Antioxidant response to ZnO nanoparticles in juvenile Takifugu obscurus: protective effects of salinity.

The extensive utilization of Zinc Oxide nanoparticles (ZnO NPs) has garnered significant attention due to their detrimental impacts on ecosystem. Unfortunately, ecotoxicity of ZnO NPs in coastal waters with fluctuating salinity has been disregarded. This study mainly discussed the toxic effects of ZnO NPs on species inhabiting the transition zones between freshwater and brackish water, who are of great ecological and economic importance among fish. To serve as the model organism, Takifugu obscurus, a juvenile euryhaline fish, was exposed to different ZnO NPs concentrations (0-200 mg/L) and salinity levels (0 and 15 ppt). The results showed that a moderate increase in salinity (15 ppt) could alleviate the toxic effect of ZnO NPs, as evidenced by improved survival rates. The integrated biomarker response index on oxidative stress also revealed that the toxicity of ZnO NPs was higher in freshwater compared to brackish water. These outcomes can be attributed to higher salinity (15 ppt) reducing the bioavailability of ZnO NPs by facilitating their aggregation and inhibiting the release of metal ions. It is noteworthy that elevated salinity was found to alleviate ZnO NPs toxicity by means of osmotic adjustment via the activation of Na+/K+-ATPase activity. This study demonstrates the salinity-dependent effect of ZnO NPs on T. obscurus, suggesting the possibility for euryhaline fish like T. obscurus to adapt their habitat towards more saline environments, under constant exposure to ZnO NPs.

Differences in DNA methylation between slow and fast muscle in Takifugu rubripes.

Fish skeletal muscle is comprised of fast muscle (FM) and slow muscle (SM), which constitutes 60% of total the body mass. Fish skeletal muscle can affect fish swimming activity, which is important for aquaculture due to its growth-potentiating effects. DNA methylation can influence gene expression level. We previously identified multiple differentially expressed genes (DEGs) between FM and SM in Takifugu rubripes. However, it is unknown if the expression levels of these DEGs are influenced by DNA methylation. In the present study, we used DNA methylation sequencing to study the DNA methylation profiles of FM and SM in T. rubripes. SM had higher overall methylation levels than FM. A total of 8479 differentially methylated genes (DMGs) and 3407 DMGs containing differentially methylated regions (DMRs) in the promoter regions between FM and SM were identified. After enrichment analysis, we found functionally relevant DMGs between FM and SM, including Kapca, Plcd3a, Plcd1, Pi3k, Tsp4b and Pgfrb in the hedgehog signaling pathway and phosphatidylinositol (PI)-related pathways. Due to the different methylation levels of these genes between FM and SM, the expression levels of Kapca, Plcd3a, Plcd1, Pi3k, and Tsp4b were higher in FM and Pgfrb was higher in SM. There were differences in the hedgehog signaling pathway and PI-related pathways between FM and SM. In SM, the cytokine-cytokine receptor interaction promoted focal adhesion, while ECM-receptor interactions promoted focal adhesion in FM. These results provide information regarding the difference between FM and SM in T. rubripes.

Iris-like eye closure of the fine-patterned pufferfish, Takifugu flavipterus.

Unlike many tetrapods and elasmobranchs, eye-closing ability is absent in bony fishes, with the single-known exception of the family Tetraodontidae. We observed the eye-closing response of the tetraodontid fine-patterned puffer, Takifugu flavipterus, which provides the first detailed data on the kinematics and mechanism of this ability in this family. During eye-closing behavior, the skin around the eye converges toward the center of the iris. This is very different to the reversing uni-directional (e.g., upward then downward) movement of the eyelids of other vertebrates. Electrical stimulation of a freshly dead specimen showed that this movement occurs due to the contraction of a sheet of muscle located just beneath the skin around the eye, which is characteristic of Family Tetraodontidae. Eye-closing is accompanied by simultaneous retraction of the eyeball away from the surface, which is initiated just before the skin of the eye begins to move. The eye-closing ability observed in this study appears to have been acquired independently in the Tetraodontidae.

Research on the drag reduction property of puffer (Takifugu flavidus) spinal nonsmooth structure surface.

Puffers show good drag reduction performance during migration. It is worth noting that spines which are different from ordinary fish scales are densely distributed on the puffer skin. Here, the special morphological structure of puffer spines was observed using microscopy techniques, accurate contour models were established based on image processing techniques and curve fitting, then feature sizes were obtained. Based on the results, the nonsmooth surface was established by orthogonal test to simulate the flow field. In addition, the influence of spinal structure on boundary layer flow field and the drag reduction property of nonsmooth surface were further analyzed. The nonsmooth surface formed by spinal structure elements can effectively reduce the wall shear stress and Reynolds stress, and there was a special "climbing vortex" phenomenon, so as to reduce the surface viscous friction resistance and achieve drag reduction. Compared with the smooth surface, the drag reduction rate of the nonsmooth surface was 12.94% when the inflow velocity was 5 m/s, which revealed and verified the drag reduction performance of the spines of puffer skin. The results lay a foundation for further research and optimization of drag reduction ability of nonsmooth surface of bionic spines. HIGHLIGHTS: The contour of the spinous process was accurately reflected by the Fourier function. The spines of puffer skin have good drag reduction effect. There was a special "climbing vortex" phenomenon to explain the drag reduction property.

Freshwater entry behaviour of a non-migratory stenohaline marine fish Takifugu snyderi.

We conducted salinity choice trials with the stenohaline marine species Takifugu snyderi to test their freshwater (FW) entry frequency in relation to starvation. The fish preferred to enter non-natal FW rather than remain in seawater. No relationship was detected between starvation and FW entry behaviour. Our results provide new empirical evidence of a stenohaline fish entering a non-natal osmotic environment. Further research on the entry of stenohaline species such as this one into lethal environments may help determine if this might help promote the evolution of diadromous life histories.

Effect of acute exposure to nitrite on physiological parameters, oxidative stress, and apoptosis in Takifugu rubripes.

In the present study, we evaluated the effects of nitrite exposure on hematological parameters, oxidative stress, and apoptosis in juvenile Takifugu rubripes. The fish were exposed to nitrite (0, 0.5, 1, 3, and 6 mM) for up to 96 h. In the high nitrite concentration groups (i.e., 3 and 6 mM), the concentrations of methemoglobin (MetHb), cortisol, glucose, heat shock protein (Hsp)-70, Hsp-90, and potassium (K+) were significantly elevated. Whereas, the concentrations of hemoglobin (Hb), triglyceride (TG), total cholesterol (TC), and sodium (Na+) and chloride (Cl-) ions were significantly decreased. Compared with those of the control groups, the concentrations of the antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and glutathione peroxidase (GPx), in the gills were considerably elevated at 12 and 24 h after exposure to nitrite (1, 3, and 6 mM), but reduced at 48 and 96 h. The increase in the antioxidant enzymes may contribute to the elimination of reactive oxygen species (ROS) induced by nitrite during early nitrite exposure, when the antioxidant system is not sufficiently effective to eliminate or neutralize excessive ROS. In addition, we found that nitrite exposure could alter the expression patterns of some key apoptosis-related genes (Caspase-3, Caspase-8, Caspase-9, p53, Bax, and Bcl-2). This indicated that the caspase-dependent apoptotic pathway and p53-Bax-Bcl-2 pathway might be involved in apoptosis induced by nitrite exposure. Furthermore, our study provides insights into how acute nitrite exposure affects the physiological responses and potential molecular mechanism of apoptosis in marine fish. The results can help elucidate the mechanisms involved in nitrite-induced aquatic toxicology in marine fish.

Tetrodotoxin functions as a stress relieving substance in juvenile tiger puffer Takifugu rubripes.

We tested whether tetrodotoxin (TTX) functions as a stress relieving substance in puffer fish. We orally administered TTX to the juveniles of hatchery-reared non-toxic tiger puffer Takifugu rubripes and measured the effects of TTX on brain corticotropin-releasing hormone (CRH) mRNA expression and plasma cortisol levels in comparison with effects in non-toxic juveniles. Firstly, the reciprocal connections of CRH and adrenocorticotropic hormone (ACTH) were confirmed by dual-label immunohistochemistry. CRH-immunoreactive (ir) cell bodies were detected in the hypothalamus and CRH-ir fibers were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Next, a TTX-containing diet (2.35 mouse units (517 ng)/g diet) or a non-toxic diet were fed to the fish for 28 days under a recirculating system. Standard length and body weight became significantly larger in the TTX-treated group. The degree of loss of the caudal fin, which is an indicator of the degree of agonistic interactions, where high values show a higher loss of caudal fin of a fish due to nipping by other individuals, was significantly lower in the TTX-treated group. Relative CRH mRNA expression levels in the brain and cortisol levels in the plasma were significantly lower in the TTX-treated group. These results indicate that TTX functions as a stress relieving substance by affecting the CRH-ACTH-cortisol axis and reducing agonistic interactions in tiger puffer juveniles.

Integrated application of multi-omics provides insights into cold stress responses in pufferfish Takifugu fasciatus.

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.

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

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

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

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

Including planocerid flatworms in the diet effectively toxifies the pufferfish, Takifugu niphobles.

Beginning with the larval stages, marine pufferfish such as Takifugu niphobles contain tetrodotoxin (TTX), an extremely potent neurotoxin. Although highly concentrated TTX has been detected in adults and juveniles of these fish, the source of the toxin has remained unclear. Here we show that TTX in the flatworm Planocera multitentaculata contributes to the toxification of the pufferfish throughout the life cycle of the flatworm. A species-specific PCR method was developed for the flatworm, and the specific DNA fragment was detected in the digesta of wild pufferfish adults. Predation experiments showed that flatworm larvae were eaten by the pufferfish juveniles, and that the two-day postprandial TTX content in these pufferfish was 20-50 µg/g. Predation experiments additionally showed flatworm adults were also eaten by pufferfish young, and after two days of feeding, TTX accumulated in the skin, liver and intestine of the pufferfish.

Heterogeneity of neuromasts in a fish without lateral line canals: the pufferfish (Takifugu obscurus) model.

Fish detect water motion with their mechanosensory lateral line. The basic functional unit of the lateral line is the neuromast. In most fish species, neuromasts are located in lateral line canals (canal neuromasts) or on the skin (superficial neuromasts). In this paper, we describe the lateral line system of the pufferfish, Takifugu obscurus If threatened, this fish inflates its body by sucking water into the esophagus. Pufferfish lack a canal system but have neuromasts located directly on the skin or in open grooves. Each groove houses tall, medium and short neuromasts, based on the height of their pedestal. One or more medium neuromasts were always located between two tall neuromasts, and the short neuromasts were scattered between them. Tall neuromasts showed phasic responses to water jets, similar to the canal neuromasts of other fish species. In contrast, the medium and short neuromasts showed tonic responses to water jets. The response properties of nerve fibers that innervated the medium and short neuromasts were similar to those of the superficial neuromasts found in other fish species. Our results suggest that each groove of a pufferfish has two functional groups of neuromasts. This may allow pufferfish to extract spatial and temporal hydrodynamic information, despite the changes in body shape that occur during and after inflation. The short neuromasts at the bottom of a groove most likely supplement the medium neuromasts when the body is maximally inflated.

Promoter analysis of the fish gene of slow/cardiac-type myosin heavy chain implicated in specification of muscle fiber types.

Vertebrate skeletal muscles consist of heterogeneous tissues containing various types of muscle fibers, where specification of the fiber type is crucial for muscle development. Fish are an attractive experimental model to study the mechanisms of such fiber type specification because of the separated localization of slow and fast muscles in the trunk myotome. We examined regulation of expression of the torafugu gene of slow/cardiac-type myosin heavy chain, MYH M5 , and isolated an operational promoter in order to force its tissue-specific expression across different fish species via the transgenic approach in zebrafish and medaka. This promoter activity was observed in adaxial cell-derived superficial slow muscle fibers under the control of a hedgehog signal. We also uncovered coordinated expression of MYH M5 and Sox6b, which is an important transcriptional repressor for specification of muscle fiber types and participates in hedgehog signaling. Sequence comparison in the 5'-flanking region identified three conserved regions, CSR1-CSR3, between torafugu MYH M5 and its zebrafish ortholog. Analysis of deletion mutants showed that CSR1 significantly stimulates gene expression in slow muscle fibers. In contrast, deletion of CSR3 resulted in ectopic expression of a reporter gene in fast muscle fibers. CSR3 was found to contain a putative Sox family protein-binding site. These results indicate that the dual mechanism causing inhibition in fast muscle fibers and activation in slow muscle fibers is essential for slow muscle fiber-specific gene expression in fish.

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

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

Mitigation of nitrite toxicity by increased salinity is associated with multiple physiological responses: A case study using an economically important model species, the juvenile obscure puffer (Takifugu obscurus).

Nitrite is a common pollutant in water and is highly toxic to aquatic animals. To reveal the mechanism of salinity in attenuating nitrite toxicity to fish, we measured the physiological responses of juvenile Takifugu obscurus exposed to nitrite concentrations (0, 10, 20, 50, and 100 mg/L) under different salinity levels (0, 10, and 20 ppt) for 96 h. Salinity increased the survival rates of juvenile T. obscurus exposed to nitrite. Changes in key hematological parameters, antioxidant system, malondialdehyde, Na+/K+-ATPase, and HSP70 indicated that nitrite induced considerable damage to juveniles; salinity mitigated the harmful effects. This finding reflects similar changing trends in both antioxidants and their gene expressions among different tissues. We applied an overall index, an integrated biomarker response (IBR), that increased under high-nitrite condition but recovered to the normal levels under salinity treatment. Analysis of the selected detection indices and IBR values showed that the overall mitigating effect of salinity on nitrite toxicity seems to be at sub-cellular level and associated with complicated physiological responses.

Effects of acute temperature and salinity changes, body length and starvation on the critical swimming speed of juvenile tiger puffer, Takifugu rubripes.

The critical swimming speed (U crit, cm s-1) of juvenile tiger puffer Takifugu rubripes was determined under different temperatures (15, 21, 25 and 30 °C), salinities (5, 10, 20, 32 and 40), body lengths (3.32, 4.08, 5.06 and 5.74 cm) and starvation days (1, 3, 6 and 9 days). Acute temperature change, body length and starvation significantly influenced the U crit of tiger puffers, whereas acute salinity change had no significant effect. The U crit increased as the temperature increased from 15 to 30 °C. The U crit increased as the body length increased from 3.32 to 5.74 cm, whereas relative critical swimming speed (U crit', body length s-1) decreased. The relationship between the body length (l, cm) and U crit or U crit' can be described by the quadratic model as U crit = - 1.4088 l 2 + 16.976 l - 11.64, R 2 = 0.9698 (P < 0.01) or U crit' = - 0.1937 l 2 + 0.9504 l + 7.7666, R 2 = 0.9493 (P < 0.01). The U crit decreased as starvation days increased from 1 to 9 days. Low temperature and starvation can reduce the swimming ability of juvenile tiger puffers. Results can be of value in evaluating the swimming ability of juvenile tiger puffers, understanding ecological processes and improving the population enhancement of tiger puffers.

Light- and circadian-controlled genes respond to a broad light spectrum in Puffer Fish-derived Fugu eye cells.

Some cell lines retain intrinsic phototransduction pathways to control the expression of light-regulated genes such as the circadian clock gene. Here we investigated the photosensitivity of a Fugu eye, a cell line established from the eye of Takifugu rubripes, to examine whether such a photosensitive nature is present. Microarray analysis identified 15 genes that showed blue light-dependent change at the transcript level. We investigated temporal profiles of the light-induced genes, as well as Cry and Per, under light-dark, constant light (LL), and constant dark (DD) conditions by quantitative RT-PCR. Transcript levels of Per1a and Per3 genes showed circadian rhythmic changes under both LL and DD conditions, while those of Cry genes were controlled by light. All genes examined, including DNA-damage response genes and photolyase genes, were upregulated not only by blue light but also green and red light, implying the contribution of multiple photopigments. The present study is the first to identify a photosensitive clock cell line originating from a marine fish. These findings may help to characterize the molecular mechanisms underlying photic synchronization of the physiological states of fishes to not only daily light-dark cycles but also to various marine environmental cycles such as the lunar or semi-lunar cycle.

Immune and physiological responses of pufferfish (Takifugu obscurus) under cold stress.

Low temperature is an important environmental factor in aquaculture farming that affects the survival and growth of organisms. In the present study, we investigated the effects of low temperature on biochemical parameters, oxidative stress and apoptosis in pufferfish. In the stress group, water temperature decreased from 25 °C to 13 °C at a rate of 1 °C/1 h. Fish blood and liver were collected to assay biochemical parameters, oxidative stress and expression of genes at 25 °C, 21 °C, 17 °C, 13 °C and 13 °C for 24 h. The results showed that low temperature could decrease total blood cell count, inhibit cell viability, and subsequently lead to DNA damage. Biochemical parameters such as plasma protein and ALP significantly declined in fish under low temperature, while a significant increase in AST, ALT, LDH and glucose was observed. The gene expression of antioxidant enzymes (SOD and CAT), HSP90 and C3 were induced by low temperature stress. Furthermore, the gene expression of apoptotic related genes including P53, caspase-9 and caspase-3 were up-regulated, suggesting that caspase-dependent pathway could play important roles in low temperature-induced apoptosis in fish. This study may provide baseline information about how cold stress affects the physiological responses and apoptosis in fish.

Combined effects of cadmium and salinity on juvenile Takifugu obscurus: cadmium moderates salinity tolerance; salinity decreases the toxicity of cadmium.

Obscure puffer Takifugu obscurus, a species of anadromous fish, experiences several salinity changes in its lifetime. Cadmium (Cd) is a toxic heavy metal that can potentially induce oxidative stress in fish. The present study aimed to detect the combined effects of Cd (0, 5, 10, 20 and 50 mg L(-1)) and salinity (0, 15 and 30 ppt) on juvenile T. obscurus. Results showed the juveniles could survive well under different salinities; however, with Cd exposure, the survival rates significantly decreased at 0 and 30 ppt. At 15 ppt, tolerance to Cd increased. Cd exposure clearly induced oxidative stress, and the responses among different tissues were qualitatively similar. Salinity acted as a protective factor which could reduce the reactive oxygen species and malondialdehyde levels. In addition, salinity could enhance the antioxidant defense system, including superoxide dismutase, catalase and glutathione. Na(+)/K(+)-ATPase activity significantly decreased under Cd exposure in gill, kidney and intestine. These findings indicated that Cd could moderate the adaptability of juvenile T. obscurus to high salinity and low salinity played a protective role upon Cd exposure. Thus, the role of salinity should be considered when evaluating the effect of heavy metals on anadromous and estuarine fishes.

Toxic Takifugu pardalis eggs found in Takifugu niphobles gut: Implications for TTX accumulation in the pufferfish.

Pufferfish (Takifugu spp.) possess a potent neurotoxin, tetrodotoxin (TTX). TTX has been detected in various organisms including food animals of pufferfish, and TTX-producing bacteria have been isolated from these animals. TTX in marine pufferfish accumulates in the pufferfish via the food web starting with marine bacteria. However, such accumulation is unlikely to account for the amount of TTX in the pufferfish body because of the minute amounts of TTX produced by marine bacteria. Therefore, the toxification process in pufferfish still remains unclear. In this article we report the presence of numerous Takifugu pardalis eggs in the intestinal contents of another pufferfish, Takifugu niphobles. The identity of T. pardalis being determined by direct sequencing for mitochondrial DNA. LC-MS/MS analysis revealed that the peak detected in the egg samples corresponded to TTX. Toxification experiments in recirculating aquaria demonstrated that cultured Takifugu rubripes quickly became toxic upon being fed toxic (TTX-containing) T. rubripes eggs. These results suggest that T. niphobles ingested the toxic eggs of another pufferfish T. pardalis to toxify themselves more efficiently via a TTX loop consisting of TTX-bearing organisms at a higher trophic level in the food web.