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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.

Aggressive behavior is important for animals to obtain limited resources. Understanding fish behavior and physiological response is of great significance to evaluate aquaculture production and fish welfare. Food is an important trigger of aggressive behavior in juvenile fish under high-density aquaculture conditions. The aim of this study was to investigate the aggressive behavior and monoamine levels of juvenile pufferfish (mean body mass of 6.29 ± 0.33 g) under normal feeding and restricted feeding. Our main results included the following: (1) The mortality and fin damage were higher and aggression was more intense of juvenile pufferfish at the 1% ration than those of the 3% ration; (2) during feeding, the velocity, body contact, and activity at the 1% ration were significantly higher than that of the 3% ration; (3) the concentrations of brain 5-hydroxyindoleacetic acid (5-HIAA) and monoamine oxidase A (MAOA) at the 1% ration were significantly lower, and dopamine (DA) concentrations were significantly higher. These results suggest that juvenile pufferfish shows serious aggressive behavior at the low ration, which may be related to the decrease of 5-HIAA and MAOA concentrations, and the increase of DA concentrations.

The present study was aimed at screening suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) in tiger puffer (Takifugu rubripes), an important aquaculture species in Asia and also a good model species for lipid research. Specifically, this reference gene screening was targeted at standardization of gene expression in different tissues (liver, muscle, brain, intestine, heart, eye, skin, and spleen) or under different nutritional conditions (starvation and different dietary lipid levels). Eight candidate reference genes (ribosomal protein L19 and L13 (RPL19 and RPL13), elongation factor-1 alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase1 (HPRT1), beta-2-Microglobulin (B2M), 18S ribosomal RNA (18SrRNA), and beta actin (ACTB)) were evaluated with four algorithms (geNorm, NormFinder, BestKeeper, and comparative ΔCt method). The results showed that different algorithms generated inconsistent results. Based on these findings, RPL19, EF1α, 18SrRNA, and RPL13 were relatively stable in different tissues of tiger puffer. During starvation conditions, ACTB/RPL19 was the best reference gene combination. Under different dietary lipid levels, ACTB/RPL13 was the most suitable reference gene combination. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in tiger puffer.

The tissue distribution pattern of lipid is highly diverse among different fish species. Tiger puffer has a special lipid storage pattern, storing lipid predominantly in liver. In order to better understand the lipid physiology in fish storing lipid in liver, the present study preliminarily investigated the tissue distribution of transcription for 29 lipid metabolism-related genes in tiger puffer, which are involved in lipogenesis, fatty acid oxidation, biosynthesis and hydrolysis of glycerides, lipid transport, and relevant transcription regulation. Samples of eight tissues, brain, eye, heart, spleen, liver, intestine, skin, and muscle, from fifteen juvenile tiger puffer were used in the qRT-PCR analysis. The intestine and brain had high transcription of lipogenic genes, whereas the liver and muscle had low expression levels. The intestine also had the highest transcription level of most apolipoproteins and lipid metabolism-related transcription factors. The transcription of fatty acid β-oxidation-related genes was low in the muscle. The peroxisomal fatty acid oxidation may dominate over mitochondrial β-oxidation in the liver and intestine of tiger puffer, and the MAG pathway probably predominates over the G3P pathway in re-acylation of absorbed lipids in the intestine. The intracellular glyceridases were highly transcribed in the brain, eye, and heart. In conclusion, in tiger puffer, the intestine could be a center of lipid metabolism whereas the liver is more likely a pure storage organ for lipid. The lipid metabolism in the muscle could also be inactive, possibly due to the very low level of intramuscular lipid.

We assessed the effects of light intensity and spectrum on the growth and survival of Takifugu rubripes larvae from 30 to 69 days after hatching. Five lighting regimes were applied using 0.5, 1.5, and 3.0 W m−2 full spectrum white (W0.5, W1.5, W3.0), 0.5 W m−2 yellow (Y0.5), and 0.5 W m−2 blue light (B0.5). At the end of the experiment, body length, wet weight, and specific growth rate from day 0 to day 39 were significantly greater in larvae reared under W3.0 than under B0.5 (P ˂ 0.05). No significant differences were observed among W0.5, W1.5, and W3.0, or among W0.5, Y0.5, and B0.5 (P > 0.05). Survival rate was significantly higher in larvae reared under W1.5 than W0.5 (P ˂ 0.05), but no significant differences were observed among W0.5, Y0.5, and B0.5 (P > 0.05). Additionally, light conditioning did not affect the total thickness of the retina. Although the ratio of the thickness of the retinal pigment epithelium layer/total thickness (TT) was significantly higher in larvae exposed to W3.0 compared with those exposed to other light conditions, and the thickness of the outer nuclear layer/TT was significantly lower in larvae exposed to W3.0 compared with those exposed to W0.5 (P

Hypoxia is reduced levels of oxygen. Especially in water, due to the complex environment, hypoxic situations often occur. Although fish can survive in low-oxygen waters, this survival ability depends on a complete set of coping mechanisms such as oxygen perception and gene-protein interaction regulation. The research on this mechanism is very meaningful. The present study was undertaken to examine the short-term effects of hypoxia on the brain in Takifugu rubripes. We sequenced the transcriptomes of the brain in T. rubripes to studied their response mechanism to acute hypoxia. Total 167 genes with adjusted P valuesT. rubripes exposed to acute hypoxia. However, hif1a, the master transcriptional regulator of the adaptive response to hypoxia, was not significantly regulated, which indicated that the T. rubripes brain might prevent the HIF-1 signaling pathway. Then Gene Ontology and KEGG Enrichment Analysis were carried out. The results indicated that hypoxia could cause metabolic and neurological changes, showing the clues of their adaptation to acute hypoxia. Overall, the sequenced transcriptomes of the brain in T. rubripes showed small changes under acute hypoxia. As the most complex and important organ, the brain of T. rubripes might be able to create a self-protection mechanism to resist or reduce damage caused by acute hypoxia stress.

Quantifying the expression of mRNAs in the gonads at the critical stage of molecular sex differentiation stage might help to clarify the regulatory network during early sex differentiation and provide new information on the role of sex-related genes in gonadal function. In this study, transcriptomic analysis of sex-related genes expression profiles in fugu gonads at 60 and 90 days after hatching (dah) was conducted firstly, and a total of 112,504,991 clean reads, encompassing 28.35 Gb of sequences were retrieved. Twenty-three thousand eight hundred ten genes were found to be expressed in juvenile fugu gonads, and we mainly focused on the differentially expressed genes that have the potential to be involved in the gonadal sex differentiation. For 60-dah juveniles, we identified 1014 genes that were upregulated in the ovary and 1570 that were upregulated in the testis. For 90-dah juveniles, we identified 1287 genes that were upregulated in the ovary and 1500 that were upregulated in the testis. The dimorphic expression patterns of 15 genes in gonads at 30 and 40 dah were further investigate using qPCR. Cyp11b and star were expressed at higher levels in XY than in XX, while cyp11a1 and cyp19a1a were expressed at higher levels in XX than in XY at 30 dah. At 40 dah, the levels of gsdf, dmrt1, dmrt3, cyp11c1, star, and hsd3b expression were higher in XY, while the levels of foxl2, cyp19a1a, wnt9b, and foxD4 expression were higher in XX. Sox9, cyp11a1, cyp17a1, cyp17a2, and nr5a2 were expressed at similar levels in XX and XY at 40 dah. This is the first report of gonadal transcriptome of fugu at early sex differentiation stage, and our results provide an archive for further study on molecular mechanism underlying sex differentiation in this species.

The critical swimming speed (U crit, cm s−1) of juvenile tiger puffer Takifugu rubripes was determined under different temperatures (15, 21, 25 and 30 °C), salinities (5, 10, 20, 32 and 40), body lengths (3.32, 4.08, 5.06 and 5.74 cm) and starvation days (1, 3, 6 and 9 days). Acute temperature change, body length and starvation significantly influenced the U crit of tiger puffers, whereas acute salinity change had no significant effect. The U crit increased as the temperature increased from 15 to 30 °C. The U crit increased as the body length increased from 3.32 to 5.74 cm, whereas relative critical swimming speed (U crit’, body length s−1) decreased. The relationship between the body length (l, cm) and U crit or U crit’ can be described by the quadratic model as U crit = − 1.4088 l 2 + 16.976 l − 11.64, R 2 = 0.9698 (P

As an important economic marine species cultured in China, Chinese tongue sole (Cynoglossus semilaevis) has interested us due to its sexual dimorphism and ZW/ZZ sex determination system. In a previous study, dmrt1 was identified as a dosage-dependent male-determining gene. In the present study, a female-specific expressed gene, cse0440, initially annotated as lrp1b-like, was identified from chromosome W of C. semilaevis. In view of the differences between cse0440 and lrp1b in terms of expression pattern, a phylogenetic analysis containing 85 LRP proteins was constructed and provided an evidence to re-annotate cse0440 as cseLRP13. In addition, two orthologues of cseLRP13 were separately identified from W and Z chromosomes: cseLRP13-W and cseLRP13-Z. The subsequent multiple sequence alignment and syntenic arrangements of LRP13 in C. semilaevis, Japanese medaka (Oryzias latipes), large yellow croaker (Larimichthys crocea), striped bass (Morone saxatilis), white perch (Morone americana) and Fugu rubripes (Takifugu rubripes) further supported this re-annotation. RT-PCR and in situ hybridization revealed that cselrp13 was exclusively expressed in the oocytes and follicles of ovaries. These results suggested that lrp13 may play important roles in female reproduction. In future, with the advancement of micromanipulation in flatfish, the detailed function of two lrp13 orthologues in C. semilaevis will be elucidated.

This study aims to investigate the genes encoding prolactin (PRL) and prolactin receptors (PRLR) and their tissue-specific expression in starry flounder Platichthys stellatus. Starry flounder PRL gene consisting of five exons encodes an ORF of 212 amino acid residue comprised of a putative signal peptide of 24 amino acids and a mature protein of 188 amino acids. It showed amino acid identities of 73 % with tuna Thunnus thynnus, 71 % with black porgy Acanthopagrus schlegelii, 69 % with Nile tilapia Oreochromis niloticus, 64 % with pufferfish Takifugu rubripes, 63 % with rainbow trout Oncorhynchus mykiss, and 60 % with mangrove rivulus Kryptolebias marmoratus. Phylogenetic analysis of piscine PRLs also demonstrated a similarity between starry flounder and other teleosts but with a broad distinction from non-teleost PRLs. PRLR gene consists of eight exons encoding a protein of 528 amino acid residues. It showed a similarity to the PRLR2 subtype as reflected by amino acid identities of 54 % with A. schlegelii, 48.1 % with K. marmoratus, 46.3 % with tilapia O. mossambicus, and 46.1 % with O. niloticus PRLR2 as compared to PRLR1 isoform having less than 30 % identities. While mRNA transcript corresponding to PRL was detected only from the pituitary, most of PRLR mRNA was detected in the gill, kidney, and intestine, with a small amount in the ovary. The level of PRL transcript progressively increased during 6 days of acclimation to freshwater and then decreased but stayed higher than that of seawater at 60 days of acclimation. An opposite pattern of changes including a decrease at the beginning of the acclimation but a slight increase in the level osmolality was found as adaptation continued. The results support the osmoregulatory role of PRL signaling in starry flounder.

Two cDNA libraries from Takifugu rubripes spermatozoa and eggs were constructed and a total of 620 expressed sequence tag (EST) clones were generated from the two libraries: 300 clones are from the spermatozoa library and 320 clones are from the eggs library. The most abundant cDNA clones in the two libraries were identified. A total of 207 'contigs' (or single) EST clones were found to share significant sequence identity with known sequences in the GenBank database, representing at least 51 different genes. In order to understand the two types of germ cells further, the expression profiles of the identified clones in these cDNA libraries were analyzed. Furthermore, the presence of specific messenger RNAs in the spermatozoa and eggs has been demonstrated with BLAST analysis; the spermatozoa and egg library can supply unique and novel cDNA sequences in the Takifugu rubripes EST project. Another aim of this study is to identify cDNA clones that can be used as molecular markers for the analysis of the spermatogenesis and oogenesis in Takifugu rubripes. Six potential clones (S1-3 from spermatozoa and E1-3 from eggs) were selected to analyze their expression patterns by reverse transcription (RT)-PCR analyses. Half of these showed a specific expression in the expected tissue. Two of the clones were found by RT-PCR and in situ hybridization to be expressed specifically in the testis or ovary, and they maybe suitable molecular markers for the analysis of spermatogenesis and oogenesis.

In vitro accumulation of tetrodotoxin (TTX) and paralytic shellfish toxin (PST) in tiger puffer fish Takifugu rubripes was investigated using liver tissue slices. When T. rubripes liver slices were incubated with Leibovitz’s L-15 medium containing 0.13 mM TTX at 20 °C in air with saturated humidity, they accumulated 21.5 ± 7.3 μg TTX g−1 liver after the incubation for 12 h and increased to 55.3 ± 8.2 μg TTX g−1 liver at 48 h. In the incubation of T. rubripes liver slices with 0.13 mM PST-containing medium, PST was detected 6.3 ± 0.9 μg g−1 liver at 12 h and reached a plateau thereafter. These results reveal the difference between TTX and PST in accumulation in T. rubripes liver tissue slices. To examine the variation in PST accumulation among fish species, the liver tissue slices from tiger puffer fish T. rubripes, parrot-bass Oplegnathus fasciatus and green ling Hexagrammos otakii were incubated at a concentration of 0.027 mM PST. The toxin contents of 3.0 μg g−1 liver were observed at 8 h regardless of fish species but were not increased subsequently, showing no variety among these three species as to accumulation patterns of PST. It is noted that the tiger puffer fish T. rubripes liver specifically accumulate TTX in preference to PST.

We isolated two types of cDNA clone encoding hemopexin-like protein, fWap65-1 and fWap65-2, from the pufferfish Takifugu rubripes. The deduced amino acid sequence of fWap65-1 showed 66–67% identity with those of goldfish and carp Wap65s, whereas the sequence of fWap65-2 did 44–46% identity. Both fWap65s showed 32–38% amino acid identity with mammalian hemopexins, in which fWap65-2 was more related than fWap65-1. While hemopexins contain two conserved histidine residues in their heme binding pockets, these residues were also conserved in fWap65-2, but not in fWap65-1. The exon-intron organization was highly conserved between fWap65s and human hemopexin gene, suggesting that Wap65s are fish orthologs of human hemopexin. The 5′-flanking regions of both fWap65s contained various putative transcriptional elements, including Cdx1, GATA-1, C/EBPβ and LyF-1. The expression patterns of fWap65s in various tissues of Fugu were examined by RT-PCR, demonstrating the dominant expression of both genes in liver followed by brain. In addition, the small quantities of fWap65-1 transcripts were also detected in eye, gill and gonad, whereas the transcripts of fWap65-2 could not be observed except for liver and brain. Although the average values for mRNA levels of both fWap65s in warm-acclimated fish tended to be higher than those of cold-acclimated fish, their differences were not statistically significant.