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Simple Summary: Cryptocaryon irritans is a primitive ciliate that parasitizes the skin of marine fish and can cause illness or even death in Fugu. In this study, we revealed an immune regulatory network of genes and non-coding RNAs co-modulating competing endogenous RNAs. Potential biomarkers of Fugu infection with Cryptocaryon irritans were analyzed by bioinformatics. Of these, the regulatory network showed that the LOC105418663-circ_0000361-fre-miR-204a-fzd3a ceRNA axis was potentially involved in modulating the immune response of Fugu against Cryptocaryon irritans infection. Our research results have laid a theoretical foundation for a deeper understanding of the immune mechanism of Fugu and proposed new treatment and prevention measures for resisting Cryptocaryon irritans infection. Cryptocaryon irritans (C. irritans) is a proto-ciliate parasite that infects marine fishes, including the cultured species Takifugu rubripes (T. rubripes), causing disease and potential mortality. In host organisms, infection by parasites triggers an immune response that is modulated by regulatory elements including proteins and non-coding RNAs. In this study, the whole transcriptome RNA sequencing of T. rubripes gill tissue before and after infection with C. irritans was performed to reveal the competitive endogenous RNA (ceRNA) regulatory network. Histomorphology revealed gill segment swelling and parasitic invasion in the infected group. The analysis identified 18 differentially expressed miRNAs (DEMs), 214 lncRNAs (DELs), 2501 genes (DEGs), and 7 circRNAs (DECs) in the infected group. Gene Ontology (GO) enrichment analysis revealed that these genes were notably enriched in the Wnt signaling pathway and mTOR signaling pathway. The co-expression networks (lncRNA/circRNA-miRNA-mRNA) were constructed based on correlation analysis of the differentially expressed RNAs. Further analysis suggested that the LOC105418663-circ_0000361-fru-miR-204a-fzd3a ceRNA axis was potentially involved in the regulation of immune responses against C. irritans infection. Finally, the expression levels of DEG, DEL, and DEM were validated. This study reveals the regulatory mechanism of a candidate ceRNA network, providing insights into the potential mechanism of T. rubripes' infection with C. irritans. [ABSTRACT FROM AUTHOR]

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Response surface methodology (RSM) was employed to optimize the hydrolysis conditions of acid-soluble collagen (ASC) from Takifugu rubripes skin for the preparation of anti-tyrosinase peptides. The anti-tyrosinase and antioxidant activity of ultrafiltration peptides with different molecular masses were tested before and after being subjected to simulated gastrointestinal digestion, and the moisture absorption and retention properties were also evaluated to explore their application potential. The results showed that neutral protease was the most suitable enzyme for releasing anti-tyrosinase peptides from T. rubripes skin collagen. The optimum process conditions were as follows: substrate concentration 2.9 g/100 mL, hydrolysis time 7.8 h, and enzyme dosage 7 862 U/g. The hydrolysate obtained under these conditions inhibited tyrosinase activity by (35.14 ± 0.03)%. The molecular mass of the hydrolysate mainly ranged from 0 to 5 kDa, and the fraction CP-1 (< 3 kDa), with the highest anti-tyrosinase activity, scavenged 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation and hydroxyl radicals with half maximal inhibitory concentration (IC50) values of 9.51, 2.03 and 7.29 mg/mL, respectively. CP-1 maintained high anti-tyrosinase and antioxidant activity after in vitro simulated digestion. In addition, CP-1 had high moisture absorption and retention properties and therefore could be used as a substitute for glycerol. This study may provide a theoretical basis for the deep processing and application of collagen peptides from T. rubripes skin.

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This study aimed to estimate the optimal light conditions for indoor cultivation of juvenile tiger puffer (Takifugu rubripes), The juvenile fish were exposed to five irradiance of green light (G1: 300 mW/ m², G2: 500 mW/ m², G3: 750 mW/ m², G4: 1 000 mW/ m² and G5: 1 500 mW/ m²), white light (W) and dark (D) for 60 days. At the end of the experiment, the retinal structure of the juvenile fish was observed, and measurements were taken for retinal thickness, expression of visual protein genes, and changes in antioxidant enzyme activity. The results showed that different irradiance of green light had no significant effect on the retinal structure of juvenile fish. Different degrees of melanin migration occurred in the retina of all treatment groups. Melanin granules were more aggregated in the G1 and D groups, while in the G4 and G5 treatment groups, the density of melanin granules was relatively high. The total retinal thickness (TT) decreased with increased green light irradiance. The ratio of the thickness of each retinal layer to the total thickness was further analyzed. The results showed that the changes in retinal thickness showed different trends among different regions, with the G3 group having the highest proportion of the photoreceptor layer (PRos/ is/ TT) (P<0. 05). The proportion of the outer plexiform layer (OPL/ TT) was significantly higher than those in other groups except G1 (P<0. 05). The proportion of outer nuclear layer (ONL/ TT) of juvenile fish in the G2 and G3 groups was significantly higher than that in the G1 and G4 groups (P < 0. 05). There was no significant difference in the proportion of pigment epithelium (RPE/ TT) and ganglion cell layer (GCL/ TT) among different treatment groups (P>0. 05). Regarding the expression of opsin genes, Rod opsin expression was the highest in groups exposed to white light (W)and dark conditions (D)(P<0. 05). SWS2 expression in the G3 group was the highest (P<0. 05). The expression of Opsin3 was the highest in the W group and the G5 group (P<0. 05). For antioxidant enzymes, the activities of Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Peroxidase (GPX) in the green light treatment groups increased regularly with the increase of light irradiance, and there was no extreme decrease. In view of the effects of different light irradiance on retinal thickness and oxidative stress in eye tissues of juvenile tiger puffer in this study, it is recommended to use a white-colored cultivation substrate and green light in indoor cultivation environments. Additionally, it is advised to control the light irradiance within the range of 500 to 750 mW/ m². [ABSTRACT FROM AUTHOR]

Copyright of Journal of Agro-Environment Science is the property of Journal of Agro-Environment Science Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copper is one of the predominant water pollutants. Excessive exposure to copper can cause harm to animal health, affecting the central nervous system and causing blood abnormalities. Cuproptosis is a novel form of cell death that differs from previous programmed cell death methods. However, the impact of copper on the intestines remains unclear. Therefore, we investigated the effects of different concentrations of copper exposure on the intestinal proteome of Takifugu rubripes (T. rubripes). Relevant biomarkers were used to detect cuproptosis. We revealed the crosstalk relationship between cuproptosis and self-rescue at different concentrations, and discussed the feasibility of using potential cuproptosis indicators as anti-infection factors. We observed intestinal damage in the three copper exposure groups, especially in T. rubripes treated with 100 and 500 μg/L copper, with shedding and breakage of intestinal villus and fuzzy and loose structure of intestinal mucosa. The presence of copper stress not only causes cuproptosis but also oxidative damage caused by reactive oxygen species (ROS). The results of quantitative proteomics by TMT showed that compared to the 50 and 100 μg/L copper exposure groups, the expression of glutaminase, pyruvate kinase, and skin mucus lectin in the 500 μg/L group was significantly increased. The positive mediators COX5A and CTNNB1, as well as the negative mediators CD4 and FDXR, were found to be differentially expressed. Using the protein expression trends of cuproptosis indicator factors FDX1 and DLAT to indicate the concentration of copper ions in the environment. In addition, we found a new effect of promoting ferroptosis: providing additional copper ions can activate the phenomenon of ferroptosis. Our results expand our understanding of the potential health risks of copper in T. rubripes. At the same time, it is of great significance for the process of copper poisoning and the development of new environmental toxicology detection reagents.

Osmoregulation is essential for aquatic organisms to balance their internal fluids and ions and adapt to environmental changes. The gills are a crucial organ for maintaining osmotic balance in fish by regulating the uptake and excretion of ions. Changes in the salinity of the aquatic environment can lead to alterations in the expression of genes within gill cells, which helps to maintain the internal salinity balance and allows the fish to adapt to different salinity levels. In this study, we used transcriptomic data of the gill from the euryhaline Takifugu obscurus and the stenohaline Takifugu rubripes, as well as their hybrid offspring (Tor1 and Tor2), to investigate how the gill contributes to osmoregulation in response to freshwater and seawater. Our study revealed 8 commonly differentially expressed genes among these four fish species. Analysis of the T. obscurus transcriptome data has demonstrated that SLC1a5, a member of the solute carrier (SLC) family, and Gper1, a G-protein coupled estrogen receptor 1-like, are enriched in many biological processes and might be important for its osmoregulation in freshwater. Furthermore, PDE8B, Lrrc9 and Cplx4 might be important osmoregulatory genes that are inherited by offspring in T. obscurus. Moreover, upon encountering changes in water salinity, T. rubripes and the hybrid offspring Tor1 reveal a marked concentration of differentially expressed genes in metabolic pathways within their gills, whereas T. obscurus and the hybrid offspring Tor2 show a more even distribution of DEGs across various signaling pathways. Finally, the Pearson correlation analysis revealed that the T. rubripes displayed a significant correlation with Tor1, while the T. obscurus was significantly related to Tor2. Overall, this study provides a theoretical foundation for understanding the salinity adaptation mechanisms in hybrid offspring of Takifugu, and holds significant implications for guiding the breeding of economically valuable Takifugu strains with improved traits.

Tiger puffer (Takifugu rubripes) is one of the most expensive farmed marine fish species in China. The high profitability of farmed tiger puffer is used to attract many new investors. However, some investments failed due to a lack of information about the tiger puffer fish farm business, poor farm and facility design, and flawed investment plans. To improve the investment efficiency and profitability of this industry, this analysis evaluated the economic performance of tiger puffer from eggs to harvest size using two different open-indoor combination aquaculture systems: a combination of recirculating aquaculture system and open net pen (RAS+ONP) and a combination of flow-through system and earth pond (FTS + EP). This analysis evaluated the financial performance of both systems using profit margin, net present value (NPV), internal rate of return (IRR), and payback period. At the same time, a sensitivity analysis was employed to measure economic risk. The results revealed that despite the RAS+ONP system being more economically profitable than the FTS + EP system, the financial indicators of the FTS + EP system granted a better economic performance in terms of NPV and IRR due to lower initial investment and operating costs. Meanwhile, the sensitivity analysis revealed that the economic performance of both systems was vulnerable to the fluctuations in fish prices and feed prices. The potential risk of earning profit was higher for the RAS+ONP if the feed price fluctuated compared to the FTS + EP system, while the risk was higher for the FTS + EP if the fish price fluctuated. Hence, the FTS + EP system could represent an opportunity for new entrants to obtain a more profitable production. [ABSTRACT FROM AUTHOR]

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection. [ABSTRACT FROM AUTHOR]

This study employed a coagulation precipitation phosphorus removal process based on polymeric ferric sulfate (PFS) to construct a coagulation precipitation phosphorus removal device, which was then applied to the removal of active orthophosphate from the tailwater of industrialized marine culture of Takifugu rubripes. The results showed that the active orthophosphate in the mariculture tailwater could be effectively removed by the chemical coagulation precipitation method, and the optimal dosage of PFS is 55 mg/L. The removal efficiency of active orthophosphate is not affected by the concentration of CODMn in the tailwater of the industrialized marine culture of Takifugu rubripes. The decrease in salinity of the tailwater and the flow rate control of the phosphorus removal device are both beneficial for the removal of active orthophosphate. When the initial concentration of active orthophosphate in the tailwater of the industrialized culture of Takifugu rubripes is 0.254 mg/L, and the flow rate of the phosphorus removal device is con- trolled at 100 L/h, the removal rate of orthophosphate reaches 78.7% after 180 min of treatment. The residual con- centration of active orthophosphate in the effluent is reduced to 0.054 mg/L, meeting the requirements for the discharge of the tailwater from mariculture. This study provides a device for the practical application of chemical coagulation precipitation phosphorus removal processes in the treatment of industrial mariculture tailwater. [ABSTRACT FROM AUTHOR]

Fishes' skeletal muscles are crucial for swimming and are differentiated into slow-twitch muscles (SM) and fast-twitch muscles (FM) based on physiological and metabolic properties. Consequently, mitochondrial characteristics (number and morphology) adapt to each fiber type's specific functional needs. However, the mechanisms governing mitochondrial adaptation to the specific bioenergetic requirements of each fiber type in teleosts remain unclear. To address this knowledge gap, we investigated the mitochondrial differences and mitochondrial homeostasis status (including biogenesis, autophagy, fission, and fusion) between SM and FM in teleosts using Takifugu rubripes as a representative model. Our findings reveal that SM mitochondria are more numerous and larger compared to FM. To adapt to the increased mitochondrial number and size, SM exhibit elevated mitochondrial biogenesis and dynamics (fission/fusion), yet show no differences in mitochondrial autophagy. Our study provides insights into the adaptive mechanisms shaping mitochondrial characteristics in teleost muscles. The abundance and elongation of mitochondria in SM are maintained through elevated mitochondrial biogenesis, fusion, and fission, suggesting an adaptive response to fulfill the bioenergetic demands of SM that rely extensively on OXPHOS in teleosts. Our findings enhance our understanding of mitochondrial adaptations in diverse muscle types among teleosts and shed light on the evolutionary strategies of bioenergetics in fishes. [ABSTRACT FROM AUTHOR]

An 8-week feeding trial was conducted to investigate the effects of dietary carbohydrate levels on growth performance, body composition, and plasma biochemical parameters, liver and gut histology, and intestinal microbiota of Takifugu rubripes. Five isonitrogenous and isolipidic experimental diets were formulated with graded levels of cassava starch: 0% (C0), 8% (C8), 16% (C16), 24% (C24), and 32% (C32). The results showed that weight gain rate, specific growth rate (SGR) and protein efficiency ratio significantly increased in C16 and C24 groups (P < 0.05), while the feed conversion ratio expressed an opposite trend. Feeding rate is decreased with the increasing of carbohydrate levels (P < 0.05). Liver lipid content was dramatically increased in C24 treatment (P < 0.05). The intestinal amylase activity and muscle glycogen content were higher than other groups (P < 0.05). Plasma glucose levels were increased in C24 and C32 groups (P < 0.05). In addition, the abundance of intestinal potentially pathogenic bacteria (Staphylococcus) decreased, and the intestinal probiotics (Lactobacillus) increased with the increasing of dietary carbohydrate level (P < 0.05). The intestinal bacteria, indicated by Sobs, Chao and Shannon indexes, were more diverse with the increasing of the carbohydrate level (P < 0.05), meanwhile the abundance of transport and metabolism of carbohydrate, lipid and amino acid were enhanced with the increasing of carbohydrate levels (P < 0.05). In conclusion, appropriate supplementation of carbohydrate in diet promoted growth performance and feed utilization of juvenile Takifugu rubripes, and increased the liver lipid and muscle glycogen content, as well as intestinal microbial diversity. Based on the quadratic regression between SGR and carbohydrate levels, the optimal dietary carbohydrate level of juvenile Takifugu rubripes is 21.6%.

Fugu (Takifugu rubripes) is a commercially important fish in aquaculture in China, Japan, and Korea. Male fugu are highly prized compared to females because the mature testes are regarded as a delicacy, thus all-male populations are preferred in aquaculture. However, the molecular mechanisms underlying sex differentiation in fugu remain largely unclear. Progestins, which are mediated by the progesterone receptor, can initiate spermatogenesis or ovarian differentiation in teleosts. To explore the effect of progestin blockage on fugu sex differentiation, the antiprogestin mifepristone (RU486) (500 μg/g of feed) was administrated to fugu larvae from 30 to 90 days after hatching (dah). Histological analysis revealed that RU486 treatment resulted in complete masculinization (100 %) of the XX fugu population at both 120 (RU486 withdrawal 30 days) and 150 (RU486 withdrawal 60 days) dah, and RU486-treated XX gonads developed testes-like structure. Gonadal transcriptomic analysis at 90 dah identified 3595 genes that were differentially expressed (DEGs) between the control and treated groups. Among them, the expression of steroid hormone biosynthesis-related genes (e.g., cyp11c1, cyp19a1a, and hsd17b1) and other key genes (e.g., gsdf, dmrt1, and foxl2) were altered in the gonads of RU486-treated groups. Most of the DEGs were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to sex differentiation, such as neuroactive ligand-receptor interaction, ovarian steroidogenesis, and steroid hormone biosynthesis pathways. The expression of cyp19a1a, foxl2, cyp11c1, gsdf, dmrt3, and dmrt1 at 50, 70, and 90 dah was further assessed. The expression levels of gsdf, dmrt1, and cyp11c1 were increased, and the expression levels of cyp19a1a and foxl2 were decreased in the gonads of XX larvae in RU486 treated groups. These results demonstrated for the first time that RU486 induced masculinization in XX fugu and provided a theoretical basis for further studies of the regulatory role of progestins in sex differentiation in teleosts.

Takifugu rubripes are warm temperate fish, suggesting that the reduced seawater temperatures in winter are likely to have a substantial impact on their survival. Considering this, there is likely to be some industrial value in breeding extremely low-temperature tolerant varieties of this fish. Here, we evaluate the expression changes in six major QTL candidate genes (tacc2, fsip1, exoc4, arhgap44a, pde10a, and unc5b) in response to reduced temperature in an effort to understand cold tolerance in T. rubripes. The expression changes of these six genes in the liver, heart, and kidney were detected using real-time quantitative PCR. This study used three groups of 8-month-old fish, all from the same family established by our research group, exposed to three different temperature gradients, where 8 ℃ and 13 ℃ acted as the minimum in the low temperature groups and 18 ℃ acted as the minimum in the control group. Our results showed that all six genes were expressed at different levels across each of these three tissues at different temperatures. The relative expression of pde10a first increased and then decreased in all three tissues, whereas the relative expression of tacc2 and exoc4 were distinctly different in the liver, kidney, and heart at 8 ℃. In this case, these transcripts first decreased and then stabilized in the liver, increased and then stabilized in the kidneys, and increased and then decreased in the heart. The relative expression of unc5b was low in the liver and heart, but high in the kidney following a second week of low-temperature growth, whereas arhgap44a expression was slightly upregulated in the liver and stable in the kidney and heart. fsip1 expression demonstrated a downward trend in the liver but seemed to first increase and then decrease in the heart and kidney. Taken together, these results demonstrate that all six of these genes are differentially expressed in different tissues of T. rubripes, with these differences exhibiting dynamic changes with respect to tissue origin and temperature. In addition, this data clearly revealed a positive correlation between cold stress and the expression of these QTL candidate genes. Thus, we can conclude that these six QTL candidate genes may play a substantial role in the low temperature adaptation of T. rubripes. This is significant because although low temperature is known to be an important factor limiting the development of the industrial utility of T. rubripes, there are still relatively few reports describing their cold stress response. This study provides a theoretical basis for the study of signaling pathways related to the low temperature tolerance response of T. rubripes and the development of low temperature tolerant varieties.

Cryptocaryon irritans (C. irritans) is a proto-ciliate parasite that infects marine fishes, including the cultured species Takifugu rubripes (T. rubripes), causing disease and potential mortality. In host organisms, infection by parasites triggers an immune response that is modulated by regulatory elements including proteins and non-coding RNAs. In this study, the whole transcriptome RNA sequencing of T. rubripes gill tissue before and after infection with C. irritans was performed to reveal the competitive endogenous RNA (ceRNA) regulatory network. Histomorphology revealed gill segment swelling and parasitic invasion in the infected group. The analysis identified 18 differentially expressed miRNAs (DEMs), 214 lncRNAs (DELs), 2501 genes (DEGs), and 7 circRNAs (DECs) in the infected group. Gene Ontology (GO) enrichment analysis revealed that these genes were notably enriched in the Wnt signaling pathway and mTOR signaling pathway. The co-expression networks (lncRNA/circRNA-miRNA-mRNA) were constructed based on correlation analysis of the differentially expressed RNAs. Further analysis suggested that the LOC105418663-circ_0000361-fru-miR-204a-fzd3a ceRNA axis was potentially involved in the regulation of immune responses against C. irritans infection. Finally, the expression levels of DEG, DEL, and DEM were validated. This study reveals the regulatory mechanism of a candidate ceRNA network, providing insights into the potential mechanism of T. rubripes’ infection with C. irritans.

Fish meal and fish oil are important sources of protein and lipid in feeds. The lack of fish meal and fish oil supply has become an important limiting factor for the aquaculture industry. The proportion of fish oil and fish meal used in commercial feeds for farmed fish and crustaceans is continually decreasing, while the carbohydrate content is increasing. Carbohydrates are one of the three major nutrients that provide energy for the body. They have a much lower production price than protein and fat. Starch is a polymeric carbohydrate made from glucose molecules; it is the most commonly used in aquatic feeds and can replace fish meal and fish oil to reduce feed cost. Therefore, it is important to fully explore the nutritional function of carbohydrates for aquaculture. Hypoxia is a common stress in aquaculture, and acute hypoxia may lead to massive mortality of cultured fish in a short period of time resulting in serious economic losses to aquaculture. Therefore, it is crucial to find ways to improve the acute hypoxia tolerance of fish. Fish mainly use glucose for energy under acute hypoxia. Hypoxia inhibits oxidative phosphorylation in mitochondria and activates the anaerobic glycolysis pathway. Glucose degrades to produce lactate and ATP. Carnivorous fishes have limited absorption and utilization capacities to feed carbohydrates, unlike omnivorous fishes. Dextrin is an intermediate starch hydrolysis product, with a molecular weight between starch and glucose; it has good adhesion properties and is more easily digested and absorbed than starch. Therefore, we hypothesized that the use of easily digestible carbohydrate in the feed is an effective way to improve the acute hypoxia tolerance of fish. Takifugu rubripes is loved by Japanese and Korean consumers owing to its delicious taste and high nutritional value. It is a characteristic species of Chinese mariculture fish. It is mainly cultured in a high-density factory, and the dissolved oxygen in the water often relies on water exchange and an oxygenation pump; its gill cover is degraded and is at risk of acute hypoxia. This study determined the effect of the addition of corn starch or dextrin (corn starch hydrolysate) to the feed on growth performance, acute hypoxia survival rate, metabolite content, and the hypoxia inducible factor (HIF) signaling pathway of T. rubripes after 8 weeks of feeding. There were no significant differences in weight gain, feed conversion ratio, hepatosomatic index, viscerosomatic index, condition factor, and body composition between the starch group and the dextrin group (P > 0.05). However, the survival rate during acute hypoxia of the dextrin group was significantly higher than that of the starch group (P < 0.05). There were no significant differences in the liver glycogen and lactate contents, and lactate dehydrogenase A4 (ldha) gene expression between the starch group and dextrin group (P > 0.05) during normoxia. However, the lactate content and ldha gene expression were significantly higher in the liver of the dextrin group than those in the starch group (P < 0.05) during hypoxia. This indicated that feeding dextrin strongly activated anaerobic glycolysis to provide more energy under hypoxia. The serum triglyceride (TG) content significantly increased in the dextrin group compared with the normoxia groups, although the TG content in the serum and liver significantly decreased in the starch group after acute hypoxia (P < 0.05). This suggested that feeding starch promoted lipid catabolism and oxygen consumption. There was no significant difference in the total soluble protein content of the muscle between the starch group and the dextrin group (P > 0.05) during normoxia. However, the total soluble protein content of the muscle decreased in the dextrin group compared with the starch group (P < 0.05) after acute hypoxia. Meanwhile, the total muscle soluble protein content, and the gene expression of liver v-akt murine thymoma viral oncogene homolog 1 (akt1), and mechanistic target of rapamycin kinase (mtor) significantly decreased in the dextrin group after hypoxia compared with the normoxia group. However, the liver mtor gene expression significantly increased in the starch group after hypoxia (P < 0.05). This data demonstrated that protein synthesis was inhibited in the dextrin group under hypoxia. Hypoxia inducible factor (HIF) is the most critical transcription factor in cellular response to hypoxic stress. The dextrin group had higher hypoxia inducible factor 1 subunit alpha like (hif-3α) gene expression in the liver and lower hypoxia inducible factor 1 subunit alpha a (hif-1α) gene expression in the muscle (P < 0.05) compared with the starch group during normoxia. The gene expression of liver hif-1α and hif-3α and muscle hif-1α and vascular endothelial growth factor A (vegfa) significantly increased in the dextrin group compared with the starch group (P < 0.05) during hypoxia. This proved that feeding dextrin strongly activated the HIF signaling pathway under hypoxia. In summary, replacing starch with easily digestible dextrin in the feed did not affect the growth performance. Instead, it activated the HIF signaling pathway and anaerobic glycolysis to provide more energy for fish. Meanwhile, feeding dextrin inhibited lipid catabolism and protein synthesis, and reduced oxygen consumption to improve the acute hypoxia tolerance of T. rubripes. The study provides important guidance for the formulation design of hypoxia-tolerant feed and healthy development of aquaculture.

Copper is an environmental pollutant, and copper in aquatic environments mainly comes from soil and water. It enters the environment through atmospheric deposition, sewage discharge, and industrial production, and enters aquatic organisms, causing toxicity. Takifugu rubripes (T. rubripes) is a marine fish with high economic value. Due to the toxic effects of heavy metals on aquatic organisms such as fish, it can affect the gut community and metabolites of fish. The gut is an important channel for fish to communicate with the outside world and a necessary pathway for the metabolism of nutrients and toxic substances in the fish body. Studies have shown that due to changes in global water emissions and the high sensitivity of aquatic organisms to the environment, copper may pose greater potential hazards to aquatic organisms. Copper poses a greater risk to aquatic species than other heavy metals and metal/metal like pollutants (such as cadmium, lead, mercury, arsenic, etc.) . In order to elucidate the effects of copper exposure on the gut of T. rubripes. In this study, we exposed T. rubripes to 0, 50, 100, or 500 μg/L of copper for three days, the effects of copper exposure on the gut microbiota structure and metabolites of the T. rubripes were investigated using 16 S rRNA gene and metabolomics techniques. The research results indicate that with the increase copper concentration, the intestinal tissue of T. rubripes undergoes significant damage. 16 S rRNA sequencing results show that copper exposure alters the structure and metabolites of intestinal microbiota. Copper exposure of 100 and 500 μg/L inhibited the colonization of the bacterial gut, disrupted the intestinal barrier, and made the fish susceptible to the pathogens. Liquid chromatography–mass spectrometry analysis showed that copper regulated the production of metabolites such as L-histidine, arachidonic acid, and L-glutamic acid, which are related to energy and immunity. Microbiome–metabolome correlation analysis showed that Subdoligranulum, Family_XIII_AD3011_group, and Clostridium_sensu_stricto_1 were the key bacteria for copper ion intervention, and they might up-regulate the levels of metabolites such as indole-3-acetic acid, 3-indoleacrylic acid, and 5-hydroxyindole in the tryptophan metabolism pathway. In summary, our research has demonstrated that copper exposure can cause pathological changes in the intestinal tissue of the T. rubripes. High concentrations of copper ions can affect the colonization of the T. rubripes microbiota in the intestine, damage the fish's immune system, and alter the structure and metabolites of the intestinal microbiota, this can lead to intestinal metabolic dysfunction. providing a reference for the evaluation of the biological toxicity effects of heavy metal elements in the marine environment. This study provides a reference for evaluating the biological toxicity effects of heavy metal elements in marine environments.

Copyright of Journal of Hydrobiology / Shuisheng Shengwu Xuebao is the property of Editorial Department of Journal of Hydrobiology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

As important environmental factors, the light spectra and tank colours have not received sufficient attention. Most fishes have the ability to perceive environment, distinguish colours, and exhibit preferences or aversions towards different environments, which can provide a reference for the design of their rearing environment. Tiger puffer (Takifugu rubripes) is an important mariculture species in China and East Asia, but its preference for illumination spectra and tank colours is unclear. This study focuses on the preferences of juvenile tiger puffers for different spectra and tank background colours in different rearing backgrounds and body sizes. The experiments were conducted in a preference testing device, and the behavioural videos were recorded and analysed using a motion behaviour tracking system (EthoVision XT 12). The results show that the puffers showed preference for short-wavelength lights ((i.e., cyan, green, etc.), avoidance of long-wavelength light (i.e., red) and less stay time in the full light spectrum and dark. For tank colours, the puffers showed a preference for light background colours (i.e., white), and avoidance of deep background colours (i.e., dark, red, etc.). Fish body sizes and original breeding environment could significantly affect the selective preference of juvenile puffer (P

Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2β, were estimated to have two domains, and X2β is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: The supply of fish oil, which provides long-chain polyunsaturated fatty acids (LC-PUFA) but is mostly wild-caught, is stagnating, resulting in limited availability and high prices. Therefore, many alternative lipid sources, mainly vegetable oils and rendered animal fats, are increasingly being applied in fish feeds. However, these lipid sources do not contain LC-PUFA, and therefore lower the fillet quality in terms of the LC-PUFA content. Feeding strategies such as alternate feeding between fish oil- and alternative oils-based diets have been applied to maintain the n-3 LC-PUFA contents in farmed fish. The present study validated the relevant efficacy. A 12-week feeding experiment was conducted to evaluate the effects of replacing fish oil (FO) with beef tallow (BT) on the fatty acid composition of farmed tiger puffer (Takifugu rubripes). Two replacement strategies were used: a standard Graded Dietary Replacement of FO with BT (GDR strategy) and Alternate Feeding between FO- and BT-based Diets (AFD strategy). The positive and negative control diets were formulated with 6% FO (FO-C group) or BT (BT-C group) as the sole added lipid source. In the GDR strategy, three experimental diets were formulated, with 25, 50 and 75% of the added FO in the FO-C diet replaced with BT, named 25BT, 50BT and 75BT, respectively. In the AFD strategy, alternated feeding patterns between the FO-C and BT-C diet—namely, 1, 2 and 3 weeks with BT-C followed by 1 week feeding with FO-C (1BT-1FO, 2BT-1FO and 3BT-1FO, respectively)—were applied. Each diet or feeding strategy was assigned to triplicate tanks. The results showed that dietary BT inclusion reduced the contents of long-chain polyunsaturated fatty acids (LC-PUFA) in both the muscle and liver (edible tissues for this species) of the experimental fish, and the liver displayed a more drastic decrease than the muscle. The LC-PUFA content linearly decreased with the decreasing dietary FO levels in the GDR strategy. However, in the AFD strategy, a linear relationship was not observed between the LC-PUFA content and the FO feeding duration. The 3BT-1FO treatment resulted in higher LC-PUFA content than 2BT-1FO. When comparing the two strategies with the same final FO administration level—namely, 50BT vs. 1BT-1FO, and in particular, 75BT vs. 3BT-1FO—the AFD strategy resulted in higher LC-PUFA contents in both the muscle and liver than the GDR strategy. In conclusion, when FO was replaced with BT in the diets, alternate feeding between FO- and BT-based diets resulted in a higher LC-PUFA content than the standard direct replacement. Three weeks of feeding with BT-C followed by one week of feeding with FO-C appeared to be a good alternate feeding pattern. This study provided a promising strategy of FO-sparing in fish farming when the LC-PUFA contents were maintained as high as possible. [ABSTRACT FROM AUTHOR]

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Simple Summary: Tiger puffer fish (Takifugu rubripes) is an economically important marine fish, in which the development of selective breeding is limited due to the long time of sexual maturation. In order to promote the breeding research of this species, we established the spermatogenic stem cell line of the tiger puffer fish. In this study, we isolated spermatogonial stem cells from the testis of the 6-month-old tiger puffer fish and cultured them stably in vitro for more than 60 generations. The characteristics of the in vitro cultured spermatogonial stem cell line showed polygonal in shape. The germ cell marker genes dnd, ddx4, piwil, dazl, stem cell marker genes sox2, myca and nanog, and spermatogonial stem cell marker genes gfra1b and ly75 were highly expressed in this cell line. The establishment of this spermatogonial stem cell line provides a dependable material for the reproduction research of tiger puffer fish. Tiger puffer fish (Takifugu rubripes) has become the main fish species cultured in China since the last century because of its high economic value. Male and female tiger puffer fish need 2 and 3 years each to reach sexual maturity, which limits the development of breeding research for this species. In recent years, in vitro culture of fish spermatogonial stem cells (SSCs) have shown potential in aquaculture. In the present study, we established a spermatogenic stem cell line from T. rubripes (TrSSCs). TrSSCs were characterized by polygonal morphology, predominantly retained 44 chromosomes, and grew rapidly at 26 °C and in L-15. TrSSCs were still able to grow stably after more than one year of in vitro culture. TrSSCs showed positive alkaline phosphatase staining. TrSSCs expressed germ cell-associated genes, including dnd, ddx4, piwil, gfra1b, sox2, myca, nanog, ly75, and dazl, as determined by semiquantitative assays, and almost all cells were found to express the germ cell genes ddx4 and gfra1b in a fluorescence in situ hybridization assay. In vitro, induction experiments demonstrated the TrSSCs possessed the ability to differentiate into other types of cells. Our research has enriched the fish spermatogonial stem cell resource bank, which will provide an efficient research model for sex determination and sex control breeding in fish, establishing a foundation for subsequent breeding research. [ABSTRACT FROM AUTHOR]

Environmental conditions regulate the growth and reproduction of fish. The increase in sea temperature during winter may have adverse effects on Takifugu rubripes. To study the mechanism of low-temperature tolerance of T. rubripes, the expression of antifreeze protein (AFP) gene, cold-induced RNA binding protein (CIRP) gene, high mobility group protein box-1 (HMGB1) gene, and Y-box binding protein (YB-1) gene in the liver, spleen, kidney, brain, heart, intestine, muscle, gonad, and skin tissues of T. rubripes obtained from different temperatures (18℃, 13℃, 8℃, and 5℃) was analyzed by quantitative real-time PCR. The results showed that the AFP gene was widely expressed in tissues, with the highest expression in the muscle (P < 0.05). With the decrease in temperature, the expression of the AFP gene in each tissue showed a significant increasing trend, reaching the highest value in the 5℃ group. The expression of the CIRP gene was the highest in the muscle (P < 0.05). With a decrease in temperature, the trend of CIRP gene expression in various tissues was different. The CIRP gene expression levels of liver, kidney, brain, heart, intestine, and skin showed a trend of initial increase, followed by a decrease, and then an increase. The expression levels in the spleen, muscle, and gonads showed an upward trend, reaching the highest value in the 5℃ group. The expression of the HMGB1 gene was the highest in muscle (P < 0.05), followed by that in the brain, liver, heart and skin. As the temperature decreased, the expression of the HMGB1 gene in all tissues except the liver increased first and then decreased, and reached the maximum value in the 8℃ group, which was significantly higher than that of the other groups (P < 0.05). The expression of the YB-1 gene was the highest in the muscle (P < 0.05), with the lowest expression level in other tissues. As the temperature decreased, the expression level of most tissues (brain, heart, intestine, kidney, liver, muscle, and spleen) increased first, then decreased, and then increased, reaching the minimum value in the 8℃ group (P < 0.05). These results show that the expression levels of the four genes are different at different temperature, reflecting the functional specificity of these four genes. Under low-temperature stress, these genes responded positively. Their expression changed to varying degrees, suggesting that the four genes may have potentially important roles in the adaptation of T. rubripes to low temperatures. In addition, by analyzing the law of gene expression, 8℃ may be the key regulatory point for T. rubripes to deal with low-temperature stress. Too low temperature may cause its regulation disorder. The results of this study can provide a relevant basis for studying the regulation mechanism of the low-temperature response of T. rubripes.

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In aquaculture, the accurate recognition of fish underwater has outstanding academic value and economic benefits for scientifically guiding aquaculture production, which assists in the analysis of aquaculture programs and studies of fish behavior. However, the underwater environment is complex and affected by lighting, water quality, and the mutual obscuration of fish bodies. Therefore, underwater fish images are not very clear, which restricts the recognition accuracy of underwater targets. This paper proposes an improved YOLO-V7 model for the identification of Takifugu rubripes. Its specific implementation methods are as follows: (1) The feature extraction capability of the original network is improved by adding a sizeable convolutional kernel model into the backbone network. (2) Through ameliorating the original detection head, the information flow forms a cascade effect to effectively solve the multi-scale problems and inadequate information extraction of small targets. (3) Finally, this paper appropriately prunes the network to reduce the total computation of the model; meanwhile, it ensures the precision of the detection. The experimental results show that the detection accuracy of the improved YOLO-V7 model is better than that of the original. The average precision improved from 87.79% to 92.86% (when the intersection over union was 0.5), with an increase of 5.07%. Additionally, the amount of computation was reduced by approximately 35%. This shows that the detection precision of the proposed network model was higher than that for the original model, which can provide a reference for the intelligent aquaculture of fishes. [ABSTRACT FROM AUTHOR]

Dissolved oxygen (DO) is an essential ecological factor encountered by fish and other aquatic animals, and hypoxia can adversely affect the physiology, biochemistry and behavior of aquatic animals. Blood is an essential tissue for oxygen transportation and plays a key role in maintaining homeostasis and adapting the organism to hypoxia. However, studies focusing on the molecular regulatory mechanisms of blood under hypoxia are scant. Therefore, to explore the hypoxia adaptive mechanisms in the blood, we used RNA-Seq technology to investigate the transcriptome changes in the blood of Takifugu rubripes under acute hypoxia. In our study, 20 fish were selected and divided into four groups, including a control group (PPM5.4) and three experimental groups (PPM4, PPM2 and PPM0). In addition to GO and KEGG enrichment analysis, we further used GSEA enrichment analysis to screen the pathways mediated in T. rubripes under hypoxia. Results revealed that hypoxia had a greater effect on blood than other tissues, and T. rubripes could inhibit cell proliferation and down-regulated immune-related pathways (including IL-17 signaling pathway, C-type lectin receptor signaling pathway, B cell receptor signaling pathway and Toll-like receptor signaling pathway). However, the blood of T. rubripes could positively modulate the genes expression involved in energy regulation, angiogenesis and oxidative stress to resist hypoxic damage. Furthermore, T. rubripes could also up-regulate apoptosis and JAK−STAT signaling pathway to conduct self-protection in response to hypoxia. In conclusion, all these results indicated that immune processes, energy metabolism, angiogenesis and oxidative stress were significantly altered in the blood of acute hypoxic T. rubripes, which contributed to maintain cellular energy balance and oxygen supply during hypoxic conditions. The key regulatory genes and pathways identified in this study not only reveal the mechanism of hypoxia tolerance in T. rubripes, but also provide a theoretical basis for breeding hypoxia-tolerant T. rubripes and other fish.

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. [ABSTRACT FROM AUTHOR]

The light‐sensitive capacity of fish larvae is determined by the structure of the retina and the opsins expressed in the retinal and nonretinal photoreceptors. In this study, the retinal structure and expression of opsin genes during the early developmental stage of Takifugu rubripes larvae were investigated. Histological examination showed that at 1 days after hatching (dah), seven layers were observed in the retina of T. rubripes larva, including the pigment epithelial layer [retinal pigment epithelium layer (RPE)], photoreceptor layer (PRos/is), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL). At 2 dah, optic fibre layer (OFL) can be observed, and all eight layers were visible in the retina. By measuring the thickness of each layer, opposing developmental trends were found in the thickness of ONL, OPL, INL, IPL, GCL and OFL. The nuclear density of ONL, INL and GCL and the ratios of ONL/INL, ONL/GCL and INL/GCL were also measured and the ratio of ONL/GCL ranged from 1.9 at 2 dah to 3.4 at 8 dah and no significant difference was observed between the different developmental stages (P > 0.05). No significant difference was observed for the INL/GCL ratio between the different developmental stages, which ranged from 1.2 at 2 dah to 2.0 at 18 dah (P > 0.05). The results of quantitative real‐time polymerase chain reaction (PCR) showed that the expression of RH1, LWS, RH2‐1, RH2‐2, SWS2, rod opsin, opsin3 and opsin5 could be detected from 1 dah. These results suggest that the well‐developed retina and early expression of the opsins of T. rubripes during the period of transition from endogenous to mixed feeding might be critical for vision‐based survival skills during the early life stages after hatching. [ABSTRACT FROM AUTHOR]

Tiger puffer (Takifugu rubripes) is one of the most expensive farmed marine fish species in East Asia. Growth traits are important economic factors in cultured fish, miRNAs and circRNAs play important roles in growth and development. However, little is known of how miRNAs and circRNAs contribute to regulating fish growth through neuroendocrine regulation. To explore the role of miRNAs and circRNAs in regulating T. rubripes growth, RNA sequencing technology was used to sequence whole brain tissue transcriptomes from fast and slow-growing T. rubripes families. A total of 14 and 261 differentially expressed (DE) circRNAs and miRNAs were identified, respectively, in brain tissues. Among these, the expression levels of circCacna1c may maintain stable neuroendocrine environments by precisely regulating intracellular calcium (Ca2+) concentrations, thereby accelerating T. rubripes growth. Notably, genes including tgfb3 , bmp2b , and bmp16 appeared to modulate T. rubripes development by coordinating neural activities during development via the Smad pathway. A miRNA-mRNA regulatory network was also identified that comprised 98 and 113 DE miRNAs and mRNAs, respectively. miR-124-4-3p and miR-124-4-5p of the miR-124 family were implicated in the regulation of neural development in T. rubripes by suppressing target genes like runx3 , hoxb3a , mdfic , flt3 , smo and syt10. In addition, 8 circRNA-miRNA-mRNA pathways were constructed including 2 circRNAs, 5 miRNAs, and 2 mRNAs. DE miRNAs and circRNAs identified here are primarily involved in nerve development and the maintenance of Ca2+ homeostasis in fast- and slow-growing T. rubripes. Overall, these results inform a better understanding of growth-related transcriptional dyanmics in T. rubripes brains, but also aid in the systematic selection and breeding of new varieties with higher qualities and yields. • Growth-related miRNAs and circRNAs are identified of brain in T. rubripes. • miRNA-mRNA network plays an important role in growth-related neuroendocrine activities. • The miR-124 family may be implicated the growth of T. rubripes by regulating neural development. • CircCacna1c may affect the release of growth hormone in T. rubripes by regulating intracellular Ca2+ concentration. [ABSTRACT FROM AUTHOR]

Estrogen exposure during early-life stages has been found to delay ovarian development in female fish, even after a long-term depuration period. However, the mechanisms underlying the disordered ovarian development remain unclear. In this study, the larvae of tiger puffer Takifugu rubripes were exposed to 0 (control) and 10 μg/L 17β-estradiol (E2) from 20 to 90 days post-hatch (dph) and maintained in clear seawater until 180 dph. Genetic females collected at 90 and 180 dph were identified by analyzing a sex-associated SNP. Then, the ovarian structure, gonadosomatic index (GSI), the maximum follicle area and the mRNA levels of genes involving in cell cycle (ckd2, ckd4, cdk6, ccna2, ccnd2, cdkn1b and cdkn2c), meiosis initiation (sycp3, rec8, spo11, and dmc1), follicle formatiaon (bmp2, hnrnpk, hmp15, gdf9, nobox and figla) and apoptosis (bax and bcl-2) were analyzed between control and E2-exposed females. The results show that, no structure difference in ovaries was observed between control and E2-treated females at 90 dph, but the primary growth follicles in E2-treated females were observed to be fewer in number than control at 180 dph. Both ovarian weight and GSI of E2-treated females were significant lower than the control at 90 and 180 dph, while there was no significant different in the maximum follicle area between control and E2-treated females at neither 90 or 180 dph. Additionally, the E2 exposure suppressed the expression of sycp3, rec8, spo11, dmc1, bmp2, hnrnpk and bcl-2 at 90 dph, but the mRNA levels of those genes in E2-treated females showed no significant different with the control at 180 dph. The reduced mRNA levels of sycp3, rec8, spo11 and dmc1 might result in disrupted meiosis, and suppression the expression of bmp2 and hnrnpk affected follicle formation. Then, abnormal meiosis initiation and follicle formation might further promote apoptosis as indicated by the decrease in mRNA levels of bcl-2, which ultimately contributed to less number of follicles and low GSI value in E2-treated females.

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

We evaluated whether bearing tetrodotoxin (TTX) affects salinity stress in the juvenile tiger puffer Takifugu rubripes. Juveniles of hatchery-reared non-toxic T. rubripes [body weight (BW): 1.7 ± 0.2 g, n = 120] were divided into six tanks and acclimatized to salinity (8.5 ppt) that is equivalent to blood osmolality. Fish in three tanks were fed non-toxic diet, and those in the other three tanks were fed a TTX-containing diet (356 ng/g diet) three times a day until satiation. In each diet treatment, salinity of one tank was kept at 8.5 ppt, and the other two tanks were adjusted to either 1.7 or 34.0 ppt, and fish were reared for another 33 days. Then, we compared survival, growth, TTX accumulation, plasma osmolality, plasma cortisol, and glucose levels among treatments. We detected TTX only in the fish in the TTX-diet groups. Survival was highest at 8.5 ppt (70%) and lowest at 1.7 ppt in the TTX-diet group (20%). The BW was greater at 8.5 ppt, and plasma osmolality was significantly higher at 34.0 ppt than at any other salinities. Plasma cortisol level was significantly higher but glucose level was lower at 1.7 ppt. Possessing TTX at a low salinity may be lethal to tiger puffer juveniles. [ABSTRACT FROM AUTHOR]

The fish oil finishing (FOF) strategy, that is, re-feeding fish with fish oil (FO)-based diet after a certain period of feeding with alternative lipid source-based diets. On tiger puffer, the present study investigated the response of intestinal microbiota to FOF. Fish were fed four diets based on FO, soybean oil, palm oil and beef tallow as lipid sources, respectively, firstly for 50 days (growing-out period), and then fed the FO-based diet for 30 more days (FOF period). The results showed that dietary terrestrially sourced oils impaired the intestinal function in the growing-out period. However, the activities of amylase, trypsin and anti-oxidative enzymes (SOD, CAT, T-AOC), as well as gene expression of inflammatory cytokines (IL-1β, TNF-α, TGF-β) and tight junction protein (Claudin4, Claudin7, Claudin18, JAM, ZO-1) in the intestine were significantly recovered by FOF. The 16S rDNA sequencing analysis showed that FOF improved the similarity of bacterial community among the groups. The MetaStat analysis confirmed that FOF regulated the abundance of butyric acid-producing bacteria (Lachnospiraceae, Eubacterium, Butyricicoccus, Clostridium and Roseburia) and bacteria related to digestion and absorption (Sphingomonas, Romboutsia and Brevibacillus). In conclusion, FOF can recover the intestine function. The intestinal microbiota probably participated in and played a key role in the recovery process. [ABSTRACT FROM AUTHOR]

Ovarian development is a complex process involving multiple genes, but the molecular mechanisms underlying this process in Takifugu rubripes remain poorly understood. This study aimed to identify genes associated with ovarian development in T. rubripes and to investigate the regulatory mechanisms of oocyte maturation. Transcriptome data were compared across four different developmental stages (stage II to V) to identify differentially expressed genes (DEGs) and perform GO and KEGG enrichment analysis. The expression patterns of randomly selected genes were then validated by qPCR. The results yielded a total of 1,289,401,820 raw data from all libraries, with 16,929 DEGs identified across all comparison groups. The DEGs were predominantly enriched in ovarian steroidogenesis, estrogen-mediated signaling, and TGF-beta signaling pathways. The qPCR analysis showed that cyp17a1 was identified as being expressed at similar levels in stage II and III. Thereafter, cyp17a1 was observed to undergo a continuous increase in expression from stage III to V. cyp19a1, nanos1, foxl2 and ar were identified as being expressed at similar levels at stage II and III, then increase in expression from stage III to IV and subsequent downregulation from stage IV to V. hsd17b1 was identified as being expressed at similar levels at stage II and IV. This study represents a transcriptomic study of ovarian development in female T. rubripes. Several essential ovarian-related genes and sex-related biological pathways were identified. The results will improve our understanding of the molecular mechanisms underlying ovarian development in this species., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests. Each author has no financial, personal, professional, or intellectual relationships that could potentially bias their work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Water temperature plays a vital role in shaping the physical conditions crucial for the growth, development and reproduction of fish species. Since limited comprehensive multi-omics analyses exploring the molecular mechanisms of temperature influences on the early life stages of fish. Here, the effects of temperature variations on the growth of Takifugu rubripes, a commercial teleost farmed in Asia were investigated. Nineteen-days-old fugu larvae were subjected to different temperature (15 °C-T15, 20 °C-T20, 25 °C-T25) for 30 days. Liver tissues were harvested at the end of the study for transcriptomic and metabolomic assessments. The T. rubripes larvae in the T15 group showed a significant decrease in total length and body weight compared to the T20 and T25 groups (p < 0.05). 1344, 416, and 2080 differentially expressed genes (DEGs) were identified in T15-vs-T20, T20-vs-T25, and T15-vs-T25 comparisons, respectively. Those DEGs were mainly enriched in metabolic, protein digestion and absorption, steroid biosynthesis, and glycerophospholipid metabolism pathways. 15 DEGs were randomly selected for RNA-seq validation, and the transcriptome results were consistent with the qPCR validation results, illustrating the accuracy of transcriptome sequencing. 340, 238, and 330 significantly different metabolites (SDMs) were identified in positive modes when comparing in T15-vs-T20, T20-vs-T25, and T15-vs-T25, respectively. Additionally, 145, 137, and 159 SDMs were identified in negative modes within the three comparisons. Those SDMs enriched in biosynthesis of secondary metabolites, glycerophospholipid metabolism, linoleic acid metabolism, and metabolic pathways. The integration of transcriptomic and metabolomic analyses indicated that DEGs and SDMs mainly enriched in metabolic pathways. These discoveries provide valuable insights into the effects of temperature on fish larvae in aquaculture, laying a foundation for future breeding approaches aimed at improving the growth of T. rubripes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Abstract Background As the critical tissue of the central nervous system, the brain has been found to be involved in gonad development. Previous studies have suggested that gonadal fate may be affected by the brain. Identifying brain-specific molecular changes that occur during estrodiol-17β (E2) -induced feminization is crucial to our understanding of the molecular control of sex differentiation by the brains of fish. Results In this study, the differential transcriptomic responses of the Takifugu rubripes larvae brain were compared after E2 treatment for 55 days. Our results showed that 514 genes were differentially expressed between E2-treated-XX (E-XX) and Control-XX (C-XX) T. rubripes, while 362 genes were differentially expressed between E2-treated-XY (E-XY) and Control-XY (C-XY). For example, the expression of cyp19a1b, gnrh1 and pgr was significantly up-regulated, while st, sl, tshβ, prl and pit-1, which belong to the growth hormone/prolactin family, were significantly down-regulated after E2 treatment, in both sexes. The arntl1, bhlbe, nr1d2, per1b, per3, cry1, cipc and ciart genes, which are involved in the circadian rhythm, were also found to be altered. Differentially expressed genes (DEGs), which were identified between E-XX and C-XX, were significantly enriched in neuroactive ligand-receptor interaction, arachidonic acid metabolism, cytokine-cytokine receptor interaction and the calcium signaling pathway. The DEGs that were identified between E-XY and C-XY were significantly enriched in tyrosine metabolism, phenylalanine metabolism, arachidonic acid metabolism and linoleic acid metabolism. Conclusion A number of genes and pathways were identified in the brain of E2-treated T. rubripes larvae by RNA-seq. It provided the opportunity for further study on the possible involvement of networks in the brain-pituitary-gonadal axis in sex differentiation in T. rubripes.

The tiger puffer (Takifugu rubripes) is an important economic fish species in northern China. However, it is a warm-temperature species, and low winter temperatures can result in high mortality in aquaculture. Understanding the mechanisms of cold resistance in tiger puffers will thus provide critical information to help cope with winter cold. In this study, we performed transcriptome analysis of livers from puffer fish kept at different temperatures (18°C, 13°C, and 8°C) to identify the key pathways and genes involved in the response to low-temperature stress. We also detected serum levels of proteases, arginine, and proline to obtain further information on the response to cold adaption. Totals of 51, 942, and 195 differentially expressed genes were identified in the 18°C vs 13°C, 18°C vs 8°C, and 13°C vs 8°C groups, respectively. Pathway analysis showed that significantly enriched pathways were mainly related to digestion, metabolism, and environmental adaptation. Most genes in the pathways related to digestion and metabolism were down-regulated, while most genes in the pathways related to environmental adaptation were up-regulated. Serum levels of proteases were significantly lower in the low-temperature groups (13°C and 8°C) compared with the control group (18°C), while arginine and proline levels were significantly higher in the 8°C group compared with the other two groups. These results suggest that low temperature caused digestive and metabolic disorders, as well as adaptive changes to low temperature in tiger puffers. On this premise, we found that some up-regulated genes in the pancreatic secretion pathway, arginine and proline metabolism pathway, and circadian rhythm pathway played important roles in the survival, growth, and development of tiger puffers under low-temperature stress. The accumulation of arginine and proline can maintain metabolism and circulation and resist cold stress. The circadian rhythm is closely related to digestion and metabolism, which is an adaptive change and plays a positive role in the resistance to low temperature. The results of this study provide new insights and a theoretical basis for the study of cold tolerance in tiger puffers.

A 12-week feeding experiment was conducted to evaluate the effects of replacing fish oil (FO) with beef tallow (BT) on the fatty acid composition of farmed tiger puffer (Takifugu rubripes). Two replacement strategies were used: a standard Graded Dietary Replacement of FO with BT (GDR strategy) and Alternate Feeding between FO- and BT-based Diets (AFD strategy). The positive and negative control diets were formulated with 6% FO (FO-C group) or BT (BT-C group) as the sole added lipid source. In the GDR strategy, three experimental diets were formulated, with 25, 50 and 75% of the added FO in the FO-C diet replaced with BT, named 25BT, 50BT and 75BT, respectively. In the AFD strategy, alternated feeding patterns between the FO-C and BT-C diet—namely, 1, 2 and 3 weeks with BT-C followed by 1 week feeding with FO-C (1BT-1FO, 2BT-1FO and 3BT-1FO, respectively)—were applied. Each diet or feeding strategy was assigned to triplicate tanks. The results showed that dietary BT inclusion reduced the contents of long-chain polyunsaturated fatty acids (LC-PUFA) in both the muscle and liver (edible tissues for this species) of the experimental fish, and the liver displayed a more drastic decrease than the muscle. The LC-PUFA content linearly decreased with the decreasing dietary FO levels in the GDR strategy. However, in the AFD strategy, a linear relationship was not observed between the LC-PUFA content and the FO feeding duration. The 3BT-1FO treatment resulted in higher LC-PUFA content than 2BT-1FO. When comparing the two strategies with the same final FO administration level—namely, 50BT vs. 1BT-1FO, and in particular, 75BT vs. 3BT-1FO—the AFD strategy resulted in higher LC-PUFA contents in both the muscle and liver than the GDR strategy. In conclusion, when FO was replaced with BT in the diets, alternate feeding between FO- and BT-based diets resulted in a higher LC-PUFA content than the standard direct replacement. Three weeks of feeding with BT-C followed by one week of feeding with FO-C appeared to be a good alternate feeding pattern. This study provided a promising strategy of FO-sparing in fish farming when the LC-PUFA contents were maintained as high as possible.

Tiger puffer fish (Takifugu rubripes) has become the main fish species cultured in China since the last century because of its high economic value. Male and female tiger puffer fish need 2 and 3 years each to reach sexual maturity, which limits the development of breeding research for this species. In recent years, in vitro culture of fish spermatogonial stem cells (SSCs) have shown potential in aquaculture. In the present study, we established a spermatogenic stem cell line from T. rubripes (TrSSCs). TrSSCs were characterized by polygonal morphology, predominantly retained 44 chromosomes, and grew rapidly at 26 °C and in L-15. TrSSCs were still able to grow stably after more than one year of in vitro culture. TrSSCs showed positive alkaline phosphatase staining. TrSSCs expressed germ cell-associated genes, including dnd, ddx4, piwil, gfra1b, sox2, myca, nanog, ly75, and dazl, as determined by semiquantitative assays, and almost all cells were found to express the germ cell genes ddx4 and gfra1b in a fluorescence in situ hybridization assay. In vitro, induction experiments demonstrated the TrSSCs possessed the ability to differentiate into other types of cells. Our research has enriched the fish spermatogonial stem cell resource bank, which will provide an efficient research model for sex determination and sex control breeding in fish, establishing a foundation for subsequent breeding research.

Tiger pufferfish (Takifugu rubripes) is one of Asia's most economically valuable aquaculture species. However, winter production of this species in North China is limited by low water temperature and unavailability of high-quality feed, resulting in high mortality and low profitability. Therefore, the aim of this study was to evaluate the effect of feeding frequency (F1: one daily meal; F2: two daily meals; F3: four daily meals; F4: continuous diurnal feeding using a belt feeder) on the growth performance, plasma biochemistry, digestive and antioxidant enzyme activities, and expression of appetite-related genes in T. rubripes (initial weight: 266.80 ± 12.32 g) cultured during winter (18.0 ± 1.0 °C) for 60 days. The results showed that fish in the F3 group had the highest final weight, weight gain rate, specific growth rate, survival rate, and best feed conversion ratio. Additionally, daily feed intake increased significantly with increasing feeding frequency. The protein efficiency and lipid efficiency ratios of fish in the F3 group were significantly higher than those of fish in the other groups. Furthermore, total cholesterol, triglycerides, and glucose levels increased with increasing feeding frequency, peaking in the F2 group and decreasing under higher feeding frequencies. The antioxidant (superoxide dismutase, catalase, glutathione, and glutathione peroxidase) and digestive (trypsin, amylase, and lipase) enzyme activities of fish in the F1 group were significantly higher than those of fish in the F3 and F4 groups. Additionally, there was a decrease in orexin expression with increasing feeding frequency. In contrast, the expression levels of tachykinin, cholecystokinin, and leptin increased with increasing feeding frequency, peaking in the F4 group. Overall, the findings of this study indicated that a feeding frequency of four meals per day was optimal for improved growth performance of pufferfish juveniles cultured during winter. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Light has long been known to have a profound influence on the growth and development of fish. The previous studies showed that different spectra had different effects on the growth of juvenile Takifugu rubripes. Among them, green light can promote the growth of Takifugu rubripes, but the influence mechanism is unknown. In this study, how different LED spectrums affect fish growth from the perspective of GH/IGF-I axis-related genes and apoptosis genes were deeply explored. In the experiment, juvenile Takifugu rubripes with an initial body length of (9.01 ± 0.70) cm and an initial body weight of (18.05 ± 3.17) g were selected as the research objects. 525 Takifugu rubripes juveniles were selected, cultured and monitored in five different LED spectrum treatment groups: white light (WL, λ 400-780nm), red light (RL, λ 625-630nm), yellow light (YL, λ 590-595nm), green light (GL, λ 525-530nm) and blue light (BL, λ 450-455nm). The photoperiod was 12L:12D, and the light intensity was set to 250 mW/m2. The effects of light spectrum on growth, melatonin synthesis, GH/IGF-I axis and relative expressions of apoptosis-related genes in juvenile Takifugu rubripes were studied, including arylalkylamine N-acetyltransferase (AANAT2), growth hormone (GH), growth hormone receptor type 1 (GHR1), growth hormone releasing hormone (GHRH), insulin-like growth factor type I (IGF-I), insulin-like growth factor type II (IGF-II), insulin-like growth factor binding protein (IGFBP), Bcl-2 protein family (Bcl-2), tumor suppressor (p53) and cysteine protease family (caspase 3, caspase 8, caspase 10) genes. The results showed that the final weight was the highest in the GL group (29.36 ± 3.78 g) and the lowest in the YL group (21.28 ± 2.56 g). The GL indeed promote the growth of Takifugu rubripes. The GHR1, IGF-I, IGF-II and IGFBP of juvenile Takifugu rubripes cultured under GL were significantly higher than those of WL (control group), BL and YL groups. The relative expression levels of GH and GHRH genes had no difference from those in the WL control group, and the relative expression levels of apoptosis genes in the GL group were significantly lower than those in the BL and YL groups. Under RL, the relative expression levels of all growth genes on the GH/IGF-I growth axis were relatively high. But at the same time, the relative expression of caspase 10 gene in juvenile Takifugu rubripes was high, and the growth state was inhibited. It is speculated that RL might disturb the endocrine system of the juvenile Takifugu rubripes, thus impeded its growth and development. Therefore, different LED spectra can affect the growth of juvenile Takifugu rubripes by affecting the expressions of GH/IGF-I growth axis and apoptosis-related genes: GL significantly promotes the growth of Takifugu rubripes, which may be due to that GL promoted the expressions of growth factors genes such as GHR1, IGF-I, and IGF-II, and decreased the expression of apoptosis-related genes, while the situation in the YL, BL and WL groups was on the contrary. RL significantly inhibited the growth of juvenile Takifugu Rubripes, which may be due to the fact that juvenile Takifugu Rubripes under RL were in a stressful state and the high expression of growth axis-related genes was not sufficient to offset the negative effects of the stress response, resulting in the inhibition of growth performance.

Basic knowledge about the effect of light on fish in needed to improve their development, survival, and growth to increase aquaculture efficiency. In this study, the effects of light photoperiods (light (L): dark (D) = 12:12 and 16:8) and spectrum (full spectrum (W), blue (B) and yellow (Y)) on Takifugu rubripes larvaes were assessed from 20 to 80 days after hatching (dah). At 50 dah, the body length of larvae exposed to Y16:8 and B16:8 was significantly higher than that in the W16:8 group (P

The tiger puffer (Takifugu rubripes) is an important economic fish species in northern China; however, cold stress during overwintering can have serious adverse effects on its aquaculture. Understanding the mechanisms of cold resistance in tiger puffers will thus provide critical information regarding its overwintering process. Here, we compared tiger puffer kidney transcriptomes at 5 °C and 23 °C to identify the key pathways and genes involved in the response to low temperature stress. We identified a total of 1679 differentially expressed genes (DEGs), including 635 up-regulated genes and 1044 down-regulated genes in the 5 °C compared with the 23 °C group. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated five significantly enriched pathways, four of which were related to metabolism. Transcriptome data and quantitative polymerase chain reaction verification showed that most of the DEGs were down-regulated, with a ratio of down-regulated to up-regulated genes of 26:6, among the three significantly enriched pathways involving amino acid and lipid metabolism. Further analysis of the up-regulated genes showed that the arginine and proline pathway was the primary signaling pathway, and that the two metabolic pathways for arginine (including nitric oxide synthase and arginase) were likely to be key factors affecting cold resistance in tiger puffers. Diet supplementation with arginine may thus improve cold tolerance in tiger puffers. These results provided new insights into the stress impact of the aquaculture environment on tiger puffers from the perspective of nutrition.

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.

Copper is one of the predominant water pollutants. Excessive exposure to copper can cause harm to animal health, affecting the central nervous system and causing blood abnormalities. Cuproptosis is a novel form of cell death that differs from previous programmed cell death methods. However, the impact of copper on the intestines remains unclear. Therefore, we investigated the effects of different concentrations of copper exposure on the intestinal proteome of T akifugu rubripes (T. rubripes). Relevant biomarkers were used to detect cuproptosis. We revealed the crosstalk relationship between cuproptosis and self-rescue at different concentrations, and discussed the feasibility of using potential cuproptosis indicators as anti-infection factors. We observed intestinal damage in the three copper exposure groups, especially in T. rubripes treated with 100 and 500 μg/L copper, with shedding and breakage of intestinal villus and fuzzy and loose structure of intestinal mucosa. The presence of copper stress not only causes cuproptosis but also oxidative damage caused by reactive oxygen species (ROS). The results of quantitative proteomics by TMT showed that compared to the 50 and 100 μg/L copper exposure groups, the expression of glutaminase, pyruvate kinase, and skin mucus lectin in the 500 μg/L group was significantly increased. The positive mediators COX5A and CTNNB1, as well as the negative mediators CD4 and FDXR, were found to be differentially expressed. Using the protein expression trends of cuproptosis indicator factors FDX1 and DLAT to indicate the concentration of copper ions in the environment. In addition, we found a new effect of promoting ferroptosis: providing additional copper ions can activate the phenomenon of ferroptosis. Our results expand our understanding of the potential health risks of copper in T. rubripes. At the same time, it is of great significance for the process of copper poisoning and the development of new environmental toxicology detection reagents. • Explore the potential environmental toxicology and immune mechanism of teleosts. • Copper exposure can cause intestinal protein changes and intestinal damage. • Revealed the crosstalk between cuproptosis and copper stress at different Cu2+ concentrations. • The selected relevant biomarkers can be used to detect copper toxicity. • A new effect of promoting ferroptosis: providing additional copper ions. [ABSTRACT FROM AUTHOR]