Tiger Pufferfish Takifugu Rubripes Research Articles

Heritability and predictive ability for the heterobothriosis resistance and growth performance in the tiger pufferfish Takifugu rubripes fed standard or low fishmeal diets

Heterobothriosis resistance and high growth performance are major concerns in the tiger pufferfish (Takifugu rubripes) farming. Our previous small-scale study suggested the feasibility of genomic selection (GS) for these traits but also identified an undesired genetic correlation between them, which could hamper simultaneous genetic improvement. Here, we sought to validate these initial findings through larger samples and crosses and also explore the potential of GS for the individuals fed a normal fishmeal diet and a low fishmeal (LFM) diet. We produced three populations: one was fed a normal diet (PopFM: 1100 individuals) and two were fed an LFM diet (PopLFM1: 993 individuals; PopLFM2: 1017 individuals); all three populations were artificially infected with Heterobothrium okamotoi, a monogenean parasite causing heterobothriosis. We recorded standard lengths of the fish after artificial infection (SLEAI) and the number of parasites on the gill cavity wall (HC) and genotyped genome-wide single nucleotide polymorphisms (SNPs) using genotyping by random amplicon sequencing, direct (GRAS-Di) technology for each sample: PopFM, 12 K SNPs; PopLFM1, 19 K SNPs; PopLFM2, 21 K SNPs. Heritability and prediction ability for each trait were estimated for each population using genomic best linear unbiased prediction (GBLUP), Bayes C, and Bayesian ridge regression methods. SNP effects on each trait were estimated by Bayes C, Bayesian ridge regression, and genome-wide association analyses (GWAS) using the same data sets. We observed moderate to high levels of heritability for HC (0.16–0.57) and SLEAI (0.50–0.77), with predictive ability of 0.25–0.55 for HC and of 0.51–0.73 for SLEAI. These values were slightly larger in LFM diet groups. As reported previously, a weak undesired genetic correlation was observed between the traits, and the correlation was more pronounced in the LFM groups. In conclusion, our results indicate that genetic gain in growth and parasite resistance traits can be expected for the tiger pufferfish through GS, especially for individuals reared on an LFM diet. However, close attention should be paid to the extent of the undesirable genetic correlation between the two traits that may be amplified in the populations fed an LFM diet.

The variation of intestinal autochthonous bacteria in cultured tiger pufferfish Takifugu rubripes.

Intestinal autochthonous bacteria play important roles in the maintenance of the physiological homeostasis of animals, especially contributing to the host immune system. In the present study, the variation of autochthonous bacterial community in the intestinal tract of 2-7 months-old tiger pufferfish Takifugu rubripes and bacterial communities in the seawater of recirculating aquaculture system (RAS) and the following offshore sea cage aquaculture system (OSCS) were analyzed during the aquaculture period from May to October 2021. Proteobacteria was found to be the most dominant phyla in both intestinal and seawater bacterial communities, which accounted for 68.82% and 65.65% of the total bacterial abundance, respectively. Arcobacter was the most core bacterial taxon in the intestinal bacterial community, with the most dominant abundance (42.89%) at the genus level and dominant positions in co-occurrence relationships with other bacterial taxa (node-betweenness value of 150). Enterococcaceae was specifically enriched in the intestinal bacterial community of pufferfishes from RAS, while Vibrionaceae was enriched in the intestinal bacterial community from OSCS. The F-values of beta diversity analysis between intestinal and seawater bacterial communities generally increased from May (6.69) to October (32.32), indicating the increasing differences between the intestinal and seawater bacterial communities along with the aquaculture process. Four bacterial taxa of Weissella sp., Akkermansia muciniphila, Dietzia sp. and Psychrobacter pacificensis had significant correlations with immune response parameters, and they were suggested to be the indicators for immune status and pathological process of pufferfish. The knowledge about the specific core bacteria, potentially pathogenic bacteria and the change of bacterial community in the intestinal tract of cultured pufferfish is of great scientific significance and will contribute to the understanding of intestinal bacterial homeostasis and biosecurity practice in pufferfish aquaculture.

Reduced lifetime fitness (growth, body condition and survivability) of hatchery-reared tiger pufferfish Takifugu rubripes compared to wild counterparts.

Tiger pufferfish Takifugu rubripes (order Tetraodontiformes, family Tetraodontidae) is a highly exploited species and stocks continue to decline, although hatchery-reared juveniles have been released since 1965 for stock enhancement. To determine why the stock has not recovered through hatchery-release practices, this study investigated and compared the population characteristics of wild and hatchery-origin fish. The length-mass relationship showed that hatchery-origin fish were skinnier, with males weighing less than 90% of the mass of wild males of the same length. The hepatosomatic index tended to be lower in hatchery-origin fish. Age was estimated using the otolith-based method, and the estimates were more accurate and precise than those obtained by the conventional vertebra-based method. At the age of 2.9 years, an age at which specimens were the most abundant in catches, hatchery-origin males weighed only 67% of wild males. The maximum observed age was 12 years for wild fish and 5 years for hatchery-origin fish. The instantaneous total mortality rates of hatchery-origin fish were more than twice as high as those of wild fish. In summary, the hatchery-origin fish had poor health status, poor growth and high mortality, and their fitness in natural environments was therefore hypothesized to be low throughout life.

Genetic Dissection of a Precocious Phenotype in Male Tiger Pufferfish (Takifugu rubripes) using Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di)

The novel non-targeted PCR-based genotyping system, namely Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di), is characterized by the simplicity in library construction and robustness against DNA degradation and is expected to facilitate advancements in genetics, in both basic and applied sciences. In this study, we tested the utility of GRAS-Di for genetic analysis in a cultured population of the tiger pufferfish Takifugu rubripes. The genetic analyses included family structure analysis, genetic map construction, and quantitative trait locus (QTL) analysis for the male precocious phenotype using a population consisting of four full-sib families derived from a genetically precocious line. An average of 4.7 million raw reads were obtained from 198 fish. Trimmed reads were mapped onto a Fugu reference genome for genotyping, and 21,938 putative single-nucleotide polymorphisms (SNPs) were obtained. These 22 K SNPs accurately resolved the sibship and parent–offspring pairs. A fine-scale linkage map (total size: 1,949 cM; average interval: 1.75 cM) was constructed from 1,423 effective SNPs, for which the allele inheritance patterns were known. QTL analysis detected a significant locus for testes weight on Chr_14 and three suggestive loci on Chr_1, Chr_8, and Chr_19. The significant QTL was shared by body length and body weight. The effect of each QTL was small (phenotypic variation explained, PVE: 3.1–5.9%), suggesting that the precociousness seen in the cultured pufferfish is polygenic. Taken together, these results indicate that GRAS-Di is a practical genotyping tool for aquaculture species and applicable for molecular breeding programs, such as marker-assisted selection and genomic selection.

Genomic selection for heterobothriosis resistance concurrent with body size in the tiger pufferfish, Takifugu rubripes

Parasite resistance traits in aquaculture species often have moderate heritability, indicating the potential for genetic improvements by selective breeding. However, parasite resistance is often synonymous with an undesirable negative correlation with body size. In this study, we first tested the feasibility of genomic selection (GS) on resistance to heterobothriosis, caused by the monogenean parasite Heterobothrium okamotoi, which leads to huge economic losses in aquaculture of the tiger pufferfish Takifugu rubripes. Then, using a simulation study, we tested the possibility of simultaneous improvement of parasite resistance, assessed by parasite counts on host fish (HC), and standard length (SL). Each trait showed moderate heritability (square-root transformed HC: h2 = 0.308 ± 0.123, S.E.; SL: h2 = 0.405 ± 0.131). The predictive abilities of genomic prediction among 12 models, including genomic Best Linear Unbiased Predictor (GBLUP), Bayesian regressions, and machine learning procedures, were also moderate for both transformed HC (0.248‒0.344) and SL (0.340‒0.481). These results confirmed the feasibility of GS for this trait. Although an undesirable genetic correlation was suggested between transformed HC and SL (rg = 0.228), the simulation study suggested the desired gains index can help achieve simultaneous genetic improvements in both traits.

Precocious maturation in male tiger pufferfish Takifugu rubripes: genetics and endocrinology

Testes of the tiger pufferfish Takifugu rubripes are a delicacy in Japan, and selective breeding for a male precocious phenotype, i.e., with early initiation of testes development, is desirable. However, it is unknown if precocious gonad development in this species is under genetic control. Here, we investigated genetic involvement in precociousness by using progeny tests with sires from two cultured populations, including a family line anecdotally known for its precociousness, and a wild population. Progeny derived from the “precocious” line consistently had greater testes weight than that from the other lines, even after accounting for effects of body weight, which indicates that precociousness is truly heritable. We also compared chronological changes in plasma steroid hormones between progenies sired by males from the precocious line and a wild population, and found that the precocious family line had higher levels of plasma estradiol-17β (E2) prior to the initiation of testicular development. Our findings suggest that selective breeding for testes precociousness in the tiger pufferfish is feasible, and that plasma E2 may be an indicator of this phenotype, which would allow for phenotype evaluation without the need to sacrifice specimens.

Genetic variation in resistance of the tiger pufferfish Takifugu rubripes to a host-specific monogenean parasite Heterobothrium okamotoi

The tiger pufferfish Takifugu rubripes is one of the most valuable aquaculture species in Japan. In this study, we investigated the genetic variation in resistance to heterobothriosis caused by a host-specific monogenean parasite, Heterobothrium okamotoi, in the tiger pufferfish, and examined the possibility of selective breeding for enhancing resistance traits. A total of 299 tiger pufferfish produced from ten males and one female caught from the wild were experimentally exposed to oncomiracidia of H. okamotoi. The number of parasites on the gills at 3 weeks post-infection ranged from 0 to 68, with great variation observed both within and between families. The estimated heritability for parasite density was small, but significantly different from zero (H2 = 0.098; 95% confidence interval: 0.025–0.201). Although we may have sampled a limited fraction of the genetic variance as the number of families was small, the results suggest that there are genetic components that affect heterobothriosis resistance in the species. A positive phenotypic correlation was observed between parasite count and standard length (r = 0.66). Further investigation of the genetic correlation between the two traits using larger families is needed to assess the impacts of selective breeding on heterobothriosis resistance in this species.

An effective microinjection method for genome editing of marine aquaculture fish: tiger pufferfish Takifugu rubripes and red sea bream Pagrus major

Genome editing technology is becoming increasingly accepted as a way to improve traits in marine fish aquaculture. In fish, microinjection is a major method for introducing RNA or protein into eggs for genome editing; however, this method has not yet been established in aquaculture fish. We successfully established microinjection methods achieving high survival rates for tiger pufferfish and red sea bream by optimizing the following three parameters: (1) the soaking solution of fertilized eggs during microinjection, (2) the elapsed time from in vitro fertilization to microinjection, (3) the elapsed time from stripping to microinjection. In tiger pufferfish, Iwamatsu solution or diluted sea water is effective as the soaking solution. In vitro fertilization can be performed at intervals of 15 min from fertilization until 2.5 h after stripping. Similarly, in red sea bream, Leibovitz’s L-15 medium or Iwamatsu solution is effective as the soaking solution and in vitro fertilization can be performed at intervals of 10 min from fertilization until 2.5 h after stripping. We anticipate our findings will contribute to effectively establish genome edited aquaculture breeds.

High concentrations of perfluorooctane sulfonate in mucus of tiger puffer fish Takifugu rubripes: a laboratory exposure study.

Distribution of perfluorooctane sulfonate (PFOS) was investigated in tissues (plasma, blood clot, mucus, skin, liver, muscle, and gonad) of tiger puffer fish Takifugu rubripes. A single dose of PFOS was intraperitoneally injected at 0.1mg/kg body weight with samples taken over a 14-day period. The highest concentration of PFOS was found in the plasma, 861ng/mL at 14days, followed by the mucus, liver, blood clot, gonads, muscles, and skin of fish. A gradual upward trend in PFOS concentration was observed in the mucus and liver whereas there was no change in the plasma, blood clot, gonad, muscle, and skin after the initial increase in PFOS concentrations following injection. No significant trend for estimated total PFOS content in whole body was observed during the experimental period. Relatively high concentrations of PFOS (690ng/g ww after 14days) were detected in body surface mucus that continuously oozes from the skin. These results may suggest that mucus is one of the elimination pathways of PFOS in tiger puffer fish.

Molecular Identification and Toxicity of Pufferfish Juveniles Contaminating Whitebait Products

Catches of whitebait, sardine fry, sometimes contains other marine animals, including fishes, mollusks, and crustaceans, and therefore boiled and dried whitebait products may contain these marine animals if sorting is incomplete. In September 2014, contamination of boiled and dried whitebait products with pufferfish juveniles became a serious food safety concern, as tiger pufferfish Takifugu rubripes juveniles are toxic and contain tetrodotoxin (TTX). The toxicity of the juveniles of other pufferfish species, however, is unclear. To evaluate the food safety of whitebait products contaminated with pufferfish juveniles, we identified the species and toxicity of pufferfish juveniles contaminating whitebait products processed between July and September, 2014. Nucleotide sequence analysis of 16S rRNA or cytochrome b gene fragments of the mitochondrial DNA indicated that partial sequences of the polymerase chain reaction products of 15 specimens were identical with those of Lagocephalus spadiceus, and partial sequence from 2 specimens were identical with those of Takifugu vermicularis. We analyzed TTX by liquid chromatography-tandem mass spectrometry. TTX was not detected in the L. spadiceus specimens and was below the quantification limits (30 ng/g) in a T. vermicularis specimen. Based on whitebait product manufacturer's research, 795 individuals and 27.2 g of pufferfish juveniles were detected in 8,245 kg whitebait product. Thus, the ratio of pufferfish to whitebait product was estimated to be 0.096 individual/kg whitebait product and 0.0033 g/kg whitebait product, respectively.

Comparison of tetrodotoxin uptake and gene expression in the liver between juvenile and adult tiger pufferfish, Takifugu rubripes

Marine pufferfish of the family Tetraodontidae accumulate high levels of tetrodotoxin (TTX). The profile of TTX accumulation is reported to differ between tiger pufferfish Takifugu rubripes juveniles and adults administered TTX. Adults mainly accumulate TTX in liver, while juveniles transfer TTX from the liver to the skin. In the present study, we investigated TTX uptake into liver tissue slices of T. rubripes juveniles (4-month-old) and adults (18-month-old) in an in vitro incubation experiment, and compared their differential gene expression profiles in the liver by suppression subtracted hybridization (SSH). The tissue culture experiment revealed that TTX uptake in the liver itself was indistinguishable between the juveniles and the adults. In SSH analysis, a total of 176 clones were upregulated in the juvenile liver, the majority of which comprised hemoglobin subunit alpha-2-like gene (53 clones), hemoglobin subunit beta-like gene (40 clones), and type-4 ice-structuring protein LS-12-like gene (20 clones). A total of 211 clones were upregulated in the adult liver, including serotransferrin-like gene (84 clones), fibrinogen beta chain-like gene (15 clones), and 14 kDa apolipoprotein gene (10 clones). Based on these and previous findings on genes related to TTX intoxication in pufferfish, serotransferrin-like gene, complement C3-like gene, water-temperature-acclimation-related-65 kDa-protein-like gene, and chymotrypsin elastase family member 2A-like gene appear to be involved in TTX toxification of the T. rubripes liver.

Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes

It is well known that perfluorooctane sulfonate (PFOS) preferentially accumulates in the plasma of wildlife and humans. Although earlier studies have suggested that this was due to binding of PFOS to a plasma protein, definite characterization of the protein in in vivo exposure studies was not conducted thus far. In this study, we conducted both in vitro and in vivo experiments to identify PFOS binding protein in the plasma of fish. For the in vivo studies, PFOS was administered intraperitoneally to tiger pufferfish, Takifugu rubripes, and the plasma was separated by ammonium sulfate fractionation. High concentrations of PFOS were found in the 65–70 percent ammonium sulfate fraction (190ng/mL). After SDS-PAGE and N-terminal amino acid sequence analysis, the PFOS-binding protein was identified as an apolipoprotein A–I, which was confirmed on the basis of a significant correlation to the PFOS concentration in each fraction. The plasma samples fractionated by ammonium sulfate from untreated pufferfish were subjected to PFOS binding assay by the equilibrium dialysis method. The results further confirmed that the 60–65 percent ammonium sulfate fraction showed a high PFOS-binding ratio, similar to that found from in vivo studies. We demonstrated that PFOS is likely bound to an apolipoprotein A–I in the plasma of tiger pufferfish in in vivo and in vitro studies.

Metabolomic investigation of pathogenesis of myxosporean emaciation disease of tiger puffer fish Takifugu rubripes

Serum biochemical analysis was undertaken to study the pathophysiological details of emaciation disease of the tiger puffer fish Takifugu rubripes (Temminck and Schlegel). Serum parameters were measured by biochemical analysis using automated dry chemistry and gas chromatography/mass spectrometry (GC/MS). Serum concentrations of albumin, amylase, calcium, creatinine, glucose and total protein were significantly lower in the emaciated fish when compared with those of normal fish. Regression analyses found close correlation between concentrations of total protein, albumin, amylase, glucose and progress of the disease. In contrast, serum alanine aminotransferase increased significantly in emaciated fish indicating liver function disorder. Further, GC/MS metabolic profiling of the puffer serum showed that the profile of the emaciated fish was distinct to that of non-infected control. The serum content of amino acids including glycine, 5-oxo-proline and proline, and ascorbic acid, fumaric acid and glycerol increased significantly in serum in moderately emaciated fish. The serum glucose, linolenic acid and tyrosine level decreased significantly in the late phase of the disease. Our results clearly show that prolonged intestinal damage caused by myxosporean infection impairs absorption of nutrients, resulting in extreme emaciation.

Isolation of collagen from tiger pufferfish parts and its solubility in dilute acetic acid

Collagen was extracted from several tissues (muscle, skin, bone, alimentary tract, gill, fin, hepatopancreas, and air bladder) of the tiger pufferfish Takifugu rubripes, and the content and solubility of the collagen extracted from each tissue were examined. Collagen content in ordinary muscle was 0.95 ± 0.07 % of wet tissue, which is lower than that reported for other fish species even though tiger pufferfish meat has a tough texture. The solubility of collagen extracted from the muscle and skin was relatively high, and collagen accounted for 47.2 ± 7.8 and 70.8 ± 8.1 % of wet tissue, respectively. In contrast, the solubility of the collagen extracted from bone was the lowest of all the tissues examined, being only 5.7 ± 0.8 % of total wet tissue. The extent of hydroxylation of proline and lysine residues was also examined. In most tissues, the extent of hydroxylation of the lysine residue in insoluble collagen was higher than that of acid-soluble collagen, indicating that hydroxylysine contributes to the stability of collagen. This is the first report of collagen contents, solubility, and extent of hydroxylation of proline and lysine residues in collagen extracted from different tissues of one organism. It is possible that hydroxylysine-derived collagen cross-links play a critical role in the stability of collagen in dilute acetic acid.

Comparison of five tuna plasma analytes measured on two automated blood analyzers

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.

Tetraodon genome confirms Takifugu findings: most fish are ancient polyploids

The genome of Tetraodon nigroviridis confirms that ray-finned fish underwent an acient whole-genome duplication.