense compound in organisms bearing it. Although TTX was thought to function as a chemical agent for defense and anti-predation and an attractant for TTX-bearing animals including pufferfish, it has recently been demonstrated that pufferfish were also attracted to 5,6,11-trideoxyTTX, a related compound, rather than TTX alone. In this study, we attempted to estimate the roles of TTXs (TTX and 5,6,11-trideoxyTTX) in the pufferfish, Takifugu alboplumbeus, through examining the location of TTXs in various tissues of spawning pufferfish from Enoshima and Kamogawa, Japan. TTXs levels in the Kamogawa population were higher than those in the Enoshima population, and there was no significant difference in the amount of TTXs between the sexes in either population. Individual differences were greater in females than in males. However, the location of both substances in tissues differed significantly between sexes: male pufferfish accumulated most of their TTX in the skin and liver and most of their 5,6,11-trideoxyTTX in the skin, whereas females accumulated most of their TTX and 5,6,11-trideoxyTTX in the ovaries and skin.

Proteomics and Phosphoproteomic Analysis to Identify Spleen of Takifugu rubripes Infected Cryptocaryon irritans.

Takifugu rubripes is important commercially fish species in China and it is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. In this study, we used proteomics and phosphoproteomic analysis to identify differentially abundant proteins in the spleen of T. rubripes infected with the Cryptocaryon irritans. We identified 5,307 proteins and 6,644 phosphorylated sites on 2,815 phosphoproteins using high-throughput proteomics analysis of the spleen of T. rubripes based on 26,421 unique peptides and 5,013 modified peptides, respectively. The 5,307 quantified host proteins, 40 were upregulated and 43 were downregulated in the infection group compared to the control group. Among the 2815 phosphoproteins, 44/120 were upregulated/downregulated, and 62/151 were upregulated/downregulated in the 6644 quantified phosphosites. Using the combination of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, screening for significantly different phosphoproteins, motif analysis and protein-protein interaction analysis, we ultimately identified three phosphorylated proteins (G-protein-signaling modulator 1-like, zinc finger protein 850-like, and histone H1-like) and three phosphorylated protein kinases (serine/threonine-protein kinase homolog isoform X2, mitogen-activated protein kinase 5-like, and protein kinase C theta type) as potential biomarkers for T. rubripes immune responses. We then screened the phosphorylation sites of these biomarker proteins for further verification. Based on our results, we speculate that phosphorylation modification of the phosphorylation sites is involved in the immunity of T. rubripes against C. irritans.

Host Hybridization Dominates over Cohabitation in Affecting Gut Microbiota of Intrageneric Hybrid Takifugu Pufferfish.

Microbial symbionts are of great importance for macroscopic life, including fish, and both collectively comprise an integrated biological entity known as the holobiont. Yet little is known as to how the normal balance within the fish holobiont is maintained and how it responds to biotic and/or abiotic influences. Here, through amplicon profiling, the genealogical relationship between artificial F1 hybrid pufferfish with growth heterosis, produced from crossing female Takifugu obscurus with male Takifugu rubripes and its maternal halfsibling purebred, was well recapitulated by their gut microbial community similarities, indicating an evident parallelism between host phylogeny (hybridity) and microbiota relationships therein. Interestingly, modest yet significant fish growth promotion and gut microbiota alteration mediated by hybrid-purebred cohabitation were observed, in comparison with their respective monoculture cohorts that share common genetic makeups, implying a certain degree of environmental influences. Moreover, the underlying assemblage patterns of gut microbial communities were found associated with a trade-off between variable selection and dispersal limitation, which are plausibly driven by the augmented social interactions between hybrid and purebred cohabitants differing in behaviors. Results from this study not only can enrich, from a microbial perspective, the sophisticated understanding of complex and dynamic assemblage of the fish holobiont, but will also provide deeper insights into the ecophysiological factors imposed on the diversity-function relationships thereof. Our findings emphasize the intimate associations of gut microbiota in host genetics-environmental interactions and would have deeper practical implications for microbial contributions to optimize performance prediction and to improve the production of farmed fishes. IMPORTANCE Microbial symbionts are of great importance for macroscopic life, including fish, and yet little is known as to how the normal balance within the fish holobiont is maintained and how it responds to the biotic and/or abiotic influences. Through gut microbiota profiling, we show that host intrageneric hybridization and cohabitation can impose a strong disturbance upon pufferfish gut microbiota. Moreover, marked alterations in the composition and function of gut microbiota in both hybrid and purebred pufferfish cohabitants were observed, which are potentially correlated with different metabolic priorities and behaviors between host genealogy. These results can enrich, from a microbial perspective, the sophisticated understanding of the complex and dynamic assemblage of the fish holobiont and would have deeper practical implications for microbial contributions to optimize performance prediction and to improve farmed fish production.

Salinity Moderated the Toxicity of Zinc Oxide Nanoparticles (ZnO NPs) towards the Early Development of Takifugu obscurus.

ZnO nanoparticles (ZnO NPs) have been applied in a wide range of fields due to their unique properties. However, their ecotoxicological threats are reorganized after being discharged. Their toxic effect on anadromous fish could be complicated due to the salinity fluctuations during migration between freshwater and brackish water. In this study, the combined impact of ZnO NPs and salinity on the early development of a typical anadromous fish, obscure puffer (Takifugu obscurus), was evaluated by (i) observation of the nanoparticle characterization in salt solution; (ii) quantification of the toxicity to embryos, newly hatched larvae, and larvae; and (iii) toxicological analysis using biomarkers. It is indicated that with increased salinity level in brackish water (10 ppt), the toxicity of ZnO NPs decreased due to reduced dissolved Zn2+ content, leading to higher hatch rate of embryos and survival rate of larvae than in freshwater (0 ppt). The irregular antioxidant enzyme activity changes are attributed to the toxic effects of nanoparticles on CAT (catalase), but further determination is required. The results of present study have the significance to guide the wildlife conservation of Takifugu obscurus population.

Recovery of Fatty Acid and Volatile Flavor Compound Composition in Farmed Tiger Puffer (Takifugu rubripes) with a Fish Oil-Finishing Strategy.

Booming fish farming results in a relative shortage of fish oil (FO) supply, meaning that alternative oils are increasingly used in fish feeds, which leads to reduction of long-chain polyunsaturated fatty acids (LC-PUFAs) and other relevant changes in fish products. This study investigated the efficacy of an FO-finishing strategy in recovering the muscle quality of farmed tiger puffer. An eight-week feeding trial (growing-out period) was conducted with five experimental diets, in which graded levels (0 (control), 25, 50, 75, and 100%) of added FO were replaced by poultry oil (PO). Following the growing-out period was a four-week FO-finishing period, during which fish in all groups were fed the control diet. Dietary PO significantly decreased the muscle LC-PUFA content, whereas in general, the FO-finishing strategy recovered it to a level comparable with that of the group fed FO continuously. The recovery efficiency of EPA was higher than that of DHA. Dietary PO also led to changes of volatile flavor compounds in the muscle, such as butanol, pentenal, and hexenal, whereas the FO-finishing strategy mitigated the changes. In conclusion, the FO-finishing strategy is promising in recovering the LC-PUFA and volatile-flavor-compound composition in farmed tiger puffer after the feeding of PO-based diets.

Chromosome-level haplotype-resolved genome assembly for Takifugu ocellatus using PacBio and Hi-C technologies.

Takifugu species serve as a model system for evolutionary studies due to their compact genomes and diverse phenotypes. The ocellated puffer (Takifugu ocellatus), characterized by special colouration, is a scarce anadromous species in the genus Takifugu. As an ornamental and tasty fish species, T. ocellatus has moderate economic value. However, the available genomic resources for this pufferfish are still limited. Here, a chromosome-level reference genome, as well as two haploid genomes, was constructed by PacBio HiFi long sequencing and Hi-C technologies. The total length of the reference genome was 375.62 Mb with a contig N50 of 11.55 Mb. The assembled sequences were anchored to 22 chromosomes with an integration efficiency of 93.78%. Furthermore, 28,808 protein-coding genes were predicted. The haplotype-resolved reference genome of T. ocellatus provides a crucial resource for investigating the explosive speciation of the Takifugu genus, such as elucidating evolutionary histories, determining the genetic basis of trait evolution, and supporting future conservation efforts.

The variation of intestinal autochthonous bacteria in cultured tiger pufferfish Takifugu rubripes.

Intestinal autochthonous bacteria play important roles in the maintenance of the physiological homeostasis of animals, especially contributing to the host immune system. In the present study, the variation of autochthonous bacterial community in the intestinal tract of 2-7 months-old tiger pufferfish Takifugu rubripes and bacterial communities in the seawater of recirculating aquaculture system (RAS) and the following offshore sea cage aquaculture system (OSCS) were analyzed during the aquaculture period from May to October 2021. Proteobacteria was found to be the most dominant phyla in both intestinal and seawater bacterial communities, which accounted for 68.82% and 65.65% of the total bacterial abundance, respectively. Arcobacter was the most core bacterial taxon in the intestinal bacterial community, with the most dominant abundance (42.89%) at the genus level and dominant positions in co-occurrence relationships with other bacterial taxa (node-betweenness value of 150). Enterococcaceae was specifically enriched in the intestinal bacterial community of pufferfishes from RAS, while Vibrionaceae was enriched in the intestinal bacterial community from OSCS. The F-values of beta diversity analysis between intestinal and seawater bacterial communities generally increased from May (6.69) to October (32.32), indicating the increasing differences between the intestinal and seawater bacterial communities along with the aquaculture process. Four bacterial taxa of Weissella sp., Akkermansia muciniphila, Dietzia sp. and Psychrobacter pacificensis had significant correlations with immune response parameters, and they were suggested to be the indicators for immune status and pathological process of pufferfish. The knowledge about the specific core bacteria, potentially pathogenic bacteria and the change of bacterial community in the intestinal tract of cultured pufferfish is of great scientific significance and will contribute to the understanding of intestinal bacterial homeostasis and biosecurity practice in pufferfish aquaculture.

CRISPR-Based Colorimetric Nucleic Acid Tests for Visual Readout of DNA Barcode for Food Authenticity.

Food authenticity is a critical issue associated with the economy, religion, and food safety. Herein, we report a label-free and colorimetric nucleic acid assay for detecting DNA barcodes, enabling the determination of food authenticity with the naked eye. This method, termed the CRISPR-based colorimetric DNA barcoding (Cricba) assay, utilizes CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR associated protein) to specifically recognize the polymerase chain reaction (PCR) products for further trans-cleavaging the peroxidase-mimicking G-quadruplex DNAzyme. Based on this principle, the presence of the cytochrome oxidase subunit I gene could be directly observed with the naked eye via the color change of 3,3',5,5'-tetramethylbenzidine sulfate (TMB). The whole detection process, including PCR amplification and TMB colorimetric analysis, can be completed within 90 min. The proposed assay can detect pufferfish concentrations diluted to 0.1% (w/w) in a raw pufferfish mixture, making it one of the most sensitive methods for food authenticity. The robustness of the assay was verified by testing four common species of pufferfish, including Lagocephalus inermis, Lagocephalus spadiceus, Takifugu bimaculatus, and Takifugu alboplumbeus. The assay is advantageous in easy signal readout, high sensitivity, and general applicability and thus could be a competitive candidate for food authenticity.

Prostaglandin E2 synchronizes lunar-regulated beach spawning in grass puffers.

Many organisms living along the coastlines synchronize their reproduction with the lunar cycle. At the time of spring tide, thousands of grass puffers (Takifugu alboplumbeus) aggregate and vigorously tremble their bodies at the water's edge to spawn. To understand the mechanisms underlying this spectacular semilunar beach spawning, we collected the hypothalamus and pituitary from male grass puffers every week for 2 months. RNA sequencing (RNA-seq) analysis identified 125 semilunar genes, including genes crucial for reproduction (e.g., gonadotropin-releasing hormone 1 [gnrh1], luteinizing hormone ß subunit [lhb]) and receptors for pheromone prostaglandin E (PGE). PGE2 is secreted into the seawater during the spawning, and its administration activates olfactory sensory neurons and triggers trembling behavior of surrounding individuals. These results suggest that PGE2 synchronizes lunar-regulated beach-spawning behavior in grass puffers. To further explore the mechanism that regulates the lunar-synchronized transcription of semilunar genes, we searched for semilunar transcription factors. Spatial transcriptomics and multiplex fluorescent in situ hybridization showed co-localization of the semilunar transcription factor CCAAT/enhancer-binding protein δ (cebpd) and gnrh1, and cebpd induced the promoter activity of gnrh1. Taken together, our study demonstrates semilunar genes that mediate lunar-synchronized beach-spawning behavior. VIDEO ABSTRACT.

Identification of Novel Antifreeze Peptides from Takifugu obscurus Skin and Molecular Mechanism in Inhibiting Ice Crystal Growth.

Foodborne hydrolyzed antifreeze peptides have been widely used in the food industry and the biomedical field. However, the components of hydrolyzed peptides are complex and the molecular mechanism remains unclear. This study focused on identification and mechanism analysis of novel antifreeze peptides from Takifugu obscurus skin by traditional methods and computer-assisted techniques. Results showed that three peptides (EGPRAGGAPG, GDAGPSGPAGPTG, and GEAGPAGPAG) possessed cryoprotection via reducing the freezing point and inhibiting ice crystal growth. Molecular docking confirmed that the cryoprotective property was related to peptide structure, especially α-helix, and hydrogen bond sites. Moreover, the antifreeze peptides were double-faces, which controlled ice crystals while affecting the arrangement of surrounding water molecules, thus exhibiting a strong antifreeze activity. This investigation deepens the comprehension of the mechanism of antifreeze peptides at molecular scale, and the novel efficient antifreeze peptides can be developed in antifreeze materials design and applied in food industry.

Accumulation of microplastics in fugu (Takifugu bimaculatus): A comparative study between fishing grounds and aquafarms.

Microplastics (MPs) in fish have attracted attention recently, for their ecological and food safety risks. However, knowledge gaps still exist regarding MPs in fugu, a special poisonous but precious seafood, especially that accumulated in its tissues. Accordingly, this study investigated the characteristics of MPs in cultured Takifugu bimaculatus which raised on three aquafarms and in wild individuals from three fishing grounds. More than 98.85 % of T. bimaculatus were contaminated by MPs and the average MPs abundance in wild fugu (4.25 ± 2.63 items/individual) was lower than that of cultured fugu (7.91 ± 2.16 items/individual). The abundance of MPs in fugu's tissues under different life patterns shows significant differences. There were marked differences in size of MPs presented in various tissues. This study adds to the knowledge on MPs accumulation in the tissues of wild and cultured fugu, providing warnings about its transmission and ecological risks in the food chain.

An almost nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an odorant for the grass puffer.

Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this TTX analog, which has been present in their body with TTX but whose function was unknown, as an odorant for chemical communication or effective TTX accumulation.

Reduced lifetime fitness (growth, body condition and survivability) of hatchery-reared tiger pufferfish Takifugu rubripes compared to wild counterparts.

Tiger pufferfish Takifugu rubripes (order Tetraodontiformes, family Tetraodontidae) is a highly exploited species and stocks continue to decline, although hatchery-reared juveniles have been released since 1965 for stock enhancement. To determine why the stock has not recovered through hatchery-release practices, this study investigated and compared the population characteristics of wild and hatchery-origin fish. The length-mass relationship showed that hatchery-origin fish were skinnier, with males weighing less than 90% of the mass of wild males of the same length. The hepatosomatic index tended to be lower in hatchery-origin fish. Age was estimated using the otolith-based method, and the estimates were more accurate and precise than those obtained by the conventional vertebra-based method. At the age of 2.9 years, an age at which specimens were the most abundant in catches, hatchery-origin males weighed only 67% of wild males. The maximum observed age was 12 years for wild fish and 5 years for hatchery-origin fish. The instantaneous total mortality rates of hatchery-origin fish were more than twice as high as those of wild fish. In summary, the hatchery-origin fish had poor health status, poor growth and high mortality, and their fitness in natural environments was therefore hypothesized to be low throughout life.

Toxicity in Takifugu rubripes exposed to acute ammonia: Effects on immune responses, brain neurotransmitter levels, and thyroid endocrine hormones.

Exposure to ammonia can cause convulsions, coma, and death. In this study, we investigate the effects of ammonia exposure on immunoregulatory and neuroendocrine changes in Takifugu rubripes. Fish were sampled at 0, 12, 24, 48, and 96 h following exposure to different ammonia concentrations (0, 5, 50, 100, and 150 mg/L). Our results showed that exposure to ammonia significantly reduced the concentrations of C3, C4, IgM, and LZM whereas the heat shock protein 70 and 90 levels significantly increased. In addition, the transcription levels of Mn-SOD, CAT, GRx, and GR in the liver were significantly upregulated following exposure to low ammonia concertation, however, downregulated with increased exposure time. These findings suggest that ammonia poisoning causes oxidative damage and suppresses plasma immunity. Ammonia exposure also resulted in the elevation and depletion of the T3 and T4 levels, respectively. Furthermore, ammonia stress induced an increase in the corticotrophin-releasing hormone, adrenocorticotropic hormone, and cortisol levels, and a decrease in dopamine, noradrenaline, and 5-hydroxytryptamine levels in the brain, illustrating that ammonia poisoning can disrupt the endocrine and neurotransmitter systems. Our results provide insights into the mechanisms underlying the neurotoxic effects of ammonia exposure, which helps to assess the ecological and environmental health risks of this contaminant in marine fish.

In Vivo Detection of Tetrodotoxin in Takifugu obscurus Based on Solid-Phase Microextraction Coupled with Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Pufferfish is nutritious and delicious, but the tetrodotoxin (TTX) that may exist in its body poses a serious safety hazard. It is important to use scientific and effective methods to detect the TTX in pufferfish, but most of the existing methods require complex pre-treatment steps and have sample lethality. The solid-phase microextraction (SPME) technology can be used for in vivo detection due to its advantages such as no solvent demand, simple operation, and fast detection speed. In this study, the GO-PAN@PNE SPME fibers were made via a dipping method, and their extraction effect was verified in the TTX aqueous and spiked fish. The established method has good reproducibility, and the limit of detection of TTX in pufferfish was 32 ng·g-1, and the limit of quantitation was 150 ng·g-1, which can meet the detection needs of pufferfish for safe consumption. This method was used to in vivo detect the Takifugu obscurus exposed to the TTX, to determine the content of TTX in the pufferfish muscle. The detection method established in this study can relatively quickly and easily realize the in vivo detection of TTX in the pufferfish, which can provide theoretical support for improvement in the food safety level of the pufferfish.

The brain of the wild toxic marine pufferfishes accumulates tetrodotoxin.

We have previously detected tetrodotoxin (TTX) in the brain of the wild toxic torafugu Takifugu rubripes by immunohistochemistry and LC/MS analysis. We have also indicated that TTX is a stress-relieving substance in the brain and reduces agonistic interactions in torafugu juveniles. Although the toxicity of marine pufferfish in the Japanese waters has been extensively examined for food hygiene, whether wild toxic pufferfish generally possess TTX in the brain has not been investigated. In the present study, we examined the presence of TTX in the brain of several wild toxic marine pufferfishes such as kusafugu T. alboplumbeus, komonfugu T. flavipterus, shosaifugu T. snyderi, okinawafugu Chelonodontops patoca, and in wild non-toxic pufferfishes such as shirosabafugu Lagocephalus spadiceus and yoritofugu Sphoeroides pachygaster. We also examined tsumugihaze Yongeichthys criniger, known to possess TTX in the skin, viscera, and gonad. TTX was extracted from the brain, liver, skin, and muscle and was analyzed by LC/MS. TTX was detected in the brain as well as in the liver, skin, and muscle in kusafugu, komonfugu, shosaifugu, okinawafugu, and tsumugihaze. In shirosabafugu, low level of TTX (0.8 mouse unit/g-brain) was detected in the brain in 1 out of 3 individuals. In yoritofugu, no TTX was detected in any of the tissues. We conclude that the brain is also an organ that contains TTX in the wild toxic marine pufferfishes.

Transcriptome Analysis Identifies Key Metabolic Changes in the Brain of Takifugu rubripes in Response to Chronic Hypoxia.

The brain is considered to be an extremely sensitive tissue to hypoxia, and the brain of fish plays an important role in regulating growth and adapting to environmental changes. As an important aquatic organism in northern China, the economic yield of Takifugu rubripes is deeply influenced by the oxygen content of seawater. In this regard, we performed RNA-seq analysis of T. rubripes brains under hypoxia and normoxia to reveal the expression patterns of genes involved in the hypoxic response and their enrichment of metabolic pathways. Studies have shown that carbohydrate, lipid and amino acid metabolism are significant pathways for the enrichment of differentially expressed genes (DEGs) and that DEGs are significantly upregulated in those pathways. In addition, some biological processes such as the immune system and signal transduction, where enrichment is not significant but important, are also discussed. Interestingly, the DEGs associated with those pathways were significantly downregulated or inhibited. The present study reveals the mechanism of hypoxia tolerance in T. rubripes at the transcriptional level and provides a useful resource for studying the energy metabolism mechanism of hypoxia response in this species.

Transcriptome analysis revealed gene expression feminization of testis after exogenous tetrodotoxin administration in pufferfish Takifugu flavidus.

Tetrodotoxin (TTX) is a deadly neurotoxin and usually accumulates in large amounts in the ovaries but is non-toxic or low toxic in the testis of pufferfish. The molecular mechanism underlying sexual dimorphism accumulation of TTX in ovary and testis, and the relationship between TTX accumulation with sex related genes expression remain largely unknown. The present study investigated the effects of exogenous TTX treatment on Takifugu flavidus. The results demonstrated that exogenous TTX administration significantly incresed level of TTX concentration in kidney, cholecyst, skin, liver, heart, muscle, ovary and testis of the treatment group (TG) than that of the control group (CG). Transcriptome sequencing and analysis were performed to study differential expression profiles of mRNA and piRNA after TTX administration of the ovary and testis. The results showed that compared with female control group (FCG) and male control group (MCG), TTX administration resulted in 80 and 23 piRNAs, 126 and 223 genes up and down regulated expression in female TTX-treated group (FTG), meanwhile, 286 and 223 piRNAs, 2 and 443 genes up and down regulated expression in male TTX-treated group (MTG). The female dominant genes cyp19a1, gdf9 and foxl2 were found to be up-regulated in MTG. The cyp19a1, whose corresponding target piRNA uniq_554482 was identified as down-regulated in the MTG, indicating the gene expression feminization in testis after exogenous TTX administration. The KEGG enrichment analysis revealed that differentially expressed genes (DEGs) and piRNAs (DEpiRNAs) in MTG vs MCG group were more enriched in metabolism pathways, indicating that the testis produced more metabolic pathways in response to exogenous TTX, which might be a reason for the sexual dimorphism of TTX distribution in gonads. In addition, TdT-mediated dUTP-biotin nick end labeling staining showed that significant apoptosis was detected in the MTG testis, and the role of the cell apoptotic pathways was further confirmed. Overall, our research revealed that the response of the ovary and testis to TTX administration was largely different, the ovary is more tolerant whereas the testis is more sensitive to TTX. These data will deepen our understanding on the accumulation of TTX sexual dimorphism in Takifugu.

Isolation and Biological Activity of 9-epiTetrodotoxin and Isolation of Tb-242B, Possible Biosynthetic Shunt Products of Tetrodotoxin from Pufferfish.

Tetrodotoxin (TTX, 1) is a potent voltage-gated sodium channel blocker detected in certain marine and terrestrial organisms. We report here a new TTX analogue, 9-epiTTX (2), and a TTX-related compound, Tb-242B (4), isolated from the pufferfish Takifugu flavipterus and Dichotomyctere ocellatus, respectively. NMR analysis suggested that 2 exists as a mixture of hemilactal and 10,8-lactone forms, whereas other reported TTX analogues are commonly present as an equilibrium mixture of hemilactal and 10,7-lactone forms. Compound 2 and TTX were confirmed not to convert to each other by incubation under neutral and acidic conditions at 37 °C for 24 h. Compound 4 was identified as the 9-epimer of Tb-242A (3), previously reported as a possible biosynthetic precursor of TTX. Compound 4 was partially converted to 3 by incubation in a neutral buffer at 37 °C for 7 days, whereas 3 was not converted to 4 under this condition. Compound 2 was detected in several TTX-containing marine animals and a newt. Mice injected with 600 ng of 2 by intraperitoneal injection did not show any adverse symptoms, suggesting that the C-9 configuration in TTX is critical for its biological activity. Based on the structures, 2 and 4 were predicted to be shunt products for TTX biosynthesis.