Takifugu Rubripes Research Articles

Bio-characteristics, tissue expression of miR-375 in hypothalamic-pituitary-ovarian axis and its regulation in reproduction-related diseases

Our study concentrated on the expression of miRNA-375 in the hypothalamic-pituitary-gonadal axis of female Hu sheep. The investigation involved cloning the precursor sequence of miR-NA-375, followed by comparison with database entries and subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In our approach, we obtained ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus, and pineal gland from fertile but nonpregnant Hu ewes. MiRNA extraction kit was used to extract miRNA from the above eight tissues. Real-time fluorescent quantitative polymerase chain reaction was used to evaluate the role of miR-375 in the hy-pothalamic-pituitary-gonadal axis. The results of miR-375 precursor sequence cloning were compared with those of Anopheles gambiae, Apis mellifera, Bos taurus, Drosophila melanogaster, Danio rerio, Fugu rubripes, Gallus gallus, Homo sapiens, Monodelphis domestica, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Tetraodon nigroviridis, Xenopus tropicalis miR-375 in miRBase database. It was found that oar-miR-375 was highly conserved. Notably, miR-375 expression in the pineal gland was significantly higher (p < 0.01) than that in the ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus. The study also involved predicting miR-375 target genes. GO and KEGG enrichment analyses of these predicted target genes revealed that miR-375 is involved in 182 biological processes, affects 186 cellular components, and participates in 184 molecular functions. In terms of pathway enrichment, miR-375 was linked to nine pathways, including the Hippo, Wnt, and mTOR signaling pathways. This study has validated the interaction between miR-375 and its target gene FZD4, which can be recognized and bound to produce effects. These findings lead to the inference that miR-375 may play a crucial regulatory role in sheep reproduction through the Hippo pathway and Wnt pathway, laying a foundation for further exploration of miR-375’s role in this domain.

Epidermal distribution of tetrodotoxin-rich cells in newly hatched larvae of Takifugu spp.

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

Whole-Transcriptome Analysis Reveals Potential CeRNA Regulatory Mechanism in Takifugu rubripes against 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.

A newly developed 20 K SNP array reveals QTLs for disease resistance to Cryptocaryon irritans in tiger pufferfish (Takifugu rubripes)

This study describes the development and application of a 20 K single nucleotide polymorphism (SNP) array in fugu (Takifugu rubripes). The main goal of this newly developed SNP array is to enhance selective breeding practice via the incorporation of genomic information. Fugu is one of the economic aquaculture species and mainly threatened by parasite disease. The performance of the array was evaluated in a farmed pufferfish population challenged with Cryptocaryon irritans and benchmarked against whole-genome sequencing of a subset of the animals. The array showed very high call rates, consistency between technical replicates, and concordance with genome-resequencing data. Heritability estimates using pedigree and SNP datasets demonstrated a significant genetic component of resistance to C. irritans, with heritability estimates ranging from 0.53 to 0.67, and prediction accuracies were significantly improved with the use of genomic data. Genome-wide association study identified two putative quantitative trait loci (QTLs) on chromosomes 3 and 17. Our findings suggest resistance to C. irritans can be rapidly increased in fugu populations by selective breeding, further expedited via the application of genetic markers. Therefore, they represent a valuable resource for selective breeding programmes aimed at enhancing disease resistance in fugu aquaculture.

Effect of Low-Proportion Replacement of Dietary Fishmeal with Neanthes japonica Meal on Growth Performance, Body Composition, Muscle Texture, Serum Biochemistry, Digestive Enzymes and Gene Expression in Juvenile Tiger Puffer Takifugu rubripes

The polychaeta Neanthes japonica is a common by-product in mariculture ponds. It is rich in essential nutrients, but has not been well-explored. Therefore, this 56-day experiment investigated the effects of substituting N. japonica meal for dietary fishmeal on juvenile tiger puffer (15.49 ± 0.02 g, n = 450 fish). The control diet (CON) contained 40% fishmeal. Freeze-dried N. japonica meal (FNM) was supplemented into CON at the levels of 3% (FNM3), 6% (FNM6), and 9% (FNM9), replacing fishmeal. The fifth diet contained 6% oven-dried N. japonica meal (ONM6), replacing fishmeal. The results indicated that no significant difference was observed in growth, feed efficiency, and somatic index among all the treatment groups. The feed intake of the FNM6 group was significantly higher compared to CON. No significant difference was detected in fish proximate composition, as well as the fatty acid composition, amino acid composition, and muscle texture. The supplementation of N. japonica meal decreased the activities of intestinal lipase and α-amylase. The addition of freeze-dried N. japonica meal significantly up-regulated the expression of the intestinal amino-glycine transporter pat1. It was concluded that adding 9% N. japonica meal to the feed had no significant effect on the growth performance and body composition of juvenile tiger puffer.

Effects of Intermittent and Chronic Hypoxia on Fish Size and Nutrient Metabolism in Tiger Puffer (Takifugu rubripes).

Intermittent and chronic hypoxia are common stresses to marine fish, but the different responses of fish to intermittent and chronic hypoxia have not been well-known. In this study, tiger puffers were farmed in normoxia conditions (NO, 6.5 ± 0.5 mg/L), intermittent hypoxia (IH, 6.5 ± 0.5 mg/L in the day and 3.5 ± 0.5 mg/L in the night), or choric hypoxia (CH, 3.5 ± 0.5 mg/L) conditions for 4 weeks, after which the growth, nutrient metabolism and three hifα isoforms expression were measured. Both intermittent and chronic hypoxia decreased the fish growth and visceral weight but increased the feed conversion ratio and blood hemoglobin content. Chronic hypoxia but not intermittent hypoxia promoted protein synthesis and whole-fish protein content by activating mtor gene expression and promoted the glycolysis pathway by activating gene expression of hif1α and hif2α. Intermittent hypoxia but not chronic hypoxia decreased the hepatic lipid synthesis by inhibiting fasn and srebf1 gene expression. Meanwhile, intermittent hypoxia reduced the monounsaturated fatty acid content but increased the n-3 polyunsaturated fatty acids percentage. The results of this study clarified the adaptive mechanism of tiger puffer to intermittent and chronic hypoxia, which provides important information about mechanisms of hypoxia adaption in fish.

Automated phenotyping empowered by deep learning for genomic prediction of body size in the tiger pufferfish, Takifugu rubripes

Although genomic selection (GS) is accelerating the genetic improvement of economically important traits in aquaculture species, the accurate phenotyping of thousands of individuals remains a bottleneck in GS programs. Computer vision (CV) technologies with the assistance of deep learning may provide a cost-efficient solution for automated phenotyping of fish body size. In this study, we designed a CV-based automated phenotyping system based on the deep learning model Mask R-CNN (region-based convolutional neural networks) and tested its ability to predict standard length (SL) of tiger pufferfish (Takifugu rubripes) fed low fishmeal (LFM) diet. Simultaneously, we investigated the impact of the automated phenotyping on genetic parameter estimation and genomic prediction for growth-related traits. We raised a test population (n = 1001) that was fed the LFM diet from three months old until harvest at 22 months. Manual phenotyping of the fish was performed at 10-, 14-, 17-, and 22-months-old. At the last two measurement times, images of each fish were collected to train Mask R-CNN for fish detection and pixel-level segmentation of the fish body excluding the tail fin. An automated phenotyping pipeline combining the trained Mask R-CNN and standard CV algorithms were designed to estimate SL. We found a high correlation (0.965–0.971) and low relative difference (0.015–0.027) between the CV-derived and manually measured SL, indicating high precision of our CV system. A high correlation (0.912–0.956) was also observed between CV-derived body area and manual body weight. The heritability estimations and genomic predictions on manual SLs were conducted using a Genomic Best Linear Unbiased Prediction (GBLUP) model using a total of 16,471 single nucleotide polymorphism (SNP). Estimated heritability at the four sampling points ranged from 0.573 to 0.622, and the predictive ability of genomic prediction for harvest size was 0.627. These values are comparable to those reported in previous studies collected from the population fed a normal fishmeal diet. Our heritability estimation and genomic prediction derived from automated SL measurements differed negligibly to those based on manual measurements. This finding indicates the utility of our CV-based automated phenotyping system for GS breeding programs.

The impact of tetrodotoxin (TTX) on the gut microbiome in juvenile tiger pufferfish, Takifugu rubripes

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

Investigation of copper-induced intestinal damage and proteome alterations in Takifugu rubripes: Potential health risks and environmental toxicology detection

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.

The potential regulatory role of the non-coding RNAs in regulating the exogenous estrogen-induced feminization in Takifugu rubripes gonad

Estrogen plays a pivotal role in the early stage of sex differentiation in teleost. However, the underlying mechanisms of estrogen-induced feminization process are still needed for further clarification. Here, the comparative analysis of whole-transcriptome RNA sequencing was conducted between 17beta-Estradiol induced feminized XY (E-XY) gonads and control gonads (C) in Takifugu rubripes. A total of 57 miRNAs, 65 lncRNAs, and 4 circRNAs were found to be expressed at lower levels in control-XY (C-XY) than that in control-XX (C-XX), and were up-regulated in XY during E2-induced feminization process. The expression levels of 24 miRNAs, and 55 lncRNAs were higher in C-XY than that in C-XX, and were down-regulated in E2-treated XY. Furthermore, a correlation analysis was performed between miRNA-seq and mRNA-seq data. In C-XX/C-XY, 114 differential expression (DE) miRNAs were predicted to target to 904 differential expression genes (DEGs), while in C-XY/E-XY, 226 DEmiRNAs were predicted to target to 2,048 DEGs. In C-XX/C-XY, and C-XY/E-XY, KEGG pathway enrichment analysis showed that those targeted genes were mainly enriched in MAPK signaling, calcium signaling, steroid hormone biosynthesis and ovarian steroidogenesis pathway. Additionally, the competitive endogenous RNA (ceRNA) regulatory network was constructed by 24 miRNAs, 21 lncRNAs, 4 circRNAs and 5 key sex-related genes. These findings suggested that the expression of critical genes in sex differentiation were altered in E2-treated XY T. rubripes may via the lncRNA-miRNA-mRNA regulation network to facilitate the differentiation and maintenance of ovaries. Our results provide a new insight into the comprehensive understanding of the effects of estrogen signaling pathways on sex differentiation in teleost gonads.

Transcriptome Analysis Reveals the Immunosuppression in Tiger Pufferfish (Takifugu rubripes) under Cryptocaryon irritans Infection.

The tiger pufferfish (Takifugu rubripes), also known as fugu, has recently suffered from severe C. irritans infections under aquaculture environment, yet the underlying immune mechanisms against the parasite remain poorly understood. In this study, we conducted a comprehensive transcriptome analysis of the gill tissue from infected and uninfected fish using PacBio long-read (one pooled sample each for seriously infected and healthy individuals, respectively) and Illumina short-read (three pools for mildly infected, seriously infected, and healthy individuals, respectively) RNA sequencing technologies. After aligning sequence data to fugu's reference genome, 47,307 and 34,413 known full-length transcripts were identified and profiled in healthy and infected fish, respectively. Similarly, we identified and profiled 1126 and 803 novel genes that were obtained from healthy and infected fish, respectively. Interestingly, we found a decrease in the number of alternative splicing (AS) events and long non-coding RNAs (lncRNAs) after infection with C. irritans, suggesting that they may be involved in the regulation of the immune response in fugu. There were 687 and 1535 differentially expressed genes (DEGs) in moderately and heavily infected fish, respectively, compared to uninfected fish. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that immune-related DEGs in the two comparison groups were mainly enriched in cytokine-cytokine receptor interactions, ECM-receptor interactions, T-cell receptor signaling pathways, Th1 and Th2 cell differentiation, and Th17 cell differentiation pathways. Further analysis revealed that a large number of immune-related genes were downregulated in infected fish relative to uninfected ones, such as CCR7, IL7R, TNFRSF21, CD4, COL2A1, FOXP3B, and ITGA8. Our study suggests that C. irritans is potentially a highly efficient parasite that may disrupt the defense mechanisms of fugu against it. In addition, in combination of short-read RNA sequencing and previous genome-wide association analyses, we identified five key genes (NDUFB6, PRELID1, SMOX, SLC25A4, and DENND1B) that might be closely associated with C. irritans resistance. This study not only provides valuable resources of novel genic transcripts for further research, but also provides new insights into the immune mechanisms underlying C. irritans infection response in farmed fugu.

Effects of dietary tryptophan levels on growth performance, serotonin metabolism, brain 5-HT and cannibalism activities in tiger puffer, Takifugu rubripes fingerlings

Essential amino acid (EAA) requirements of aquaculture fish species can be used as a guideline for commercial feed formulation. Several EAA requirements have been quantified in tiger puffer (Takifugu rubripes). However, as the third limiting amino acid (AA) of many plant protein sources, tryptophan requirement of this species has yet to be quantified. This study was conducted to determine dietary tryptophan requirement of tiger puffer, and investigate the effect of dietary tryptophan supplementation on the cannibalism, brain serotonin (5-hydroxytryptpamine, 5-HT) metabolism and serum cortisol levels. Six experimental diets were prepared using fish meal, gelatin and corn protein concentrate as intact protein, and then supplemented with crystalline AA premix. The analyzed tryptophan levels in the experimental diets were 3.5, 4.1, 4.5, 5.5, 7.0 and 11.8 g/kg of dry diet. Experimental diets were randomly fed in triplicate to tiger puffer (initial average weight of 17.5 g) for 58 days. The weight gain (WG), feed conversion ratio (FCR) and protein efficiency ratio (PER) were significantly affected by dietary tryptophan, and the break points were observed at 5.07, 4.82 and 4.99 g/kg of dry diet, respectively. Compared to the basal diet, the cannibalism (the rate of bite mark appearance) was significantly lower and survival rate was significantly higher when fish were given diets containing 7.0 and 11.8 g/kg tryptophan. The concentrations of free tryptophan and phenylalanine in the brain exhibited a nonlinear increase in response to the incremental levels of dietary tryptophan. Brain 5-HT concentration was significantly affected by dietary tryptophan levels, but did not show a significant dose-dependent response. The expression of genes related to 5-HT metabolism (tph1a, ddc and aldh) and serotonin reuptake (sert) in the brain varied significantly and exhibited a nonlinear relationship with the graded levels of dietary tryptophan. In addition, serum cortisol levels showed a decreasing trend till the 5.5 g/kg tryptophan inclusion level, at which point there appeared to be the lowest level. In conclusion, based on nonlinear regression analysis of the WG, FCR and PER, dietary tryptophan requirement of tiger puffer was determined to be range between 4.82 and 5.07 g/kg of dry diet (10.48–11.03 g/kg of dietary protein). Dietary tryptophan supplementation above requirement could mitigate cannibalism and improve the survival of tiger puffer, which may be attributed to regulating the synthesis and catabolism of 5-HT and its availability in the synaptic cleft, and subsequently the reduction of serum cortisol content.

Pufferfish gasdermin Ea is a significant player in the defense against bacterial pathogens

Gasdermins (GSDMs) are proteins cleaved by caspase (CASP) to trigger pyroptosis. In teleosts, pyroptosis is mediated by gasdermin E (GSDME). The Pufferfish, Takifugu rubripes, possesses two GSDME orthologs: named TrGSDMEa and TrGSDMEb. TrGSDMEa is cleaved by CASP3/7 to liberate the N-terminal (NT) domain that can trigger pyroptosis in mammalian cells. However, the biological function of TrGSDMEa in pufferfish is unknown, and TrGSDMEb is poorly studied. We found that TrGSDMEb was cleaved by CASP1/3/6/7/8, but the resulting NT domain, despite its similarity to TrGSDMEa-NT domain in sequence and structure, failed to induce pyroptosis. TrGSDMEa and TrGSDMEb exhibited similar expression patterns in pufferfish under normal physiological conditions but were up- and downregulated, respectively, in expression during Vibrio harveyi and Edwardsiella tarda infection. Bacterial infection induced the activation of TrGSDMEa and CASP3/7 in pufferfish cells, resulting in pyroptosis accompanied with IL-1β production and maturation. Inhibition of TrGSDMEa-mediated pyroptosis via TrCASP3/7 reduced the death of pufferfish cells and augmented bacterial dissemination in fish tissues. Structure-oriented mutagenesis identified 16 conserved residues in teleost GSDMEa that were required for the pore formation or auto-inhibition of GSDMEa. This study illustrates the role of GSDMEa-mediated pyroptosis in teleost defense against bacterial pathogens and provides new insights into the structure-based function of vertebrate GSDME.

Teleost-specific TLR23 in Takifugu rubripes recruits MyD88 to trigger ERK pathway and promotes antibacterial defense

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes, found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes, bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost.

Gill transcriptomes analysis of Takifugu obscurus, Takifugu rubripes and their hybrid offspring in freshwater and seawater

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.

Identification of growth-associated miRNAs, circRNAs, and their corresponding regulatory networks in fast- and slow- growing families of Takifugu rubripes

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.

Distribution of pufferfish saxitoxin- and tetrodotoxin-binding protein homolog and tetrodotoxin in the brain and pituitary of juvenile tiger puffer Takifugu rubripes

Pufferfish saxitoxin- and tetrodotoxin (TTX)-binding protein (PSTBP) is considered to transfer TTX between tissues. The immunohistochemical distribution of PSTBP-homolog (PSTBPh) and TTX in the brain and pituitary of hatchery-reared juvenile tiger puffer Takifugu rubripes was investigated. PSTBPh was observed mainly in the pars intermedia of the pituitary. TTX was only detected in a TTX-fed fish in the neurohypophysis of the pituitary and in several other brain regions. The relationship between PSTBPh and TTX is discussed.

High Diversity of Long Terminal Repeat Retrotransposons in Compact Vertebrate Genomes: Insights from Genomes of Tetraodontiformes.

This study aimed to investigate the evolutionary profile (including diversity, activity, and abundance) of retrotransposons (RTNs) with long terminal repeats (LTRs) in ten species of Tetraodontiformes. These species, Arothron firmamentum, Lagocephalus sceleratus, Pao palembangensis, Takifugu bimaculatus, Takifugu flavidus, Takifugu ocellatus, Takifugu rubripes, Tetraodon nigroviridis, Mola mola, and Thamnaconus septentrionalis, are known for having the smallest genomes among vertebrates. Data mining revealed a high diversity and wide distribution of LTR retrotransposons (LTR-RTNs) in these compact vertebrate genomes, with varying abundances among species. A total of 819 full-length LTR-RTN sequences were identified across these genomes, categorized into nine families belonging to four different superfamilies: ERV (Orthoretrovirinae and Epsilon retrovirus), Copia, BEL-PAO, and Gypsy (Gmr, Mag, V-clade, CsRN1, and Barthez). The Gypsy superfamily exhibited the highest diversity. LTR family distribution varied among species, with Takifugu bimaculatus, Takifugu flavidus, Takifugu ocellatus, and Takifugu rubripes having the highest richness of LTR families and sequences. Additionally, evidence of recent invasions was observed in specific tetraodontiform genomes, suggesting potential transposition activity. This study provides insights into the evolution of LTR retrotransposons in Tetraodontiformes, enhancing our understanding of their impact on the structure and evolution of host genomes.

Functional role of TrIL-1β in Takifugu rubripes defense against Cryptocaryon irritans infection

The Japanese puffer, Takifugu rubripes, is a commercially important fish species in China that is under serious threat from white spot disease (cyptocaryoniasis), which leads to heavy economic losses. We previously found that interleukin-1β (IL-1β), an important cytokine with a potential role in resistance against pathogens, was one of the most significantly differentially up-regulated proteins in the gills and spleen of T. rubripes infected by the protozoan parasite Cryptocaryon irritans. In this study, we assessed the potential function of T. rubripes IL-1β (TrIL-1β) in fish infected with C. irritans. Phylogenetic analysis indicated that the TrIL-1β protein sequence was most closely related to that of Atlantic salmon (Salmo salar) (67.2 %). The incubation experiments revealed that TrIL-1β may reduce trophont activity by destroying membranes. Immunofluorescence experiments showed that recombinant TrIL-1β promoted the expression of endogenous IL-1β, which penetrated and disrupted the cell membranes of trophonts. Transmission electron microscopy showed that the IL-1β group had less tissue damage compared with control groups of fish. IL-1β-small interfering RNA and IL-1β overexpression experiments were performed in head kidney primary cells, and challenge experiments were performed in vitro. Quantitative RT-PCR results showed that TrIL-1β regulated and activated MyD88/NF-κB and MyD88/MAPK/p38 signaling pathways during C. irritans infection. TrIL-1β also promoted the differential expression of IgM, showing that it was involved in humoral immunity of T. rubripes. The cumulative mortality experiment show that TrIL-1β could protect fish against C. irritans infection. These results enrich current knowledge about the molecular structure of TrIL-1β. They also suggested that recombinant TrIL-1β could be used as an adjuvant in a subunit vaccine against C. irritans infection, which is of profound importance for the prevention and control of parasitic diseases in T. rubripes.

Database construction and comparative genomics analysis of genes involved in nutritional metabolic diseases in fish

Nutritional metabolic diseases in fish frequently arise in the setting of intensive aquaculture. The etiology and pathogenesis of these conditions involve energy metabolic disorders influenced by both internal genetic factors and external environmental conditions. The exploration of genes associated with nutritional and metabolic disorder has sparked considerable interest within both the aquaculture scientific community and the industry. High-throughput sequencing technology offers researchers extensive genetic information. Effectively mining, analyzing, and securely storing this data is crucial, especially for advancing disease prevention and treatment strategies. Presently, the exploration and application of gene databases concerning nutritional and metabolic disorders in fish are at a nascent stag. Therefore, this study focused on the model organism zebrafish and five primary economic fish species as the subjects of investigation. Using information from KEGG, OMIM, and existing literature, a novel gene database associated with nutritional metabolic diseases in fish was meticulously constructed. This database encompassed 4583 genes for Danio rerio, 6287 for Cyprinus carpio, 3289 for Takifugu rubripes, 3548 for Larimichthys crocea, 3816 for Oreochromis niloticus, and 5708 for Oncorhynchus mykiss. Through a comparative systems biology approach, we discerned a relatively high conservation of genes linked to nutritional metabolic diseases across these fish species, with over 54.9 % of genes being conserved throughout all six species. Additionally, the analysis pinpointed the existence of 13 species-specific genes within the genomes of large yellow croaker, tilapia, and rainbow trout. These genes exhibit the potential to serve as novel candidate targets for addressing nutritional metabolic diseases.