Over the past 50 years, red sea bream Pagrus major has been selectively bred for more than 10 generations to improve its growth rate. However, the effect of genetic factors on growth under low- or non-fish meal diet is still unclear. Here, we analyzed 251 individuals randomly mating 21 females (dam) and 16 males (sire) to determine the difference in growth between non-fish meal (i.e., treatment group) and fish meal-based diet (i.e., control group), if any. The kinship coefficient between each group of individuals and their parents was estimated by the KING (Kinship-based Inference for GWASs) software package. The presumed parents of 74 individuals in the treatment group and 57 individuals in the control group could be effectively identified. Notably, one specific female was consistently related to high- rather than low-growth individuals in both groups. In addition, more than one parent in each group was related to either high- or low-growth individuals. Parents of both high-growth individuals in the treatment group and low-growth individuals in the control group were also identified. Although further study is required on various growth factors, this study suggests the possibility of selective breeding under non-fish meal diet.

We have reported the production of myostatin (Pm-mstn) complete knockout red sea bream (Pagrus major) using CRISPR/Cas9, and the Pm-mstn mutant exhibited a 17% increase in skeletal muscle. However, important characteristics in aquaculture production, such as the growth rate and the amount of feed required for growth have not been clarified. In this study, we conducted a feeding trial using apparent satiation feeding during the juvenile stage and compared growth performance metrics including; weight gain, feed efficiency, apparent protein and lipid retention rates of the Pm-mstn mutants with wild-type fish (WT). Experimental fish were produced via artificial insemination from single male and female broodfish of heterozygous Pm-mstn mutants and reared in one tank until the start of the trial. Genotypes of 356 full sib fish were identified, and the WT and the homozygous mutant (HM) were used for the trial. Seventeen fish from WT and HM (mean body weight 41.1 ± 0.3 g and 42.7 ± 0.3 g, respectively) were randomly distributed into each of 100 L circular tanks and set in triplicate for each genotype. Fish were fed twice daily to apparent satiation with each respective diet at 08:00 and 13:00 six days per week for 8 weeks. Weight measurement of all experimental fish was conducted bi-weekly. Five fish at day zero from each genotype and 5 fish from each tank at the final day of the trial were sampled for whole-body proximate analysis. At the end of the trial, weight gain, specific growth rate, and feed efficiency were significantly higher in the HM group than that in the WT group, and there was no significant difference in the daily feed intake. The protein efficiency and apparent protein retention were significantly higher in the HM than for the WT. These results suggest that HM fish feed similarly to that of WT fish during the juvenile stage. However, HM fish have a higher ability to convert feed efficiently and accumulate ingested protein, resulting in better overall growth.

© 2020 Elsevier B.V. We have reported the production of myostatin (Pm-mstn) complete knockout red sea bream (Pagrus major) using CRISPR/Cas9, and the Pm-mstn mutant exhibited a 17% increase in skeletal muscle. However, important characteristics in aquaculture production, such as the growth rate and the amount of feed required for growth have not been clarified. In this study, we conducted a feeding trial using apparent satiation feeding during the juvenile stage and compared growth performance metrics including; weight gain, feed efficiency, apparent protein and lipid retention rates of the Pm-mstn mutants with wild-type fish (WT). Experimental fish were produced via artificial insemination from single male and female broodfish of heterozygous Pm-mstn mutants and reared in one tank until the start of the trial. Genotypes of 356 full sib fish were identified, and the WT and the homozygous mutant (HM) were used for the trial. Seventeen fish from WT and HM (mean body weight 41.1 ± 0.3 g and 42.7 ± 0.3 g, respectively) were randomly distributed into each of 100 L circular tanks and set in triplicate for each genotype. Fish were fed twice daily to apparent satiation with each respective diet at 08:00 and 13:00 six days per week for 8 weeks. Weight measurement of all experimental fish was conducted bi-weekly. Five fish at day zero from each genotype and 5 fish from each tank at the final day of the trial were sampled for whole-body proximate analysis. At the end of the trial, weight gain, specific growth rate, and feed efficiency were significantly higher in the HM group than that in the WT group, and there was no significant difference in the daily feed intake. The protein efficiency and apparent protein retention were significantly higher in the HM than for the WT. These results suggest that HM fish feed similarly to that of WT fish during the juvenile stage. However, HM fish have a higher ability to convert feed efficiently and accumulate ingested protein, resulting in better overall growth.

To improve livestock and aquaculture-raised fish as food, targeted mutagenesis using genome editing technologies is becoming more realizable. Myostatin (Mstn), which functions as the negative regulator of skeletal muscle growth, is one of the major targets to improve the edible ratio of livestock and farmed fish. We previously reported that the deficiency of Pm-mstn, one of Myostatin paralogs, improves muscle growth and changes body shape in a finfish species, red seabream (Pagrus major), as a result of editing the gene by means of CRISPR/Cas9. In this study, we established Pm-mstnb-deficient red seabream, which is a null-allelic mutant of another paralogous gene of Myostatin in the species, and analyzed their phenotype in terms of growth traits and body shape. A comparison of all growth traits between Pm-mstnbwt/wt and Pm-mstnb-5/-5 revealed no significant differences. In addition, all metrics for body shape, defined as the ratios of body depth, body width, and depth of the caudal peduncle to body length, respectively, were also similar in Pm-mstnbwt/wt and Pm-mstnb-5/-5. Therefore, we concluded that Pm-mstnb does not function as a negative regulator of skeletal muscle growth in red seabream.

© 2019, Japanese Society of Fisheries Science. The aquaculture of greater amberjack Seriola dumerili is of considerable research interest worldwide. The larviculture methods employed to culture this species, however, are still under development, and the majority of farms still rely on wild-caught juveniles. One of the problems associated with the hatchery production of this species is the optimal selection of broodstock to ensure a stable supply of high-quality eggs. Specifically, no reliable low-stress sex-discrimination technique is currently available for selecting broodstock of this species. This study investigated the efficacy of a hormone-based sex-discrimination method in full-cycle cultured S. dumerili, ranging in age from 412 to 1150 days after hatching (DAH). Plasma concentrations of the female hormone 17β-estradiol (E2) and the male hormone 11-ketotestosterone (11-KT) were measured in both spawning and non-spawning seasons, and the optimal threshold levels for sex discrimination were estimated using a receiver operating characteristic curve. Sex discrimination using E2 produced several false positives in younger fish, and had an overall accuracy of 78.7%. However, sex discrimination using 11-KT had an accuracy of 96.7%, even in 412 DAH fish. This study demonstrated that sex discrimination using 11-KT is a reliable method for optimizing the sex ratio of S. dumerili broodstock, even before the broodstock mature.

© 2018, Japanese Society of Fisheries Science. 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.

© 2018 Elsevier B.V. Genome editing is a powerful tool as a new breeding technology including for aquaculture because of the high efficiency of gene targeting without the requirement for exogenous gene integration. CRISPR/Cas9 system, a genome editing tool, has been widely used in various species due to its efficiency and flexibility. We demonstrate the establishment of a new breed of myostatin (Pm-mstn) complete knockout red sea bream (Pagrus major) using CRISPR/Cas9. This is the first report of the establishment of a new breed in aquaculture marine fish using genome editing. The mutations were formed by deletions in the first exon of the Pm-mstn, which cause disruption of the C-terminal active domain of MSTN. The breed exhibited a 16% increase of skeletal muscle, that is, an increase of edible parts. The breed showed the phenotype of short body length and small centrum, which is not observed in mice and other teleost fish. We established the homozygous gene disrupted breed in 2 years, which is far shorter than the conventional breeding method. Our study indicates that genome editing can accelerate the speed of aquaculture fish breeding.

© Copyright 2017, Mary Ann Liebert, Inc. 2017. Serotonin (5-hydroxytryptamine [5-HT]) is a bioactive monoamine that acts as a neurotransmitter in the central and peripheral nervous system of animals. Teleost fish species have serotonergic neurons in the raphe nuclei of the brainstem; however, the role of 5-HT in the raphe neurons in teleost fish remains largely unknown. Here, we established a medaka (Oryzias latipes) strain with targeted disruption of tryptophan hydroxylase 2 (tph2) gene that is involved in the 5-HT synthesis in the raphe nuclei. Immunohistochemistry and mass spectrometry analysis revealed that the homozygous mutants (tph2Δ13/Δ13) lacked the ability to synthesize 5-HT in the raphe neurons. To investigate the effects of 5-HT deficiency in adult behaviors, the mutant fish were subjected to five behavioral paradigms (diving, open-field, light-dark transition, mirror-biting, and two-fish social interaction). The homozygous mutation caused a longer duration of freezing response in all examined paradigms and reduced the number of entries to the top area in the diving test. In addition, the mutants exhibited a decreased number of mirror-biting in the males and an increased contact time in direct social interaction between the females. These results indicate that this tph2-knockout medaka serves as a good model to analyze the effects of 5-HT deficiency in the raphe neurons.

© 2017 Wiley Periodicals, Inc. In marine aquaculture fish, excessive supplement of vitamin A (VA) to zooplanktons for larval culture and experimental exposure of larvae to retinoic acid (RA: active form of VA) have been known to cause vertebral deformity. However, the tissues in the developing vertebral column that are affected by RA and the progression of vertebral deformity remain undetermined. To examine these questions, we histologically traced the progress of vertebral deformity induced by RA in Japanese flounder (Paralichthys olivaceus). Larvae were exposed to RA for 3 days at mid-metamorphosis (G-stage), a critical stage for vertebral deformity. Intervertebral ligament, which is known to form intervertebral joints in cooperation with the notochord, was severely degenerated by RA, leading to fusion of centra. During further development to adult, growth of centra was severely suppressed in an anterior–posterior direction in RA-treated fish and the notochord tissue was lost from fused centra, resulting in complete loss of intervertebral joints and fusion of centra. We conclude that RA initially damages the intervertebral ligaments, and these defects lead to fusion, narrowing of centra, and loss of intervertebral joints in the vertebral column. The cumulative effect of these modifications is a truncated body form.

© 2014 Elsevier B.V. Hatcheries often employ disinfection by electrolysis for water; however, this produces free oxidants that pose practical problems in the production of flounder, Paralichthys olivaceus. Rearing juvenile flounder in electrolyzed seawater (EL-SW) causes pseudo-albinism. To elucidate the mechanism of the onset of EL-SW-induced pseudo-albinism, we compared thyroid hormone (TH) metabolism, melanoblasts, and skin differentiation in flounder reared in normal seawater (normal SW) and EL-SW and determined the critical period for the onset of pseudo-albinism. The number of thyroxin (T4)-positive thyroid follicles increased toward the metamorphic climax. The expression of deiodinase 1 (. dio1), which converts plasma T4 to triiodothyronine (T3), in the liver peaked synchronously with the increase in T4-positive thyroid follicles. EL-SW exposure had no effect on TH metabolism. Instead, EL-SW inhibited differentiation of adult-type melanoblasts in the skin of the ocular side together with a loss of structural asymmetry in the bilateral skins. Although EL-SW does not affect TH metabolism, our data indicate that it does inhibit the signaling system that controls differentiation of the blind and ocular lateral halves during the establishment of metamorphic asymmetry. The early-metamorphic stage, E- and F-stages, was demonstrated to be the critical period for the onset of pseudo-albinism in EL-SW-reared larvae. These results suggest that long-term exposure to free oxidants, even at low levels, acts as a chronic stressor on flatfish metamorphosis.

The adult-type chromatophores of flounder differentiate at metamorphosis in the skin of ocular side to establish asymmetric pigmentation. In young larva and before metamorphosis, adult-type melanophores that migrate to the ocular side during metamorphosis reside at the base of the dorsal fin as latent precursors. However, the migration route taken by these precursor cells and the mechanisms by which lateralization and asymmetric pigmentation develop on the ocular side are unknown. To further investigate this migration and lateralization, we used in situ hybridization with gch2 probe, a marker for melanoblasts and xanthoblasts (precursors of adult type chromatophores), to examine the distribution of chromatophore precursors in metamorphosing larvae. The gch2-positive precursors were present in the myoseptum as well as in the skin. This finding indicated that these precursors migrated from the dorsal part of the fin to the skin via the myoseptum. Additionally, there were much fewer gch2-positive cells in the myoseptum of the blind side than in the skin and myoseptum of the ocular side, and this finding indicated either that migration of the precursor cells into the myoseptum of blind side was inhibited or that the precursors were eliminated from the myoseptum of the blind side. Therefore, we propose that the signals responsible for development of asymmetric pigmentation in flounder reside not only in the skin but on a larger scale and in multiple tissues throughout the lateral half of the trunk. © 2013 Wiley Periodicals, Inc.

In order to better understand the endocrine aberrations related to abnormal metamorphic pigmentation that appear in flounder larvae reared in tanks, this study examined the effects of continuous 24-h illumination (LL) through larval development on the expression of tyrosine hydroxylase-1 (th1), proopiomelanocortin (pomc), α-melanophore-stimulating hormone (α-MSH) and melanin concentrating hormone (MCH), which are known to participate in the control of background adaptation of body color. We observed two conspicuous deviations in the endocrine system under LL when compared with natural light conditions (LD). First, LL severely suppressed th1 expression in the dopaminergic neurons in the anterior diencephalon, including the suprachiasmatic nucleus (SCN). Second, pomc and α-MSH expression in the pars intermedia melanotrophs was enhanced by LL. Skin color was paler under LL than LD before metamorphic pigmentation, and abnormal metamorphic pigmentation occurred at a higher ratio in LL. We therefore hypothesize that continuous LL inhibited dopamine synthesis in the SCN, which resulted in up-regulation of pomc mRNA expression in the melanotrophs. In spite of the up-regulation of pomc in the melanotrophs, larval skin was adjusted to be pale by MCH which was not affected by LL. Accumulation of α-MSH in the melanotrophs is caused by uncoupling of α-MSH synthesis and secretion due to inhibitory role of MCH on α-MSH secretion, which results in abnormal metamorphic pigmentation by affecting differentiation of adult-type melanophores. Our data demonstrate that continuous illumination at the post-embryonic stage has negative effects on the neuroendocrine system and pituitary in flounder. © 2012 Elsevier Inc.

The gastric glands of marine aquaculture fish species are known to start secretion of pepsinogens (Pep) during larval development, far after first feeding. Although characterization of gastric gland development is considered useful for managing larval feeding, the timing of initial Pep synthesis by the gastric gland has only been reported for a few species. Consequently, this study employed in situ hybridization (ISH) to characterize the onset of expression of genes encoding the gastric enzymes Pep and Chitinase (Chi), as well as the complete development of gastric glands in the larval stomach of Japanese flounder (Paralichthys olivaceus), spotted halibut (Verasper variegatus), seven-band grouper (Epinephelus septemfasciatus) and greater amberjack (Seriola dumerili). The expression of both Chi and Pep was observed to begin in the cardiac region of the larval stomach at 25. days post-hatching (dph) in Japanese flounder, 36. dph in spotted halibut, 38. dph in seven-band grouper and 13. dph in greater amberjack. The gastric glands of these species were fully developed in the stomach approximately 5-12. days after the initiation of Pep expression. Development of gastric glands was observed to coincide with external manifestations of metamorphosis, such as the development of adult coloration in Japanese flounder and spotted halibut of the Pleuronectiformes, while it finished prior to adult-type coloration in seven-band grouper and greater amberjack of the Perciformes. © 2011 Elsevier B.V.

The bilateral symmetry of flounder larvae changes through the process of morphogenesis to produce external asymmetry at metamorphosis. The process is characterized by the lateral migration of one eye and pigmentation at the ocular side. Migration of the left or right eye to produce either dextral or sinistral forms, respectively, is usually fixed within a species. Here we propose a mechanism for the mediation of lateralization by the nodal-lefty-pitx2 (NLP) pathway in flounders, in which pitx2, the final left-right determinant of the NLP pathway, is re-expressed in the left habenula at pre-metamorphosis. After the initiation of left-sided pitx2 re-expression, the eye commences migration, when the habenulae shift their position on the ventral diencephalon rightwards in sinistral flounder (Paralichthys olivaceus) and leftwards in dextral flounder (Verasper variegatus). In addition, the right habenula increases in size relative to the left habenula in both species. Loss of pitx2 re-expression induces randomization of eye-sidedness, manifesting as normal, reversed or bilateral symmetry, with laterality of the structural asymmetry of habenulae being entirely inverted in reversed flounders compared with normal ones. Thus, flounder pitx2 appears to be re-expressed in the left habenula at metamorphosis to direct eye-sidedness by lateralizing the morphological asymmetry of the habenulae. © 2009 Japanese Society of Developmental Biologists.

Flounders form left-right asymmetry in body coloration during metamorphosis through differentiation of adult-type melanophores and xanthophores on the ocular side. As the first step in investigating the formation of flounder body coloration asymmetry, in this study, we aimed to determine where the precursors of adult-type chromatophores distribute in larvae before metamorphosis. In Paralichthys olivaceus and Verasper variegatus, GTP cyclohydrolase 2 (gch2), a common marker of melanoblasts and xanthoblasts, was found to be transiently expressed in cells located along the bilateral skeletal muscles at the basal parts of the dorsal and anal fins of premetamorphic larvae. When V. variegatus larvae were fed with a strain of Artemia collected in Brazil, this gch2 expression was abolished and the differentiation of adult-type melanophores was completely inhibited, while the density of larval melanophores was not affected. In a cell trace test in which the cells at the basal part of the dorsal fin were labeled with DiI at the premetamorphic stage, adult-type melanophores labeled with DiI were found in the skin on the ocular side after metamorphosis. These data suggest that, in flounder larvae, adult-type melanophores are distributed at the basal parts of the dorsal and anal fins as unpigmented precursor cells. © 2008 The Authors.

The lefty gene encodes a member of the TGF-β superfamily that regulates L-R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-β superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L-R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression. © 2006 Elsevier B.V. All rights reserved.

To perform genome editing on fish and crustaceans, the microinjection method (MI) on fertilized eggs is usually used. However, few species of fish and crustaceans have been established as model organisms, and it is difficult to systematically obtain fertilized eggs in non-model organisms. Therefore, we tried to develop a genome editing method without MI for fertilized eggs. We focused Vitellogenin system. In this system, proteins generated in liver are brought into eggs via blood system. We could bring GFP from liver into yolk-sac in eggs,but failed into cytoplasm of eggs.

In aquaculture, selective breeding has not developed yet. The selective breeding takes much time to establish a new breed, so it is not adequate for aquaculture where new breeds are required immediately. We tried to develop new breeding method for aquaculture using genome editing technology, CRISPR/Cas9. With red sea bream and tiger puffer, the single guide RNAs were designated on the genome sequence in exon 1 of myostatin gene and microinjected into the one-cell stage eggs which are artificially inseminated. As results, the myostain gene of both fish has successfully disrupted and new breeds with enhanced muscle mass have established.

ヒラメ・カレイの変態後の頭部左右性形成の傾き方向の決定には「左手綱核のNodal経路遺伝子pitx2の特異的発現」が関与している。ヒラメ体節期胚をNodal受容体阻害剤SB431542により一過的に浸漬処理した。これらの個体では孵化前後のNodal経路遺伝子の左体側特異的発現が完全に抑制されており、その後の心臓や腸の捻転方向といった左右性がランダム化した。浸漬仔魚を変態が完了するまで常法で飼育したところ、20%の変態中仔魚では左間脳手綱核におけるpitx2が正常に発現したが、80%の仔魚では発現が消失していた。また変態後の稚魚の60%が正常な左ヒラメ、40%が眼位逆位すなわち右ヒラメとして発生していた。このうち正常にpitx2を発現した20%の個体は正常な左ヒラメとなり、発現が消失した80%の個体は眼球の偏りを一方向へと指定するスイッチ機構が働かなくなり、半分の40%は右ヒラメ、残りの半分は左ヒラメへと変態したと推察している。以上の実証実験は胚期における左右軸決定因子が数週間という時間を隔たり、変態時に眼位の偏り方向を指定するために再度機能するという異体類特有のメカニズムが存在することを強く示唆している。 ヒラメ・カレイの非対称な体色は、成魚型色素胞前駆細胞の左右非対称な分化によって顕在化すると推察されていたが、非対称な分化のタイミングや前駆細胞の遊走経路は不明であった。変態中仔魚の鰭基部および体幹部筋肉間(筋間中隔)の細胞を蛍光色素により標識しその遊走を観察し、色素合成酵素の遺伝子発現により分化過程を観察した。その結果、変態完了以前の有眼側体幹内部を遊走中の前駆細胞が色素の生合成を始動した段階で既に体側部の左と右の違いが生じている事が明らかになった。本実験は体幹部の深部における前駆細胞の挙動が有眼側と無眼側で異なることを示した初めての例である。