To improve the production efficiency of yellowtail Seriola quinqueradiata aquaculture, we measured changes in the activities of two lipid metabolism-related enzymes, carnitine palmitoyltransferase 2 (CPT2) and glucose-6-phosphate dehydrogenase (G6PDH), in the dark muscle and livers of 0- and 1 year-old fish over the entire culture period. Concomitantly we also investigated two factors that regulate these enzymes, namely, water temperature and daylength, under natural and controlled light conditions. In 0 year-old fish, high CPT2 activity was observed with increased water temperature/longer daylength, whereas high G6PDH activity was observed with decreased water temperature/shorter daylength. The activities of these enzymes were inversely correlated with each other. The changes in CPT2 and G6PDH activities were similar in 1- and 0 year old fish cultured with and without light control. To determine the major regulatory factors of CPT2 and G6PDH activities, two experiments were performed, starting on the vernal and autumnal equinox days, respectively. Fish were reared under natural and controlled light conditions (light:dark, 12:12 h) in both experiments. CPT2 activity did not differ according to light conditions or water temperature. G6PDH activity did not differ according to light conditions, but an increase in G6PDH activity was confirmed upon lowering of the water temperature. In summary, the activities of these two lipid metabolism-related enzymes changed seasonally and the main regulating factor may be water temperature. These results provide information for determining the appropriate lipid level and fatty acid composition of the seasonal diet for cultured yellowtail.

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.

© 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.

© 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.

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 15min from fertilization until 2.5h 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 10min from fertilization until 2.5h after stripping. We anticipate our findings will contribute to effectively establish genome edited aquaculture breeds.

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.

Six isoenergetic diets were formulated as follows: fish meal (FM) 700gkg(-1) (control, C), FM 300gkg(-1) + soy protein concentrate 300gkg(-1) (SPC), FM 300gkg(-1) + enzyme-treated SPC 300gkg(-1) (ESC), FM 170gkg(-1) + soy protein isolate 300gkg(-1) (SPI), FM 160gkg(-1) + enzyme-treated SPI 300gkg(-1) (ESI) and FM 150gkg(-1) + conglycinin 300gkg(-1)(CG). Forty fish (3.9g) were randomly distributed into each of eighteen 300-L tanks, fed twice daily until satiation for 8weeks. The final body weight, specific growth rate and condition factor did not show significant differences among the fish fed with diets C, SPC, ESC and ESI (p>.05). The survival was significantly lower in fish fed with diets SPI and CG. Feed efficiency was significantly higher in fish fed with diets SPC and C than in fish fed with other diets (p<.05). There were no significant differences in nutrients retention efficiencies in fish fed with diets C, SPC, ESC and ESI. A significantly higher phosphorus retention efficiency in fish fed with soymilk protein diets resulted in lower phosphorus discharge to the environment (p<.05). These results suggest that the soymilk proteins can comfortably replace 570-770gFMkg(-1) diet of red sea bream juvenile, which will ensure significant ecological benefits through reducing phosphorus load to the environment.

The effects of delayed first feeding on the nutritional condition of tiger grouper, Epinephelus fuscoguttatus (Forsskål, 1775), larvae were examined under controlled conditions. Larval gut epithelium development and morphometric changes of the larvae fed at different first times (0, 6, 12, 18 and 24 h after mouth opening stage h AMO) were compared. Gut epithelium height (14.81 ± 0.24 μm) of larvae first fed at 0 h AMO was significantly higher (P < 0.05) compared to other treatments and gut was morphologically well developed. A continuous reduction of gut epithelium height was observed in larvae first fed beyond 0 h AMO and severe damage on connective tissue surrounding larval gut was observed in larvae fed at 24 h AMO. All morphometric growth on each body proportion of larvae first fed at 0 h AMO was gradually increased as they developed, while larvae first fed at 6, 12, 18 and 24 h AMO experienced slow development and degradation of entire body proportions. This study concludes first feeding at mouth opening stage to the tiger grouper is essential to enhance larval nutritional condition that is important to maximize larval survival and growth at subsequent stage.

Kudoa rayformis n. sp. (Myxozoa; Multivalvulida) was observed in the trunk muscle of Pacific sierra Scomberomorus sierra caught off the coast of Tonosi, Panama. The species formed pseudocysts in myofibers and infection was subclinical. The myxospores possessed four polar capsules and spore valves, one of which had a distinct filamentous extension. This unique morphological characteristic of the myxospore validated this as a new species of Kudoa. Genetically, K. rayformis n. sp. is closest to K. inornata, with 98% and 91% similarity in 18S and 28S rDNA, respectively, but its spore shape was clearly distinct. The 18S rDNA and concatenated sequences from K. rayformis were used in molecular phylogenetic analyses of kudoids to examine the congruence of phylogeny with infection site tropism, spore morphology and cyst/pseudocyst formation. The results demonstrated that the phenotypic traits were correlated with the phylogeny of Kudoidae, and that the biological features of K. rayformis originated from the ancient Kudoidae as exhibited by the non-specific infection site tropism and the ability to infect muscle and form pseudocysts. (C) 2016 Elsevier Inc. All rights reserved.

Presently there are 23 identified tuna stocks in the world: 14 fully exploited, 8 overexploited or depleted, and only a single skipjack stock deemed to be under exploited. Wild juveniles of ABFT (. Thunnus thynnus), PBFT (. Thunnus orientalis), SBFT (. Thunnus maccoyii), and YFT (. Thunnus albacares) are used to stock tuna ranches for "fattening" (grow-out, from advanced juvenile to slaughter size). Ranched tunas are taken from diverse stocks managed under various legal regimes and monitoring agencies, but all serve the fresh and frozen sushi/sashimi-grade tuna market. Because Japan commands the largest part of this market, it sets fishing and ranching trends throughout the world. In any country, as the level of affluence rises, food preferences tend to shift from those centered on starches to ones centered on protein, and Asian countries have a strong predilection for this protein to be provided by seafood products. The rise of China as an economic power already has shifted its status from being a net exporter to that of net importer of seafood products, and this change is becoming evident in global tuna markets. Consequently, all available information points to a significant and growing impact of human consumption, especially in Asia, on global tuna resources. Catch restrictions and the banning of international tuna trade clearly have failed to achieve management of tuna stocks for sustainability so, it is now incumbent to offer proactive strategies rather than reactive approaches. Such strategies need to be ones that academic institutions, international tuna markets, fishing fleets, and tuna farmers as well as regulatory agencies and environmentalists can endorse. Such is the justification for this chapter, which offers content relevant to both ecological and economic perspectives of tuna aquaculture. The solutions presented are straightforward and the science we now have in hand is strong enough to pursue them. As Roger Payne put it: "Act - that's what's so important.".

The effects of different aeration rates at night to prevent sinking syndrome-related death (SSRD) of the tiger grouper, Epinephelus fuscoguttatus were examined. The aeration rates were fixed at 300 mL min(-1) at daytime (07: 00-19: 00 hours) and regulated to 0, 300 and 900 mL min(-1) at night (19: 00-07: 00 hours). Larval survival, growth, feeding intake, sinking velocity, distribution and behaviour, stress level, surface tension-related death (STRD) and flow velocity distribution were assessed. The occurrence of SSRD in the tiger grouper was observed through the accelerated sinking velocity (Vl) (from 0.15 +/- 0.09 cm s(-1) at 4 days AH to 0.41 +/- 0.09 cm s(-1) at 12 days AH) coupled with larval passive swimming behaviour at night-time. On the final day of experiment (15 days AH), larvae reared in 900 mL min(-1) at night had attained significantly higher (P < 0.05) survival (34.4 +/- 5.5%), growth (5.8 +/- 0.5 mm) and feeding intake (60.46 +/- 6.98 ind. larva(-1)). A favourable flow field for the tiger grouper was produced in 900 mL min(-1) at night-time, in which larvae were transported 15-25 cm above the tank bottom and 1.0 cm beneath the water surface. Under these night-time rearing conditions, larval stress level and number of STRD reared in 900 mL min(-1) compared with those observed in 300 mL min(-1) remained insignificant, indicating that strong turbulence of flow velocity was not detrimental for larvae. Our findings recommend aeration at 900 mL min(-1) at night as this could improve larval survival by reducing SSRD.

We studied the epidemiology of Flavobacterium psychrophilum in ayu from Lake Biwa for establishing control measures to bacterial cold water disease (BCWD). During 1998-2011, 12,743 wild ayu were collected from coastal (set net and gill net), offshore (offshore scoop net) and inflow river (fishing weir) areas of Lake Biwa. We employed a nested-PCR technique targeting 16S rRNA gene for the detection of F. psychrophilum in the gills and kidney of ayu. Prevalence of the bacterium was greatest when the water temperature of Lake Biwa was 18-21°C, which coincides with the optimum temperature for multiplication of F. psychrophilum. The prevalence of F. psychrophilum in the gills was higher in fish from fishing weirs and set nets maintained in net cages than in fish just after catch with the other fishing techniques. This suggests that rearing ayu in net cages at high densities following catch by fishing weirs and set nets can advance infection with the bacterium among the captured fish which will be used for seedlings in aquaculture or releasing in rivers. Control measures for the captured ayu must be conducted in reliable ways to prevent spread of the disease after release and in aquaculture.

Kisspeptin (Kiss) and its cognate receptor (Kiss1R), implicated in the neuroendocrine control of GnRH secretion in mammals, have been proposed to be the key factors in regulating puberty. However, the mechanisms underlying the initiation of puberty in fish are poorly understood. The chub mackerel Scomber japonicus expresses two forms of Kiss (kiss1 and kiss2) and two Kiss receptor (kissr1 and kissr2) genes in the brain, which exhibit sexually dimorphic changes during the seasonal reproductive cycle. This indicates that the kisspeptin system plays an important role in gonadal recrudescence of chub mackerel; however, the involvement of the kisspeptin system in the pubertal process has not been identified. In the present study, we examined the mRNA expression of kiss1, kiss2, kissr1, kissr2, and gnrh1 (hypophysiotropic form) in the brain of a chub mackerel during puberty. In male fish, kiss2, kissr1 and kissr2 levels increased significantly at 14 weeks post-hatch (wph), synchronously with an increase in type A spermatogonial populations in the testis; kiss2 and gnrh1 levels significantly increased at 22 wph, just before the onset of meiosis in the testes. In female fish, kiss2 increased significantly at 14 wph, synchronously with an increase in the number of perinucleolar oocytes in the ovary; kiss1 and kiss2 levels significantly increased concomitantly with an increase in the kissr1, kissr2, and gnrh1 levels at 24 wph, just before the onset of vitellogenesis in oocytes. The present results suggest positive involvement of the kisspeptin-GnRH system in the pubertal process in the captive reared chub mackerel. (C) 2014 Elsevier Inc. All rights reserved.

This study presents the development of DNA vaccines against nocardiosis, using the Antigen 85-like (Ag85L) gene in Nocardia seriolae. An expression plasmid encoding Ag85L (pAg85L(wt)) and codon-optimized Ag85L (pAg85L(opt)) was intramuscularly injected into amberjack Seriola dumerili. Survival rates of the pAg85L(wt), pAg85L(opt) vaccinated group and PBS-injected group as a negative control were 88%, 98% and 51%, respectively, at 40 days after the N. seriolae challenge. In addition, the N. seriolae bacterial count in the spleen was significantly lower in the pAg85L(wt) and pAg85L(opt) vaccinated fish than in the PBS-injected fish (P < 0.05). These results suggest that the DNA vaccines pAg85L(wt) and pAg85L(opt) conferred protective efficacy against N. seriolae infection in amberjack.

Manipulation of stocking densities (10, 20, 30, 40 and 50 larvae L-1), each with or without the presence of shelter was conducted to determine the effects on survival, cannibalism and growth performances of larval bagrid catfish Mystus nemurus (Valenciennes 1840) from 2 to 14 days after hatching. This study revealed that stocking density significantly affected survival, cannibalism, total length, feed intake, specific growth rate and final weight of bagrid catfish larvae. Significantly higher survival was observed at moderate stocking density of more than 20 but less than 50 larvae L-1. Survival was significantly low beyond this threshold and was the lowest at 10 larvae L-1, coincides with the highest cannibalism. Total length, feed intake, specific growth rate and final weight were significantly higher at 10 larvae L-1. Shelter significantly improved total length and feed intake. No significant effects of stocking density and shelter were observed on the apparent feed conversion ratio and coefficient of variation. There was also no significant interaction between stocking density and shelter in all parameters. This study suggests that bagrid catfish larvae could be cultured at more than 20 but less than 50 larvae L-1 with the availability of shelter for optimal larviculture condition.

Taurine is often added to artificial fish diets to compensate for a reduction in fish meal (FM). However, the taurine content of FM-based diets is typically lower than in diets consisting of raw fish, even in diets where FM is the only protein source. We evaluated the effects of dietary taurine in FM-based diets on epidermal thickness and scale detachability in red sea bream Pagrus major. We compared the effect of diets containing 0% (control), 0.3% (Tau-0.3%), 0.6% (Tau-0.6%) and 1.0% (Tau-1.0%) taurine. Red sea bream (average body weight, 39 g) were fed these diets for 7 weeks. Taurine supplementation had no effect on growth, feed intake, feeding efficiency, or survival. However, the epidermal thickness was higher in fish in the Tau-0.6% and Tau-1.0% groups than in the control and Tau-0.3% groups. Similarly, scale loss was significantly higher in the control group than in the Tau-0.6% and Tau-1.0% groups. Our results suggest that supplementation with > 0.6% taurine (1.0% in diet) improves skin condition.

Understanding puberty is important for establishing aquaculture in fish. In this study, we analyzed the timing and completion of pubertal development along with changes in pituitary gonadotropin genes (fshb and lhb) in cultured chub mackerel (Scomber japonicus). At 45 days post-hatching (dph), gonadal sex differentiation was observed. The onset of puberty occurred at 192 dph in females with the start of vitellogenesis, whereas it occurred at 164 dph in males, with the beginning of spermatogenesis (proliferation and differentiation of germ cells). The completion of puberty was at 326 dph in females when vitellogenesis completed, and it was at 338 dph in males during spermiation. All fish sampled during the spawning season completed pubertal development. In the pituitary of female fish, fshb expression was activated during early secondary growth and was maintained high throughout vitellogenesis, whereas lhb expression was highest at the completion of vitellogenesis. In male fish, fshb and lhb expression were activated from the onset of spermatogenesis and further activated during late pubertal development; fshb remained high between late spermatogenesis and spermiation, whereas lhb was highest during spermiation.

Flow field control via aeration adjustment for the enhancement of larval survival of the kelp grouper Epinephelus bruneus was examined. Aeration rate of 300mLmin(-1) was introduced during daylight (07:00-19:00hours) and adjusted to 0, 300 and 900mLmin(-1) at night (19:00-07:00 hours). Larval sinking velocity +/- SD increased from 0.08 +/- 0.05 to 0.26 +/- 0.24cmsec(-1) from 4 to 12days after hatching (DAH), indicating their susceptibility to sink. Larvae reared in 300mLmin(-1) attained the highest survival rate at 24.9 +/- 3.4%, but remained significantly smaller in growth: 4.54 +/- 0.56mm compared with 4.82 +/- 0.53mm in 900mLmin(-1). The flow field in 300 and 900mLmin(-1) was at 10-20 and 15-25cm above the bottom of the tank and 8.0 and 1.0cm beneath the water surface. A favourable rearing condition was observed in 300mLmin(-1) as larvae were away from the bottom and surface areas, thus preventing them from dying due to sinking and surface tension-related death (STRD). Although sinking death was decreased with an increasing aeration rate, the stronger flow had increased larval susceptibility to STRD. Our findings suggest that aeration at 300mLmin(-1) could enhance larval survival by reducing both sinking death and STRD.

Manipulation of photoperiod: 24h light (24L), 12h light:12h dark (12L:12D) and 24h dark (24D); and feeding schedules: day and night feeding (DNF), day feeding (DF) and night feeding (NF) was conducted to determine effects on survival, cannibalism and growth of larval bagrid catfish Mystus nemurus 2-14days after hatching (dAH). Photoperiod insignificantly affected all parameters. Feeding schedule significantly affected survival and total length at 6 (P<0.049; P<0.009), 10 (P<0.033; P<0.000) and 14 dAH (P<0.013; P<0.000), respectively, but affected cannibalism at 10 (P<0.043) and 14 dAH (P<0.013). Survival for DNF was significantly higher than DF. Cannibalism for DNF was significantly lower than NF at 10 and 14 dAH. Total length for DNF was significantly higher than DF and NF at 10 and 14 dAH. At 14 dAH, feeding schedule significantly affected feed intake, final weight and coefficient of variation. For feed intake and final weight, DNF was significantly higher than DF and NF. For coefficient of variation, NF was significantly higher than DF. This study suggests that larval bagrid catfish can be reared at 24L, 12L:12D or 24D but should be fed day and night for improved growth, survival and reduced cannibalism.

We investigated the usefulness of acceleration loggers in aquaculture by examining net-cage use and metabolic rates in red sea bream, Pagrus major. First, the fish's metabolic rate (mg O-2 kg(-1) min(-1)) was measured with the logger in a swim tunnel at designated water velocities. We found that metabolic rate could be expressed by using a linear regression model of the activity rate index (unitless min(-1)) derived from acceleration data. Using this equation, the field metabolic rates of three fish in a net cage were monitored and were estimated at 14.1-15.0 kcal kg(-1) day(-1). The results suggested that 15-19% of energy from satiation feeding ration was consumed for metabolism and activity in the net cage. The loggers showed orderly net-cage use by the fish. Tagged individuals used the whole cage from surface to bottom, but individual fish that preferred the surface area rarely used the bottom, and vice versa. Metabolic rate increased significantly with distance of the fish from their preferred depths. The logger provided information on the physiological and behavioral responses of fish in a given breeding system, and its use should contribute to the design of practical aquaculture systems.

Taurine supplementation, to compensate for the reduction of fish meal (FM) in fish diets, has been the subject of numerous investigations. However, the taurine contents of FM-based diets are still lower than in diets of raw fish, even in diets where FM is the only protein source. In this study, the effect of taurine supplementation to commercial feed (containing 57% FM) on skin thickness and scale detachability in red sea bream Pagrus major was investigated. Three different levels of taurine were used: 0% taurine (control), 1% taurine (Tau-1%), and 2% taurine (Tau-2%). Red sea bream (average body weight, 107 g) were fed these diets for 60 days. No effects of taurine supplementation on growth and feeding efficiency were observed. However, skin thicknesses of fish from the Tau-1% and Tau-2% groups were significantly higher than for the control group, and scale detachability of the control group was significantly higher than for the Tau-1% and Tau-2% groups. These results suggested that taurine supplementation of commercial feed contributes little to growth performance, but does improve skin condition, in red sea bream.

Marble goby Oxyeleotris marmoratus is an important aquaculture freshwater (FW) species in Southeast Asia. To improve hatching technique, eggs from a river population in Sabah, Malaysia were incubated in FW and seawater (SW) diluted to 5, 10, 15, 20 and 30 psu, comparing hatching rates and larval deformation. Egg development, hatching rates, hatching periods, larval deformation and survival at 10 days after fertilization (dAF) were then compared in FW and 10 psu SW. Salinities from FW to 15 psu SW were tolerated, with the highest hatching observed at 10 psu SW (60.0±2.0%, mean±SD). Significantly higher hatching rates and lower deformation rates were observed at 10 psu SW than in FW. In FW, embryonic developed at similar rate with 10 psu SW, but hatching was delayed and all larvae died by 10 dAF. Peak of hatching in 10 psu SW observed in 48-60 hours after fertilization (hAF) (33.1±5.6%) while hatching in FW was delayed and peaked 72-84 hAF (10.6±3.4%). Larvae that hatched later had higher deformation rates. The eggs incubated in 10 psu SW had a shorter hatching period, higher hatching rate and better larval survival than those in FW.

The effects of delayed first feeding on the survival and growth of tiger grouper, Epinephelus fuscoguttatus (Forsskal 1775), larvae were examined under controlled conditions. The total length, yolk sac volume, oil globule volume, yolk sac absorption time and nutritional transition period (NTP) of the larvae fed at different first times (0, 6, 12, 18 and 24 h after the mouth opening stage; h AMO) were compared. Larval first feed intake was observed at 54 h after hatching (h AH) at 27.5 +/- 0.5 degrees C. The yolk sac was consumed more rapidly with an increase in delayed first feeding and was significantly different among treatments (P<0.05). Larvae first fed at 0 h AMO had the longest yolk sac absorption (72 h AH) and NTP (20 h) times and had the highest survival and growth rates at the end of the experimental period (360 h AH), being significantly higher (P<0.05) than the other treatments. First mortality was observed at 69 h AH, approximately 2 h after point of no return (PNR) occurred. This study suggests that first feeding of tiger grouper larvae should commence at 0 h AMO for enhancement of larval survival and growth.

Mycobacteriosis, caused by the intracellular parasitism Mycobacterium sp., causes economic damages to aquaculture production in Japan, particularly in seriola fish production. Antibiotics are not effective against Mycobacterium sp. and so a potent vaccine is needed. We previously reported that BCG vaccine (Mycobacterium bovis BCG) induces adaptive immunity against Mycobacterium sp. in Japanese flounder, Paralichthys olivaceus. In a phylogenetic tree, the genes for a major antigen, the Ag85 complex, in Mycobacterium sp. TUMSAT-Msp001 are closely related to homologues in Mycobacterium ulcerans. M. bovis BCG was detected until 7 days post-injection at the injection site (muscle) and 28 days post-vaccination in spleen. Cumulative mortality of amberjack, Seriola dumerili vaccinated intramuscularly (i.m.) and intraperitoneally (i.p.) with M. bovis BCG was 32.3% and 59.5% respectively, at 24 days post-infection of Mycobacterium sp., compared to 97.8% in PBS-injected fish. The bacterial counts of Mycobacterium sp. in spleen of both i.m.-and i.p.-vaccinated fish (6.2 x 10(3) and 1.3 x 10(4) CFU/mg tissue, respectively) at 20 days post-infection were significantly lower (P < 0.01) than those of PBS-injected fish (8.0 x 10(6) CFU/mg). Furthermore, Immersion challenge with Mycobacterium sp. TUMSAT Msp-001 showed 50% RPS value in BCG i.m.-vaccinated fish at the end of the experiment. These results support our previous study using Japanese flounder and suggest that BCG vaccine is also effective against Mycobacterium sp. infection in amberjack. (C) 2010 Elsevier Ltd. All rights reserved.

In order to test the effects of shielding from sunlight on the body color of red sea bream, we characterized three phases. In phase 1, juvenile red sea bream (50 days after hatching) were bred with/without a black curtain for 50 days. In phase 2, each group was further subdivided into two groups which were bred with/without a black curtain for 1 year. Finally, all groups were shielded with a black curtain for 3 months (phase 3). No significant difference in L* value (used as a criteria for body color) was observed between the shaded and unshaded groups after phase 1. The results after phase 2 indicate that shielding in phase 1 also had little effect on the later body color. In phase 3, the L* value significantly increased within 2 months in all body sites examined in all groups. We also tested the effects of shielding suntanned red sea bream which had been bred without shielding for 2 years. After 2-4 weeks of shielding, the color in various body sites was significantly improved. We conclude that shielding of juvenile fish is unnecessary because the sun-tanning that occurs during juvenile stages can be improved later by shielding.

Male and female teleost seabream (Pagrus major) were examined for seasonal variation of eumelanin, pheomelanin, 11-ketotestosterone (11KT, fish androgen), lightness (L* value) and Gonad Somatic Index (GSI: gonad mass / body mass × 100). In males, levels of pyrrole-2,3,5-tricarboxylic acid (a marker of eumelanin), 11KT and the GSI increased sharply from September and plateaued in March and April when the fish are sexually mature. These results are consistent with the lightness of their body color. Using the data from males, a high correlation was observed for all combinations of those four variables (PTCA, 11KT, lightness and GSI). In females, little change was observed in those variables except for the GSI. 4-Amino-3-hydroxyphenylalanine (a marker of pheomelanin) was also analyzed, but it was below the detection limit at all times. Oral treatment of juvenile red seabream with synthetic androgen methyl-testosterone for 2 months induced eumelanin accumulation about 3 times higher than the control. These data show that there is a close relationship between androgen levels and eumelanin accumulation in teleosts. This is the first report that androgen affects melanin accumulation in a dose-dependent manner. © 2010 Elsevier Inc. All rights reserved.

Twenty-three new polymorphic microsatellite markers were isolated in the Pacific bluefin tuna, Thunnus orientalis. Each locus comprised three to 34 alleles. The expected and observed heterozygosities ranged between 0.46 and 0.96 and between 0.44 and 0.97, respectively. The Kto9, Kto11, and Kto42 markers demonstrated significant deviation from Hardy-Weinberg equilibrium; high null allele frequencies (0.08-0.14) were observed in the deviating group. From the results of simulation of parentage assignment, a combination of four loci (i.e. Kto15, Kto23, Kto38, and Kto39) was considered the best for parentage assignment.

The tight junction (TJ) is a specialized cell-cell adhesion structure in epithelial and endothelial sheets unique to the chordates and functions as a barrier of fluidal diffusion across the cell sheets. In order to study the dynamics of TJ formation in vivo during embryogenesis, we have generated a transgenic medaka line that expresses claudin-7 protein fused to enhanced green fluorescent protein under the regulation of the red seabream beta-actin promoter in transparent medaka embryos. Claudins contain four transmembrane domains and have been identified as the key molecules that dictate the function of TJs. This transgenic medaka line will thus be useful for imaging of TJs in living embryos and hence in screening for mutations affecting cell-cell adhesion.

The daily expression profile of growth hormone (GH) mRNA in juvenile Pacific bluefin tuna (Thunnus orientalis) was investigated under aquacultured conditions. Total RNA from pituitaries (n=5) was sampled from 10 am to 12 pm the next day at 1 h intervals. The expression levels of GH mRNA were evaluated using real-time PCR normalized against the beta-actin gene. The expression level of GH transcripts reached a peak at 3-4 am at which time it was about 10 times higher than at any other time period. Considering their high growth rate compared to red seabream (Pagrus major) and other aquacultured species which shows continuous CH mRNA expression patterns, it can be concluded that the pulsed expression of GH mRNA just before daybreak is a key for the extremely high somatic growth rate of Pacific bluefin tuna. (C) 2008 Elsevier B.V. All rights reserved.

Stable reproduction is essential for supplying artificially hatched fish to tuna aquaculture. We observed testes maturation in reared Pacific bluefin tuna (PBT) Thunnus orientalis at 2+ years of age. The incidence of males with mature testes was 25.0%, and 40% of the males had developing testes that contain spermatozoa, while oocytes of the same aged females were not mature. These fish were wild-caught at 0+ years old in August 1997 and the gonads were examined in October 1998 and January-February 2000. Therefore, the age at examination in 2000 was estimated to be 2 years and 7-10 months old considering the spawning season of the wild PBT and the size when captured. Histological examination of the matured and developing testes showed that they contained spermatozoa, spermatids, spermatocytes, and spermatogonia. All the spermatozoa were observed to be motile in sea water under light microscopy. From the results of this and previous studies, matured males are probably fertile for at least 5 months a year in Kushimoto. The testes maturation observed at young age in captivity is considered promising to reduce the cost of broodstock maintenance for the juvenile production of PBT, especially if the sperm are cryopreserved.

Transgenic technology has been widely applied to a variety of freshwater fish species. However there are few reports on the use of this technology in commercially important marine species. In this study, the construction of expression vectors containing the beta-actin promoter region for use in the red sea bream Pagrus major, a species of considerable importance to the aquaculture industry in Japan is reported. The beta-actin gene was cloned from a red sea bream genomic DNA library. Recombinant plasmids were constructed by linking the 5' flanking region of the beta-actin gene to the green fluorescent protein reporter gene, followed by the poly A signal sequence of simian virus 40 or the 3' flanking region the beta-actin gene. Expression of these constructs was examined following microinjection into zebrafish and red sea bream embryos, and compared to that of the expression vector pXI-GFP driven by the Xenopus elongation factor la. The results indicated that the construct consisting of the beta-actin 5'-and 3' flanking regions was the most efficacious. In future studies, it is planned to investigate the efficient condition for integration into chromosomes of the transgene.

Gonadal maturation of Pacific bluefin tuna (PBT) Thunnus orientalis reared in a net cage at Amami Island was investigated. The investigation was carried out during the period from 2 to 4 years old. Histological observation of the gonad indicated that male PBT reached sexual maturation at 2 or 3 years old, while female PBT started to spawn from 4 years old. The gonad-somatic index was high in July, with the highest values of ca. 3% for 4-year-old female and male PBT. The maturation of PBT might be promoted by the elevation of water temperature and/or the elongation of day length.

The cell surface is a functional interface between the inside and the outside of the cell. Moreover, cells have systems for anchoring surface specific proteins and for confining surface proteins to particular domains on the cell surface. For use in bioindustrial processes applied to oral vaccination, we consider that cell-surface display systems must be useful and that the yeast Saccharomyces cerevisiae, the most suitable microorganism for practical purposes, is available as a host for genetic engineering because it can be subjected to many genetic manipulations. In particular, the rigid structure of the cell makes the yeast suitable for several of the applications. In this study, we describe the expression of one of the target antigens, 380R, from the red sea bream iridovirus (RSIV), which is one of the most common viral diseases in the cultured marine fish Pagrus major in Japan, using the arming yeast system and aiming at its application for oral vaccination. We first performed the molecular cloning and expression of the 380R antigen from RSIV in Escherichia coli. The nucleotide sequence of the 380R antigen was composed of an open reading frame (ORF) of 1360 bp encoding a protein of 453 residues. To prepare a specific antibody against the 380R antigen, the recombinant protein was overexpressed and purified in E. coli. As a result of indirect immunofluorescence with the specific antibody, we could observe the expression of the 380R antigen on the surface of the yeast cells. Thus, we have successfully prepared the source of an oral vaccine using cell-surface display technology in yeast.

A previous study elucidated that an extreme hypoxia during somitogenesis induced the most frequent skeletal malformation centrum defects in red sea bream (RSB), Pagrus major. In this study, details of the hypoxic conditions to induce them in RSB, dissolved oxygen (DO) concentration and exposure time to hypoxia, were investigated. Fertilized eggs were exposed to seawater of six DO concentrations (0%, 10%, 25%, 50%, 75% and 100% of saturation) for seven different periods (5, 10, 30, 60, 120, 240 and 360 min) during somitogenesis. Somitic disturbances in newly hatched larvae were induced by exposure to 0% and 10% DO concentration for 10 and 120 min and longer respectively. Rearing eggs exposed to hypoxic condition of 10% DO for 240 min for 40 days post-hatch showed that the location and the frequency of somitic disturbances in larvae and centrum defects in juveniles were significantly correlated (P < 0.01). Dissolved oxygen concentration of the interstitial water in the egg high density layer formed at the water surface in a stationary state abruptly decreased to 3.7% within 7 min. Centrum defect induction by exposure of eggs to extreme low DO concentrations for a short period, which is the probable situation in the practical juvenile production, suggests that careful maintenance of DO concentration is important in the incubating water of fertilized eggs during egg sorting and transportation, where eggs are made into a pile and undergo hypoxia, for the prevention of centrum defects.

Growth, and seasonal changes in the gonadosomatic index (GSI) and KG values were investigated in captive chub mackerel, Scomber japonicus, kept at the Fisheries Laboratory, Shirahama Experimental Station, Kinki University. Mean monthly water temperatures ranged between 12.6 and 29.3°C. At the start of the experiment, the mean fork length and body weight of 30-days-old hatchings were 10.5±0.8 cm and 11.0±2.9g, respectively; while at the end of the two year experiment, these fish measured 33.0±0.8 cm and 353.0±50.0g, respectively. In one-year old fish, GSI and female KG values increased during February and June. In two-year old fish, GSI and female KG values increased in January. The GSI and KG values were high on average between March and July, with the highest GSI and KG values being 14.9, and 10.3, respectively. In April all males had mature spermatozoa and some females had mature eggs. The results suggested that males and females matured at one and two years, respectively, after hatching.

Effects of low temperature on the color of red sea bream (Pagrus major) were investigated. In colorimetric evaluation using L^*a^*b^* system the fish reared under low-temperature (10°C) for two months lost their L^* value compared to control (bred under 22°C) while no change was observed in a^* and b^* value. No significant difference was found in blood test (GOT, GPT, ALP, gamma-GTP, total bilirubin, direct bilirubin, indirect bilirubin and bile acid) . We also examined the properties of the "yellowed" juvenile red seabream which sometimes occur in winter season in Fisheries Laboratory of Kinki University. The b^* value was significantly lower than that of control while no significant difference could be found in L^* value. The blood test showed no clear difference in direct and indirect bilirubin conc as well as in other measurements. We also examined the concentration of yellow melanin (pheomelanin), however, no significant gap could be found in nomal and yellowed red seabream, indicating that the cause of yellowness is not bilirubin and pheomelanin accumulation.

Human melanocytes respond to UV irradiation by increasing the synthesis of melanin. While much is now understood of the pathways governing this process and the nature of the melanin synthesized, little is known of melanins produced by lower vertebrates and their capacity to respond to UV. Here we report that a fish, red seabream, can undergo 'suntanning'. Histological, colorimetric and chemical assays were performed for suntanned red seabream fish bred in net cages to analyse the melanins and compared with shaded or wild red seabream fish. For color evaluation, the L* values of suntanned fish were dramatically lower than those in the other two groups. Pyrrole-2,3,5-tricarboxylic acid (PTCA), an indicator of eumelanin, was detected in suntanned fish at five times higher levels than in shaded or wild fish while 4-amino-3-hydroxyphenyl-alanine (4-AHP), a marker for pheomelanin, could not be detected in any of the samples. Histological analysis showed that melanocytes in the suntanned skin enlarged and increased in number to form a monolayer at the surface of the skin. Analysis of L* values and PTCA levels showed quite a high correlation coefficient (r = -0.843). When comparing shaded and wild red seabream fish, the scores were closer but some significant differences were still found in some body areas. These results indicate that eumelanin accumulates in suntanned fish during the increase in skin color, which is induced by sunlight, presumably by ultraviolet radiation.

To elucidate drug deposition and metabolism in cultured marine fishes, in a previous study we isolated and purified the GSTs (glutathione S-transferases) from the hepatopancreas of the red sea bream Pagrus major that contained 25 and 28 kDa GST subunits. The 25 kDa GST subunits encoded by two genes (GSTA1 and GSTA2) have been identified as Alpha-class GSTs. In the present study, we performed the molecular cloning and characterization of the GSTR1 gene encoding the 28 kDa GST subunit from the Pa. major hepatopancreas. The nucleotide sequence of GSTR1 was composed of an ORF (open reading frame) of 675 bp encoding a protein of 225 residues with a predicted molecular mass of 25.925 Da. A search of the BLAST protein database revealed that the deduced amino acid sequence of GSTR1 was structurally similar to that of GSTs derived from other fishes such as largemouth bass (Micropterus salmoides) and plaice (Pleuronectes platessa). The genomic DNA containing the GSTR1 gene was found to consist of six exons and five introns quite distinct from mammalian Theta-class GSTs. We have purified and characterized the recombinant GSTR1 enzyme (pmGSTR1-1) which showed activity only towards 1-chloro-2,4-dinitrobenzene, although it had no detectable activity towards cumene hydroperoxide, 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal and p-nitrobenzyl chloride. Moreover, pmGSTR1-1 revealed remarkable heat instability (melting temperature T m = 30.3 ± 0.11°C). Collectively, our results indicated that the characteristic GST genes including GSTR1 have been conserved and functional in fishes. Therefore we designate them 'Rho-class', a new class of GSTs. © 2005 Biochemical Society.

Two distinct cDNAs corresponding to GSTA1 and GSTA2 genes encoding glutathione S-transferases (GSTs) from the hepatopancreas of red sea bream, Pagrus major were cloned and sequenced. A comparison of the nucleotide sequences of GSTA1 and GSTA2 revealed 98% identity and their derived amino acid sequences had 96% similarity. Both genes could be classified as α-class GSTs on the basis of their amino acid sequence identity with other species. Genomic DNA cloning showed that both GSTA1 and GSTA2 genes consisted of six exons and five introns. In a comparison of genomic DNAs, the structures of GSTA1 and GSTA2 differed. In addition, Southern-blot analysis indicated that at least two kinds of α-class GSTs existed in the P. major genome. In order to biochemically characterize the recombinant enzymes (pmGSTA1-1 and pmGSTA2-2), both clones were highly expressed in Escherichia coli. The purified pmGSTA1-1 and pmGSTA2-2 exhibited glutathione conjugating activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide, while neither pmGSTs show detectable activity toward 1,2-dichloro-4-nitrobenzene, ethacrynic acid, 4-hydroxynonenal, or p-nitrobenzyl chloride. Despite their high level of amino acid sequence identity, the pmGSTs had quite different enzyme-kinetic parameters. © 2005 Elsevier Inc. All rights reserved.

We observed the spawning of chub mackerel Scomber japonicus eggs under a light microscope in 2002. The chub mackerel were kept at the Fisheries Laboratory, Kinki University, Shirahama Experimental Station since 1999. Mature males and females (BL: 33.5±2.3 cm BW: 577.9±144.8g, n=50) were kept in 50 ton spawning tank between May to July. A total of 8.6×10⁴ fertilized eggs from broodstocks were obtained from spawning over a 46 day period. The egg diameter was 1.09±0.02mm in the pre-spawning period, and 0.90±0.02mm in the post spawning period. The eggs hatched 39-40h after fertilization at 20-21°C, and the hatching rate was 10.0-96.7%. The newly hatched larvae measured 3.56±0.08mm in total length. The larvae fed 3 days after hatching on the rotifer Brachionus rotundiformis. On the 7th, 9th and 11th day after hatching, we fed the larvae Artemia Franciscan and formulated food. The total length of the larvae reached 12.60±0.91mm after 10 days, 58.90±6.02mm after 20 days, and 96.02±4.89 mm after 30 days.

A histological examination was made of the ontogenetic development of the digestive and immune systems of the larval and juvenile kelp grouper Epinephelus bruneus reared in the laboratory The liver, gall bladder, pancreas and the demarcating region between the intestines and rectum were formed within 3 days post-hatch (dph). During the preflexion phase (within 16 dph), revolution of the intestine concluded, and pharyngeal teeth and the mucous cells of the esophagus were differentiated. In the transitional period to the juvenile stage (25 dph), the blind sac of the stomach, gastric glands and pyloric caeca began to form. From the viewpoint of the differentiation phase of the adult-type digestive system, the kelp grouper is similar to Heterosomata, hitherto reported. The primordial thymus, kidney and spleen were present at 12, 1 and 6 dph, and the small lymphocytes in these lymphoid organs appeared at 21, 30 and 33 dph, respectively The developmental sequence of the lymphoid organs and the appearance ages of the lymphoid organs and small lymphocytes in the lymphoid organs in the kelp grouper are similar to those of other marine fish previously reported, except for the Pacific bluefin tuna Thunnus orientalis.

The gonadal sex differentiation in red sea bream, Pagrus major, which is one of the most important species for aquaculture in Japan, was revealed histologically. The suitable conditions for induction of all-male groups in the fish were investigated, and functional males were induced by the conditions of oral administration of 17alpha-methyltestosterone. The sex determination of this fish was also discussed.

Serial sections, prepared from 0.5 to 30 days post-hatch (dph) larval and juvenile Thunnus orientalis (Temminck & Schlegel 1844), were stained with haematoxylin and eosin and examined by light microscopy for immune organ development. The early kidney was present at 0.5 dph, undifferentiated stem cells began to appear at 2 dph, and by 7 dph occasional small lymphocytes were present. The thymus was first obvious at 5 dph, located above the fourth branchial arch, small lymphocytes appeared at 7 dph, and by 15 dph an outer thymocytic zone and an inner epithelioid zone were visible. The progenitor spleen was present at 2 dph, located close to the gut, and by 12 dph it consisted of a mass of sinusoids filled with red blood cells, and remained mainly erythroid throughout the period studied. These results suggest that development of immune organs in this species is precocious relative to other marine teleosts.

The gonadal sex differentiation in red sea bream, Pagrus major, which is one of the most important species for aquaculture in Japan, was revealed histologically. The suitable conditions for induction of all-male groups in the fish were investigated, and functional males were induced by the conditions of oral administration of 17alpha-methyltestosterone. The sex determination of this fish was also discussed.

Red sea bream, Pagrus major, is one of the most important fish cultured in Japan. Two clones of red sea bream were produced, Eggs from a mitotic gynogenetic diploid (mitotic-G2N) red sea bream were inseminated either with sperm from a mitotic-G2N male to produce a heterozygous clone (hetero-clone), or with UV-irradiated sperm of Japanese parrot fish (Oplegnathus fasciatus) and the second meiotic division suppressed by cold shock to produce a homozygous clone (homo-clone). Normal diploids were also produced from one male and female as a control. The clonal status of the Fish was confirmed by multilocus DNA Fingerprinting. The fingerprinting patterns differed between individuals within the normal diploids. However, there was no variation between individuals within hetero- or homo-clones. The patterns of the homo-clones and the mother were identical, and all the bands of homo-clones were also observed in hetero-clones. Thus, the clonal status of homo- and hetero-clones was confirmed and the production of clones from the broodstock of mitotic-G2N was achieved. The hatching rates, survival rates and growth of the hetero- and homo-clones were recorded for a brief comparison with results of diploid controls. (C) 2002 Elsevier Science B.V. All rights reserved.

Two types of gynogenetic diploids were artificially induced in the red sea bream (Pagrus major Temminck et Schlegel), either by suppressing the first cell cleavage (mitotic-G2N) or by retaining the second polar body (meiotic-G2N). The eggs of red sea bream were inseminated with UV-irradiated (3000 erg mm(-2)) sperm of Japanese parrot fish (Oplegnathus fasciatus Temminck et Schlegel), and hydrostatic pressure shock of 700 kg cm(-2) for 5.5 min at 46 min after insemination (naitotic-G2N) and cold shock of 1 degreesC for 30 min at 3 min after insemination (meiotic-G2N) were applied to the eggs, sequentially, The total hatching rate and hatching rate of normal larvae of the normal diploid, meiotic-G2N and mitotic-G2N were 86.5 and 94.9%, 38.1 and 45.8%, and 12.8 and 35.0%, respectively. The induction of mitotic-G2N was confirmed by isozyme marker analysis. The standard deviations, variances and coefficients of variation of the body weight, standard length and body depth in 91-day-old juveniles were always large in mitotic-G2N, small in normal-2N and intermediate in meiotic-G2N. The variances in the number of pectoral fin rays and caudal fin rays of mitotic-G2N were significantly higher than those of normal-2N. The incidences of deformities were highest in the mitotic-G2N group. The survival rates and growth performance of the meiotic- and mitotic-G2N were significantly lower than those of normal-2N. Both G2N survived for 3 years to the adult stage.

The necessary dose and growth stage for oral administration of 17_α-methyltestosterone (MT) suitable for induction of all-male groups were investigated in red sea bream, Pagrus major. Oral administration of MT (0.01-1.0 mg MT/kg BW/day) for 16 weeks to 281-day-old meiotic gynogenetic diploids resulted in 100% functional males in the following spawning season. MT treatment (0.1 mg/kg BW/day) to fish of different ages (55, 141, and 893 days after hatching) for 16 weeks induced males, and testicular tissue was observed in the gonads of all MT-treated fish. While functional sperm were obtained from the fish treated with MT from 141 and 893 days-of-age, no sperm was produced in fish treated from 55 days-of-age.

The growth and morphological development of larval and juvenile Epinephelus bruneus were examined in a hatchery-reared series. Average body length (BL) of newly-hatched larvae was 1.99 mm, the larvae growing to an average of 3.96 mm by day 10, 6.97 mm by day 20, 12.8 mm by day 30, 22.1 mm by day 40 and 24.7 mm by day 45 after hatching. Newly-hatched larvae had many mucous cells in the entire body epidermis. By about 4 mm BL, the larvae had developed pigment patterns peculiar to epinepheline fishes, including melanophores on the dorsal part of the gut, on the tips of the second dorsal and pelvic fin spines, and in a cluster on the ventral surface of the tail. Spinelets on the second dorsal and pelvic fin spines, the preopercular angle spine and the supraocular spine, had started to develop by about 6 mm BL. The notochord tip was in the process of flexion in larvae of 6-8 mm BL, by which time major spines, pigments and jaw teeth had started to appear. Fin ray counts had attained the adult complement at 10 mm BL. After larvae reached 17 mm BL, elements of juvenile coloration in the form of more or less densely-pigmented patches started to appear on the body. Squamation started at 20 mm BL. Major head spines had disappeared or became relatively smaller and lost their serrations by 20-25 mm BL.

The process of gonadal sex differentiation of red sea bream, Pagrus major, of a selected strain was histologically studied using fish reared in the laboratory. Central cavities were observed in gonads in all of the fish aged 3 and 4 months. The gonads in 34 of 40 fish aged 7 months had oocytes at the pen-nucleolus stage. From 8 months to 12 months after hatching, about half of the fish had bisexual gonads. Gonads of fish from 1 year to 1 year and 4 months old were ovaries or bisexual gonads, while those of the 1-year-old fish after September and 2-year-old fish were ovaries, bisexual gonads or testes. Bisexual gonads were not seen in 3-year-old fish. Therefore, the pattern of gonadal sex differentiation in this selected strain may be summarized as follows. Differentiation to ovaries progresses in all fish until 7 months after hatching. Then half of fish continues to develop their ovaries, while the rest half starts to develop testis instead until 1 to 2 years old.

Development of digestive system and changes in the activity of trypsin-like enzyme, pepsin-like enzyme and amylase were studied in larval and juvenile Bluefin tuna, Thunnus thynnus reared in the laboratory. Liver, gall bladder, pancreas, and the demarcating region between intestine and rectum formed within 36 h after hatching. After feeding commenced trypsin-like enzyme and amylase activities increased as the larvae grew. During the preflexion phase (within 10 days after hatching), revolution of the intestine concluded; and pharyngeal teeth and mucous cells of esophagus differentiated. During the flexion phase (11 to 17 days after hatching), functional jaw teeth were found and blind sac, gastric glands and pyloric caeca begin to form. Pepsin-like enzyme activity increased and functions of stomach and pyloric caeca developed from the postflexion phase to the transitional period to the juvenile (17 to 25 days after hatching) . The rate of percentage of preanal length to standard length was constant (around 40%) until 11 days after hatching, then increased to 65% at day 26, and did not change from 26 days to 30 days. These results suggest that the developments of the digestive system until 11 days (preflexion phase) are mainly qualitative and those from then to 26 days (flexion phase to postflexion phase) are both qualitative and quantitative. This quantitative and qualitative development of the digestive system might contribute to the rapid growth in the juvenile stage.

We attempted to improve the breed of red sea bream Pagrus major by means of selective breeding in order to establish red sea bream seedling for aquaculture capable of growing more quickly than natural red sea bream. In about 1964, natural young fish were reared into broodstock and, among young fish obtained from these broodstock, those growing quickly were selected and reared into broodstock. This selective breeding has been repeatedly carried out for more than 25 years. The following results were obtained. (1) As the selective breeding was repeated, the body weight of broodstock aged 4 years showed an increase. (2) The selected red sea bream seedling grew at an obviously elevated rate as the selective breeding was repeated. Namely, the time (days) required for attaining a commercially available fish size (about 1 kg) was shortened. (3) The average realized heritability, which was determined by the average body weight of 4-year-old broodstock and body weight of 4-year-old fish in the growth curve of each generation, was 0.33 +/- 0.28.

The qualitative requirements and deficiency signs of water-soluble vitamins were studied in tiger puffers Takifugu rubripes having mean body weight of 2.9 g. The qualitative requirements of tiger puffer for water-soluble vitamins indicate that the requirement of this species for vitamin B6 is lower than that of other fish species such as red sea bream, yellowtail, and Japanese parrot fish, while that for vitamins related to lipid metabolism, such as choline, nicotinic acid, pantothenic acid, biotin and inositol, is comparatively high.

Control and stressed groups of juvenile Japanese parrot fish Oplegnathus fasciatus were fed diets (moist pellet) supplemented with 0, 75, and 300 mg of ascorbic acid (AsA) per 100 g. Fish in the stressed group were intermittently exposed to decreasing water oxygen levels every 3 or 4 days for 16 weeks. In the fish given a diet containing no AsA supplement, AsA-deficiency symptoms were manifested earlier, and to a greater extent, in the stressed fish than in the non-stressed ones. Similarly, the growth of stressed fish fed on a diet supplemented with 75 mg AsA per 100 g was inferior to the growth of the control group, and in the stressed fish there was a reduction in AsA levels in the plasma, kidneys, and gills. On the other hand, fish fed on a diet supplemented with 300 mg of AsA per 100 g were barely affected by the stressor. These results indicate that, in Japanese parrot fish under these experimental conditions, exposure to intermittent hypoxic stress not only induced AsA-deficiency disease early, but also increased the AsA requirement. It was also shown that high doses of AsA increased the ability of these fish to resist the stressor.

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.

Green microalgae such as Nannochloropsis oculata are widely used for fish seedlings in aquaculture industry as Green Water. In this study, the abundance of Roseobacter clade is being focused, as some strains are known to produce inhibitory substances against fish pathogenic bacteria. In N. oculata culture, Roseobacter clade (RCA) were various and came to be abundant. Interestingly, the combination of Roseobacter clade and phytoplankton reinforced the killing effect of RCA against some V. anguillarum.

前年度の研究で,transposase認識配列を付加した発現ベクターをマダイ受精卵に導入することで,transposaseのmRNAの共インジェクションの有無にかかわらず12.4～14.7%の個体の鰭からGFP遺伝子が検出され,共インジェクションした場合には斃死個体から高率でGFP遺伝子が検出された。このようにメダカのtransposase認識配列をマダイが認識して転移する能力が示唆されたが,マダイ由来の転移があることからtransposaseシステムは安定的なトランスジェニック系統作出の技術として問題があることが考えられた。そこで本年度は18塩基の配列を認識可能なmeganucreaseの利用について検討した。 骨格筋で強く発現するα-アクチンプロモーターに緑色蛍光タンパク質(GFP)遺伝子を連結したDNA断片の両端にmeganucrease I-SceIの認識配列を付加したプラスミドベクターを作成した。2009年12月に秋季から冬季にかけて産卵する香港系統のマダイから採卵し,上記発現ベクターおよびI-SceIの共インジェクションを試みたが,卵質が悪く発生しなかった。そこで,急遽マダイ4歳魚を陸上水槽に収容して蛍光灯による長日処理および加温によって成熟を誘導したところ,1月中旬から産卵を開始した。産卵が安定するのを待って,2月11日にマダイ受精卵へのマイクロインジェクションを再度行った。上記の発現ベクター100ng/μlとI-SceIを3U/μlおよび1U/μlの濃度で共インジェクションする試験区と,発現ベクターのみをインジェクションする対照区を設定し,各試験区とも1,000粒以上の卵にマイクロインジェクションした。 その結果,3U区で230尾,1U区で315尾,対照区で309尾の孵化仔魚が得られ,3U区のみに骨格筋にGFPを発現する個体が認められた。GFP発現観察後はそれらの仔魚を飼育した。

腸内細菌叢をコントロールすることでマダイのエドワジェラ症を予防する技術を開発する

日長調節によってブリやマダイなどの海水養殖魚の成熟を抑制し，養殖生産効率の向上を図る

By the injection of 1, 10-phenanthroline, a specific inhibitor for metalloproteinase, meat tenderization of red seabream was significantly suppressed, suggesting that matrix metalloproteinase is involved in the post-mortem meat tenderization of red seabream. Then, Genes encoding matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) were isolated from red sea bream. Actually, recombinant MMP degraded collagen, a main component of the muscle connective tissue, and the activity was inhibited by the addition of TIMP. These facts suggested MMP-TIMP system plays a very important role in the post-mortem tenderization of red seabream muscle. These findings suggested the transgenic expression of TIMP might suppress the post-mortem meat tenderization. We made a transgenic medaka overexpressing TIMP. Histological analysis demonstrated the effectiveness of the transgenic expression of TIMP for the suppression of collagen breamdown. To apply this technology to cultured fish, we adopted red seabream, an important species for fish culture. For the construction of the expression vector, three distinct α-actin genes (two skeletal muscle types and one cardiac muscle type) were also isolated. These actin genes are composed of 8 exons and having E Box and CArG Box in the up-stream region, but the expression pattern in the fish was clearly distinct. We made the expression vectors harboring the promoter regions of these genes with GFP as a reporter gene. As a result, all vectors were revealed to be useful for making transgenic red seabream. By using these vectors, we introduced TIMP gene into red seabream. We are now keeping 200 fish possibly having TIMP gene.