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.

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.

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.

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.

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.

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.

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.

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.

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.

A trans-species missense SNP in Amhr2 is associated with sex determination in the tiger pufferfish, Takifugu rubripes (fugu).

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.

Effects of crude oil and dispersed crude oil on the critical swimming speed of puffer fish, Takifugu rubripes.

In order to examine the effects of crude oil and dispersed crude oil (DCO) on the swimming ability of puffer fish, Takifugu rubripes, the critical swimming speeds (U crit) of fish exposed to different concentrations of water-soluble fraction (WSF) of crude oil and DCO solution were determined in a swimming flume. WSF and DCO significantly affected the U crit of puffer fish (p < 0.05). The U crit of puffer fish exposed to 136 mg L(-1) WSF and 56.4 mg L(-1) DCO decreased 48.7 % and 43.4 %, respectively. DCO was more toxic to puffer fish than WSF. These results suggested that crude oil and chemically dispersed oil could weaken the swimming ability of puffer fish.

Identification and lateral membrane localization of cyclin M3, likely to be involved in renal Mg2+ handling in seawater fish.

The kidney of marine teleosts is the major site of Mg(2+) excretion and produces urine with a high Mg(2+) concentration. However, the transporters involved in Mg(2+) excretion are poorly understood. The cyclin M (Cnnm; also known as ancient conserved domain protein) family comprises membrane proteins homologous to the bacterial Mg(2+) and Co(2+) efflux protein, CorC. To understand the molecular mechanism of Mg(2+) homeostasis in marine teleosts, we analyzed the expression of the Cnnm family genes in the seawater (SW) pufferfish, torafugu (Takifugu rubripes), and the closely related euryhaline species, mefugu (Takifugu obscurus). Database mining and phylogenetic analysis indicated that the Takifugu genome contains six members of the Cnnm family: two orthologs of Cnnm1, one of Cnnm2, one of Cnnm3, and two of Cnnm4. RT-PCR analyses indicated that Cnnm2, Cnnm3, and Cnnm4a are expressed in the kidney, whereas other members are mainly expressed in the brain. Renal expression of Cnnm3 was upregulated in SW mefugu, whereas renal expression of Cnnm2 was upregulated in freshwater (FW) mefugu. No significant difference was observed in renal expression of Cnnm4a between SW and FW mefugu. In situ hybridization and immunohistochemical analyses of the SW mefugu kidney revealed that Cnnm3 is expressed in the proximal tubule, and its product localizes to the lateral membrane. When Cnnm3 was expressed in Xenopus laevis oocytes, whole cellular Mg(2+) content and free intracellular Mg(2+) activity significantly decreased. These results suggest that Cnnm3 is involved in body fluid Mg(2+) homeostasis in marine teleosts.

Molecular neuroendocrine basis of lunar-related spawning in grass puffer.

Grass puffer, Takifugu niphobles, exhibits unique spawning behavior: it spawns on beach in semilunar cycles during spring tide in early summer. The fish aggregate at certain seashore locations several hours before high tide every two weeks. To explore the molecular and neuroendocrine mechanisms underlying the regulation of the lunar-related spawning rhythm, seasonal and cyclic variations in gene expression for hypothalamic neuropeptides related to reproduction were examined by quantitative real-time PCR. The expression levels of genes for gonadotropin-releasing hormone, kisspeptin, LPXRFamide peptide and PQRFamide peptide in the hypothalamus varied differently depending on reproductive stage and gender, suggesting their specific roles in reproduction. In the spawning period, the expression levels of LPXRFamide peptide and its receptor genes showed diurnal and circadian variations in association with the expression of four subtypes of melatonin receptor genes. Together with the nocturnal secretion of melatonin from the pineal gland, melatonin may play an important role in transmitting the photoperiodic information of moonlight to the reproductive neuroendocrine center in the hypothalamus of grass puffer.

Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology.

Marine fish drink seawater and eliminate excess salt by active salt transport across gill and gut epithelia. Euryhaline pufferfish (Takifugu obscurus, mefugu) forms a CaCO(3) precipitate on the luminal gut surface after transitioning to seawater. NBCe1 (Slc4a4) at the basolateral membrane of intestinal epithelial cell plays a major role in transepithelial intestinal HCO(3)(-) secretion and is critical for mefugu acclimation to seawater. We assayed fugu-NBCe1 (fNBCe1) activity in the Xenopus oocyte expression system. Similar to NBCe1 found in other species, fNBCe1 is an electrogenic Na(+)/HCO(3)(-) cotransporter and sensitive to the stilbene inhibitor DIDS. However, our experiments revealed several unique and distinguishable fNBCe1 transport characteristics not found in mammalian or other teleost NBCe1-orthologs: electrogenic Li(+)/nHCO(3)(-) cotransport; HCO(3)(-) independent, DIDS-insensitive transport; and increased basal intracellular Na(+) accumulation. fNBCe1 is a voltage-dependent Na(+)/nHCO(3)(-) cotransporter that rectifies, independently from the extracellular Na(+) or HCO(3)(-) concentration, around -60 mV. Na(+) removal (0Na(+) prepulse) is necessary to produce the true HCO(3)(-)-elicited current. HCO(3)(-) addition results in huge outward currents with quick current decay. Kinetic analysis of HCO(3)(-) currents reveals that fNBCe1 has a much higher transport capacity (higher maximum current) and lower affinity (higher K(m)) than human kidney NBCe1 (hkNBCe1) does in the physiological range (membrane potential = -80 mV; [HCO(3)(-)] = 10 mM). In this state, fNBCe1 is in favor of operating as transepithelial HCO(3)(-) secretion, opposite of hkNBCe1, from blood to the luminal side. Thus, fugu-NBCe1 represents the first ortholog-based tool to study amino acid substitutions in NBCe1 and how those change ion and voltage dependence.

Species discrimination among three kinds of puffer fish using an electronic nose combined with olfactory sensory evaluation.

Species discrimination among three kinds of puffer fish, Takifugu obscurus, Takifugu flavidus and Takifugu rubripes, was conducted using an electronic nose combined with olfactory sensory evaluation. All data were treated by multivariate data processing based on principal component analysis (PCA) and discriminant factor analysis (DFA). The results showed the discriminant model by PCA method and DFA method. Using PCA and DFA, it was shown that the electronic nose was able to reasonably distinguish between each of the eleven puffer fish groups, with a discrimination index of 85. The olfactory sensory evaluation was undertaken in accordance to Sensory analysis-Methodology-Initiation and training of assessors in the detection and recognition of odors (BS ISO 5496-2006), and the results showed that the evaluation was able to identify puffer fish samples according to their species, geographical origin and age. Results from this analysis demonstrate that the E-nose can be used to complement the discrimination of odors by sensory evaluation from the three species of puffer fish studied here.

Differential expression of Na+-Cl- cotransporter and Na+-K+-Cl- cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes.

The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na(+)-K(+)-Cl(-) cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na(+)-Cl(-) cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.

The plasmablast-like leukocyte in the kidney of fugu (Takifugu rubripes).

In teleosts, the kidney is the major immune organ. From the kidney of fugu (Takifugu rubripes), we isolated a unique leukocyte population. This population shows properties similar to those of mammalian plasmablasts. First, adherent cells expressing IgM protein on their surface were obtained from the fugu kidney. Flow cytometry (FCM) showed that these cells were mainly composed of two cell populations: IgM+CD8α⁻ cells and IgM+CD8α+ cells. Further characterization of the IgM+CD8α⁻ population by RT-PCR demonstrated that the cells expressed secretory-type IgM as well as Bcl-6 and Blimp-1, developmental marker genes for the B cell lineage. Western blotting also showed that the cells secreted IgM protein. These results indicate that the IgM+CD8α⁻ cells are similar to cells at the plasmablast stage in mammals. This is the first report isolating plasmablast-like leukocytes in fish species. Our data also suggests that the teleosts kidney is a organ where B cells terminally differentiate into the plasma cells.

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.

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.