Program

In these days, much attention has been paid for heathy food stuffs, such as fish meat, all over the world. In the contrast to crops and domestic animals, however, any efforts have not made to breed beneficial traits of seawater aquaculture fish because the selection and breeding of fish in water is labor intensive and time-consuming work. The newly coming technology, genome editing technology, enables to targeted gene alternation precisely even in non-model creature including aquaculture fish and has potential to facilitate breeding in aquaculture.
In my talk, I will introduce our trial to make new traits harboring double muscle phenotype in red sea bream (Pagrus major) and Japanese puffer fish (Takifugu rubripes). Using CRISPR/Cas9 system, the most popular genome editing tool, myostain gene (mstn) has efficiently been disrupted with a frame-shift deletion in founder generation (F0 generation). The fish with high mutation rate estimated by heteroduplex mobility assay using genomic DNA extracted from fins showed a high value of condition factor [1000 x body weight / (body length)³], indicating the increase of skeletal muscle mass. As for red sea bream, both male and female F0 fish matured at two years after hatch and were mated each other to produce F1 generation. The frame-shift mutation in mstn has inherited to F1 generation and we successfully obtained mstn KO individuals (bialllic mutant in mstn). The KO fish exhibited significantly higher value of condition factor compared with that of wild-type fish, indicating that the induced phenotype has also inherited to the next generation. As for Japanese pufferfish, only male matured at two years after hatch and the inheritance of the mutation in mstn has observed.
Our data clearly showed that the genome editing is a powerful tool to generate new traits in aquaculture within a short period.

Large yellow croaker (Larimichthys crocea) is an important maricultured species in southeast China. To lay a solid foundation for improving its economical traits, we have done systematic studies on genomics and breeding technology of this species in recent years. For instances, a high-density genetic map and an optical physical map have been constructed. And a high-quality chromosome-level reference genome has been generated with a second generation of gynogens. On this basis, Genotyping-by-Sequencing (GBS) technology was adopted for SNP discovery in a reference population. And genomic selection was then applied in the genetic improvement of quantitative traits, such as n-3 HUFA content. Several large effect QTLs were mapped using the linkage or association analysis in sex determination, growth rate and n-3 HUFA, etc. The predictive accuracy of a variety of statistical methods were assessed in genomic prediction in the croaker populations. To make genomic selection more plausible in fish breeding, we explored to only include individuals with extreme phenotypes to assemble reference populations, and then predicted genomic estimated breeding values (GEBV) of candidates. When 50% individuals with extreme phenotypes were used to predict GEBV, the predictive accuracy was very similar to that estimated with all individuals. Furthermore, we also explored to only use top associated SNPs in the prediction. As a result, when ~10 top SNPs associated with the n-3 HUFA content were used to predict the GEBV of candidates, the accuracy was even higher than that estimated with genome-wide SNPs. A multiplex PCR technology was exploited to genotype the SNPs with large effects at very low costs. Taken together, the strategies we developed significantly reduce the costs but not obviously lower the predictive accuracy, and can definitely promote the application of genomic selection in the breeding of the large yellow croaker and other fish.

Pacific bluefin tuna, Thunnus orientalis, has a high market value, but its wild populations have decreased in recent years. Complete aquaculture technology has recently been established for this species, and artificial seeds are beginning to be used for production. The creation of broodstock with commercially valuable traits, such as rapid growth, is therefore of great interest. Genetic linkage map-based identification of markers associated with quantitative trait loci (QTL) facilitates marker-assisted selection (MAS) breeding and efficient genetic improvement of broodstock. Single nucleotide polymorphism (SNP)-based genetic linkage map construction using the genotyping-by-sequencing (GBS) method can increase the number of mapped markers to the reference genetic linkage map (Uchino et al., 2016) and help identify growth-related QTLs. In this study, we have developed SNP-based new genetic linkage maps and analyzed initial growth-related QTLs in Pacific bluefin tuna.
For a genetic linkage map construction and QTL analysis, male and female parents and 93 progenies were used. GBS library preparation and sequencing on an Illumina HiSeq4000 platform were performed. After trimming of low-quality reads, SNPs were identified using Stacks. For microsatellite (MS) markers, a fragment analysis was carried out on a 3730xl DNA analyzer. MapDisto was used to construct sex-specific maps. Genetic distances and genotypes of each MS and SNP marker and total lengths were used to perform composite interval mapping using R-QTL.
Sex-specific maps for 24 linkage groups consisting of 677 SNP and 651 MS markers were constructed. The total lengths of 93 progenies in the mapping population followed a normal distribution, with an average length of 9.4 mm. QTL analysis revealed one significant QTL in LG10 on the female linkage map. The new genetic linkage map generated for Pacific bluefin tuna and the growth-related QTLs detected in this study will be useful for tuna aquaculture MAS programs.

Red sea bream iridoviral disease (RSIVD) is a major viral disease in fish farming in Japan, and a new broodstock strain showing resistance to the disease has been required. In 2012, a catastrophic outbreak of RSIVD occurred at a farm, and using DNA parentage analysis we identified one novel male broodstock individual that was significantly associated with surviving individuals. The RSIVD-resistant trait of the male individual was dissected by genome-wide linkage analysis using newly developed microsatellite DNA markers with one immune-related gene, MHC class IIβ. One locus linked to an RSIVD-resistant trait was found in linkage group 2 of the male, which can be explained with highly phenotypic variance (31.1%). MHC class IIβ was highly associated with the phenotype and is considered a candidate gene. In addition, based on the sequencing analysis of the β1 region of MHC class IIβ, one novel amino-acid substitution was found at the 74 amino-acid residue involved in the pocket-4 structure, a mutation which has also been reported in other vertebrates associated with disease resistance. The F1 population was selected using marker-assisted selection (MAS), and the F2 population (MAS population) was produced. The MAS population hypothetically inherited the resistant allele (+) shown as follows after determining the Mendelian sampling variance: +/+ in 25%, at +/− in 50%, and at −/− in 25%. This population was cultured during the summer in 2016 at a fish farm where RSIVD has often occurred, and the survival rates in each genotype were retrospectively estimated. The individuals inheriting the resistant-allele in +/+ and +/− had a high survival rate (both 87.6%), but individuals in −/− showed a lower survival rate (50.0%). These data indicate that the resistant allele dominantly affects the RSIVD-resistant trait, which is highly available for marker-assisted breeding of an RSIVD-resistant strain.

Marine fish of the genus Seriola, commonly known as yellowtails or amberjacks, are important for aquaculture in Japan. In yellowtail aquaculture production, Benedenia disease is a serious parasitic disease caused by infection by the monogenean parasite Benedenia seriolae. Previous quantitative trait locus (QTL) analyses have identified a major QTL associated with resistance to Benedenia disease in linkage group Squ2 of the Japanese yellowtail Seriola quinqueradiata. Although yellowtails thus use the some genetically regulated immune mechanisms to reject and/or protect against B. seriolae infection, the bioregulatory mechanisms underlying Benedenia disease resistance are unclear.
To reveal the molecular mechanisms for Benedenia disease resistance, we screened and bacterial artificial chromosome (BAC) clones carrying genomic DNA of the QTL region of linkage group Squ2 from a yellowtail BAC library and constructed a minimal tiling path of seven BAC clones covering the QTL region. We sequenced these seven BAC clones on Illumina sequencing and identified a gene putatively responsible for the Benedenia disease resistance trait. Complete sequencing of the QTL region of linkage group Squ2 revealed that an immuno-related gene was located there. The expression of this candidate gene was detected in skin tissue parasitized by B. seriolae. A search for single nucleotide polymorphisms (SNPs) in the open reading frame of this immuno-related gene revealed that one SNP was associated with infection levels of B. seriolae.
Our findings suggest that the immuno-related gene control Benedenia disease resistance in Japanese yellowtail and represent a first step toward identification of molecular mechanisms for resistance against monogenean parasite infection in marine aquaculture fish. The polymorphism in the candidate gene may be useful as a DNA marker in marker-assisted selection for Benedenia disease resistance in Japanese yellowtail and other members of the genus Seriola.

The study of mass selection responses was conducted to compare the correlation between growth characteristics and their relation in order to obtain small head and large body in Climbing Perch, Anabas testudineus. Two populations from Thailand: pre-selected population from the best six sources growing groups (25 males and 25 females) and fourth generations post-selected populations(100 males and 100 females) were used. The correlations among mixed sexes, female and male of eight growth traits including weight(W), total length(TL), head length(HL), head width(HW), head depth(HD),body length(BL), body width(BW) and body depth(BD) showed 0.35 to 0.99 for pre-selected and 0.30 to 0.99 for post-selected, respectively. The correlation of HL and BL showed 0.62, 0.89 and 0.45 for pre-selected and 0.55,0.46 and 0.62 for post-selected, respectively. The correlation of BW and BL showed 0.77,0.98 and 0.78 for pre-selected and 0.50, 0.32 and 0.77 for post-selected, respectively. The correlation of BD and BL showed 0.57,0.72 and 0.56 for pre-selected and 0.49, 0.36 and 0.57for post-selected, respectively. The results suggests that mass selection has effects on the use of correlation between growth and the proportion of the shape desired Climbing Perch.

This study examines whole nacre thickness and monthly growth of pearls of Akoya pearl oyster, Pinctada fucata, which came from Japanese and hybrid strains and relate them to the water temperature of the aquaculture site in Ago Bay. Whole nacre thickness of the pearls was significantly different between Japanese and Hybrid strains (Student’s t-test, p-value < 0.01), pearls of Japanese strain tend to be thicker than those of Hybrids. This is due to different nacre growth rate between the two strains especially in summer, Japanese strain grew faster than Hybrid in August until November (Student’s t-test, p-value < 0.01), even though there was no significant difference in December when both strains showed very slow growth of pearl nacre due to the low water temperature. Monthly nacre tablet thickness of pearls of Japanese strain was thicker than that of Hybrid in August, September and November (Student’s t-test, p-value < 0.01) but no significant difference in November and December. The result of this study shows that pearl nacre growth and thickness are related to the water temperature of the aquaculture site. The fact that both oysters have different range of optimum temperature to grow leads to the possibility of different quality of pearls from both strains since whole nacre thickness, nacre growth and nacre tablet thickness can determine the quality of pearls.

Gonad maturation was achieved out of season by the use of appropriate controlled photoperiods, temperatures, and abundant feeding. Nowadays, many research showed immature fish brought to maturity in the laboratory were spawned with suitable hormone injections and the time of spawning could be accurately predicted. Here we investigate whether oral administration is essential in order to achieve the same effect with injections of hormone into the fish. The tilapia of sbtLH (southern bluefin tuna Luteinizing Hormone) treatment group start to spawn and produce a numerous of larvae at 8th days after feeding. We find that the gonad of feeding LH treatment group is five times mature of cold-treatment group and control group, and the expression level of cyp19a becomes highest in female tilapia. The increases of gonadosomatic index (GSI) in female following LH treatment group, relative to the cold-treatment group and control group .The techniques developed of gonad maturation reagent by oral administration may have application to ornamental aquarium fish, mariculture and in more general research on marine fish reproduction.

Catfish farming (Clarias sp.) in Indonesia is growing rapidly, requiring adequate and sustainable seed supply throughout the year. Characteristics of breeding in catfish that generally only occurs in the rainy season, causing the scarcity of seeds, especially in the dry season. The hormonal reproductive manipulation and the addition of turmeric powder into the artificial feed have been made to induce the reproduction of catfish. Female and male catfish with a weight of 400-600 grams/piece given the feed containing a combination of hormone and cucurmin (Cucurma longa) powder. Fish were given a combination of Pregnant Mare's Hormone Serum Gonadotropin (PMSG) hormone and domperidone (Oodev) at doses of 0, 0.25, 0.50 ml/kg fish/two weeks; and cucurmin powder of 0, 0,25, 0,50 gram/kg of feed. Fish fed 2% of body weight/day for eight weeks. Experiment was designed factorially with 50 fish for each treatment. Mature fish gonad then spawned by artificial spawning. The results show that the female fish given the combination of 0.25 Oodev + 0.25 cucurmin powder is the best, which has periods for gonad maturation is three weeks (twice as short), number of mature females 84% and significantly has a high amount for spawning success, degree of fertilization, hatching rate, survival rate and length of seed are better than control. While in male fish, the best results were shown by treatment of 0.5 Oodev + 0.25 cucurmin powder, which had a maturation period of 2 weeks, matured males 88%, and high motility of spermatozoa compared with controls. The combination of PMSG + domperidone and cucurmin powder through artificial feed on catfish can accelerate gonadal maturation and improve reproductive performance. These results provide hope that the fish will still be able to reproduce even in the dry season.

Bluefin tuna aquaculture is predominantly based on grow-out or fattening of wild-caught juveniles, and studies have shown the negative impacts of this type of aquaculture on the management of natural stocks. To sustain stable food provision and conserve wild tuna stocks, full-cycle aquaculture systems need to be established. However, a major problem that remains is the unstable spawning of bluefin tuna in sea cages due to fluctuating environmental factors, especially water temperature. To develop a stable supply system of fertilized eggs, we demonstrated spawning induction in Pacific bluefin tuna Thunnus orientalis (PBT) by manipulations of water temperature and photoperiod in land-based tanks. In May and June 2013, 126 PBT (average total length, 99.4 cm) reared from fertilized eggs for 2 years at Amami laboratory, Seikai National Fisheries Research Institute, FRA, Japan, were transferred into two land-based tanks (diameter, 20 m; depth, 6 m; volume, 1880 m3) at Nagasaki headquarters. The water temperature and day length of both tanks during the experimental period were maintained at 17.5–28.5°C and 10.5–15.0 h, respectively. Seventy-five live broodstocks were present in total; the first spawning in both the tanks was observed in the middle of May 2014, and continual spawning events were observed over 3 months. A total of 55,273,388 eggs were collected during the spawning season. The mean normal hatching rate was 87.9%. These results indicate that sexual maturation and spawning of 3-year-old PBT was successfully induced by the manipulation of the environmental factors in the land-based tanks. We propose that the technique for stable egg production in land-based tanks established in the present study will prove to be essential in PBT aquaculture. This study was supported by a Grant-in-Aid “Development of Sustainable Aquaculture Technology Independent of Wild Fishery Resources” from the Ministry of Agriculture, Forestry and Fisheries, Government of Japan.

According to a recent report with respect to tuna farming, there are 160 tuna farms and 1432 cages of bluefin tuna in Japan. For the bluefin tuna farmer in Japan, to determine the number of farmed fish is one of the key issues to monitor the number of fish in addition to feed waste, escapement, behavior, and dead fish. However, there presently exists no reliable method to count the number of bluefin tuna in a cage. The most popular type of counter is currently the underwater stereoscopic camera system which has mainly been used by a diver for counting. However, this kind of counting method is not only labor-intensive, but its accuracy is low in dark or turbid water. The purpose of this study is to develop an accurate counting method for the farmed bluefin tuna using the multi-transducer sonar and pinger. This newly developed a multi-transducer sonar system is based on counting the individual fish that has passed through “the sound curtain” consisting of 15 transducers (460 kHz). In addition, a pinger was used to clarify the behavior and swimming speed of the caged fish. As a result, it was found that all of the fish regularly swim in a concentric circle in the cage space, and the lap time in each lane of 1m from cage center for one round was estimated by a linear regression equation. The number of fish in each lane for one round could be calculated by multiplying this lap time and the number of fish that passed through “the sound curtain” per unit time. The total number of fish could then be calculated by adding up the number of fish in each lane. It was considered that this approach is effective for counting caged bluefin tuna with the objective of practical use.

Introduction
We reported opsin genes from longtooth grouper (LTG), which were mostly expressed 4 days post-hatching (dph) of the larvae. However, the relationship between behavior and feeding and the light spectrum remains unknown. This study determined the effects of LED wavelengths on growth performance in larval LTG.
Materials and Methods
Experiment I: Larvae were kept in black 100-L tanks irradiated with UV (375nm), blue (460 nm), green (530 nm), yellow (580 nm), and white (450–680 nm) LED lights at 15 log photons/cm2·sec. Larvae were fed with rotifers for one day and the number of rotifers in gut was measured. Experiment II: Larvae at 8 dph were kept in 2-L beakers not exposed to light and only exposed to the five LED treatments mentioned above. Experiment III: The larvae at 3, 8, 16 and 26 dph were kept in 2-L beakers under the same light-conditions as Experiment II. The frequency of surface death and the water depth of fish were measured. Experiment IV: Larvae were reared in 200-L tanks irradiated with white LED, white and UV LEDs, white LED and chlorella at 14.7 log photons/cm2 sec until 10 dph.
Results and Discussion
Experiment I: Feeding rate was higher under white, UV and blue light than under other colors. Experiment II: Feeding rate was higher under white, UV and yellow light than under other colors. Experiment III: The frequency of surface death was highest under green LEDs. Larvae under white and green LEDs were distributed close to the surface layer at 16dph. Experiment IV: Feeding and growth rate were highest under the white and UV lights group. Although LTG larvae showed positive phototaxis for medium wavelengths, it is possible that they use short wavelengths for food consumption. Additionally, exposing larvae to UV irradiation is predictably effective for rearing LTG larvae.

Microalga Nannochloropsis oculata has been recognized as an important live feed of rotifer to enrich eicosapentaenoic acid (EPA) content. Enrichment of EPA to polar lipids of rotifers results in better growth and survival of finfish larvae. We found that EPA proportion of polar lipids of N. oculata increased in response to nutrient salt starvation during the cultivating period. However, it is unclear how the EPA is accumulated into polar lipids. In this study, we assessed the organelle development of N. oculata cells related to EPA accumulation. N. oculata was cultured with a batch style using f medium under continuous aeration and irradiance. The cell population were harvested at two different phases when nutrient salts were repleted (NR phase) and depleted (ND phase). Fatty acid analysis was conducted in each phase. Nucleus and mitochondria stained with each fluorescent dye were observed by confocal laser microscopy (CLM). Intracellular ultrastructure was also observed under transmission electron microscopy (TEM). Morphological change of the organelles among those phases was evaluated. EPA proportion in polar lipids increased during ND phase. The observation using CLM showed that, from NR to ND phases, the proportion of cells including only one nucleus increased up to 97% of the growth population. During NR phase the cells including more than five mitochondria existed, whereas such cells disappeared during ND phase and the proportion of cells including less than two mitochondria increased up to 80%. It is likely that the cell activity reduced due to lower energetic productivity by mitochondria. According to TEM image, a lot of vesicles containing lamella was observed instead of chloroplast during ND phase. This morphological change triggered by lacking cell activity may be linked to essential accumulation of EPA into polar lipids of N. oculata.

Diatoms have been widely used for shrimp nursing. However, most of farmer using diatom for feeding shrimp from zoea stage until postlarva 5. So this study aimed to investigate the effect of Thalassiosira weissflogii and Amphora sp. on the growth of white shrimp postlarva. The experiment consisted of control group (T1), added T. weissflogii (T2), Amphora sp. (T3), each treatment contained 3 replications. T. weissflogii (1.0 × 106 cell/ml) and 30 grams of Amphora sp. were added to nursing tank every day. The lenght of shrimp that added Amphora sp. (2.17±0.19 cm.) (P<0.05) was significant higher than group fed with T. weissflogii (1.99±0.15 cm.) and control group (1.83±0.95 cm.). However, the highest survival rate was found in T2 (added T. weissflogii). Lipid analysis showed no significant difference between treatment and control group. The results from this study suggested that supplemented Amphora sp. and T. weissflogii could significantly increase growth performances of Litopeneaus vannamai larvae.

White shrimp culture in the sea is a break through for increasing production and product quality. In contrast to the pond, ocean dynamics i.e. currents and waves are higher that complicate feeding the shrimp, so that it necessary feeding tray. This research aims to determine the production performance i.e. growth, survival rate, diversity coefficient, feed conversion ratio and productivity of white shrimp cultured under sea floating net cages (FNC) with a varying number of feeding tray. The research was designed using completely randomized design with 3 treatments feeding tray number i.e. 1, 2 and 3 units/ cage, and each involves the repeated 3 times. Feeding tray to be used measuring 1 m x 1 m made of 1-inch PVC pipe for the frame and hapa nets for containers. White shrimps (0,58±0,02 g/shrimp) were reared in FNC of 3x3x1,5 m with density of 450 individuals/m2 and fed pelleted artificial feed with a frequency of 5 times a day. A half portion of feed were placed on a feeding tray and remain were stocked gradully to the cage. White shrimps were cultured for 120 days and every 20 day 30 shrimps were sampled for determining growth rate, and the end of rearing the number of shrimp were calculated. The best production performance took place on the provision of feeding tray 3 units/cage i.e. daily growth rate of 0,14±0,00 g/day, specific growth rate of 2,92±0,01%, survival rate of 61,26±7,96%, feed conversion ratio of 1,79±0,22 and productivity of 4,71±0,54 kg/m2. The role of natural food were also discussed.