Program

Genomic technologies have shifted from being an expensive research approach to becoming one of the most significant drivers of new knowledge in all aspects of biological sciences. A rapid growth in technologies available to determine the genome, transcriptome, proteome, peptidome, metabolome and microbiome of diverse organisms, coupled with continual drop in the cost of such analysis means that all fields of biology, from medicine to agriculture, from oceans to deserts, are benefiting from the explosion of data available about all aspects of life.

The Aquaculture industry is no exception, and in this talk some of the key genomic technologies will be introduced together with case studies where they have already made, or are making, both scientific and commercial impacts. In the context of health, rapid sequencing of pathogens enables the development of diagnostic kits as well as the identification of the origin and route of infection. In the context of genetic programs, traditional use of microsatellites is now being replaced or complimented with genotyping by sequencing technologies, which enable not only the identification of the sire and dam, but also the association of short nucleotide polymorphisms (SNPS) with traits of interest. Understanding and manipulation of reproductive processes is advanced through the study of the transcriptomes and proteomes of neural and reproductive tissues, leading to the discovery of new and novel peptides, and DNA barcoding is used for species identification and ensuring traceability and authenticity of seafood products, to name a few. The genomic revolution is here.

1.Department of Aquaculture, Faculty of Fisheries and Marine Sciences, Bogor Agricultural University (IPB), Bogor 16680, Indonesia
2.Graduate Student, Aquaculture Study Program, Department of Aquaculture, Faculty of Fisheries and Marine Sciences, Bogor Agricultural University (IPB), Bogor 16680, Indonesia

ABSTRACT

Commercial value of betta is determined by its body color and size. Indonesian betta (Betta imbellis) transgenic F0 carrying stripped catfish (Pangasianodon hypopththalmus) growth hormone cDNA (PhGH) have been produced in our previous study using transfection method. Present study was conducted to evaluate the PhGH transmission, mRNA expression level and production performance of betta transgenic first generation (F1). Three males and females of transgenic F0 were mated with non-transgenic, and three pairs of non-transgenic betta were used to produce F1 generation. PhGH gene transmission were analyzed using PCR method, mRNA expression was analyzed by RT-PCR and production performance was analyzed based on fertilization rate (FR), hatching rate (HR) body length and growth. At two months after hatching males were individually, while females were communally reared for three months. The results showed that percentage of F1 transgenic fish carrying PhGH gene was 62.5 ± 5.89%, and mRNA expression varied between individuals F1 fish. FR (91.7±7.5%) and HR (91.3±9.4%) transgenic fish was higher than non-transgenic control fish (FR: 70.0±10.0%; HR: 70.8±4.5%). At five-month-old, the mean body weight of males and females transgenic F1 was 1.47 and 1.76 times, body length was 1.32 and 1.25 times higher than non-transgenic fish, respectively, while the survival was the same. Thus, PhGH gene can be transmitted and enhanced the growth of betta transgenic F1 generation.

Turbot Scophthalmus maximus is an important cultured marine fish in North China. The induction of tetraploid turbot is important in germplasm enhancement for large-scale applications of triploid. The tetraploid fish was induced by hydrostatic pressure shock to prevent embryo cleavage. The optimal initiation time for pressure shock was 15 min before the first cleavage at 14.8-15.5°C. The optimal treatment pressure and treatment duration were 67.5MPa and 6 min. The hatching rate and tetraploid rate could reach 72% and 90%, respectively. Hydrostatic pressure shock induced higher deformity rate of embryo which could not survive to hatching stage. The rest tetraploid embryo had a prolonged hatching period, but showed no difference in morphology with diploid embryo and larvae. Although the embryo and early larvae with high tetraploid rates can be acquired, the survival rates of which are very low. Two obvious peak period of mortality were observed in gastrula stage and hatching stage. The expression of two cyclin-dependent protein kinase genes (cdk1 and cdk6) in turbot was studied using real-time RT-PCR. The expression levels cdk1 were showed significant difference with control group at blastula stage and gastrula stage, and cdk6 expression level at the gastrula stage was the highest. Trend variation pertaining to cdk1 and cdk6 expression levels in the tetraploid induction group was similar to that in the control group, except for the expression level, which was lower in the treatment group than in the control group. The tetraploid rate of larvae was also observed by flow cytometer. The tetraploid rate kept stable until 3dph and sharply decreased to 3% at 20dph. In this period, the ploidy of some tetraploid larvae decreased into diploid. The exact mechanism of ploidy decrease needs further study.

[Purpose] Yellow drum (Nibea albiflora) is one of the most important species for commercial fisheries and a promising candidate for aquaculture in China. This fish exhibits significant sexually dimorphic growth and the efficiency and profitability of its aquaculture could be maximized by all-female production. Consequently, we developed effective protocols for inducing meiotic gynogenesis in yellow drum and further producing pseudo-male using gynogenetic fish.
[Materials and Methods] Spermatozoa from rock bream (Oplegnathus fasciatus) were used and the optimal UV-irradiation dose for genetically inactivating the spermatozoa was investigated. The critical parameters for preventing extrusion of the second polar body were optimized to increase the yield of diploid gynogenotes. Flow cytometric analysis and microsatellite markers was used to verify the success of meiotic gynogenesis. Immersion treatments with 17a-methyltestosterone (MT) were applied to genotypically female gynogenetic yellow drum to induce phenotypic sex reversal.
[Results] The most effective genetic inactivation of rock bream sperm was observed at a UV dose of 45 mJ/cm2. Optimal cold-shock conditions were found to consist of cold-shock at 4ºC for a period of 8 min commencing 2 min post-fertilization. Flow cytometry and microsatellite analyses demonstrated that our protocol successfully obtained 100% gynogenetic individuals. Histological observation of the gonads revealed that treatment of gynogenetic fry (30dph) with 17ɑ-MT applied as 2-h immersion per day at a dose of 0.2 ppm induced 100% sex reversal. Our findings could potentially be applied to produce monosex yellow drum on a large scale to meet demands in the aquaculture industry.

Cryopreservation of fish embryos is a promising tool not only for aquaculture but also for conservation of endangered species, but so far it has been impossible to achieve. This is in great part due to the presence of structural barriers in the embryos, such as the chorion and other membranes, that limit the penetration and homogeneous distribution of cryoprotective agents (CPAs) inside the embryos. In this study, we tested the usefulness of microinjection to deliver extenders and CPAs directly and swiftly to specific locations inside the embryo, e.g. the perivitelline space (PS) and within the yolk mass (YM), of Japanese whiting embryos of different developmental stages. Embryos at the tail elongation stage that were injected in the YM with 2.1, 15.6, 24.4, and 33.5 nl of an extender (Yamamoto solution) had hatching rates of about 81, 67, 36, and 15%, respectively. Injection in the PS was tolerated well only up to 2.1 nl whereas larger volumes caused sharp declines in hatching rates. Embryos at six developmental stages (2~32 cells, morula, blastula, gastrula, somites and tail elongation) were punctured in the PS and the YM and the highest and lowest survival rates were obtained with the tail elongation and gastrula stages, respectively. The extender distributed swiftly within minutes from injection in both the PS and YM as visualized with the aid of the dye trypan blue. In addition to Yamamoto solution, embryos tolerated also other extenders such as fish Ringer, phosphate buffered solution, and ¼ diluted artificial sea water. We also compared the toxicity of three common CPAs, dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG) delivered by injection into the PS and YM of embryos and found that EG was the least toxic. Tail elongation embryos were more tolerant to the CPAs than those of other developmental stages.

The development of germ cell cryopreservation and transplantation techniques has led to the conservation of endangered species. Among the 16 bitterlings native to Japan, eight are listed as critically endangered and five as endangered on the Red List, including Kyushu bitterling (Rhodeus atremius). Previously, we have established allogeneic transplantation of spermatogonia in Chinese rosy bitterling. Here, we developed inter-species transplantation using Kyushu bitterling as a donor and Chinese rosy bitterling as a recipient. Approximately 3000 testicular cells were transplanted into the peritoneal cavity of 4-day-old triploid (3n) and diploid (2n) recipients. To trace donor cells in the recipient gonads, they were stained with the fluorescent membrane dye PKH26. Sixteen days post transplantation (dpt), the incorporated donor-derived germ cells were observed using fluorescence microscopy. The remaining recipients were reared until they reached maturity. Among the transplanted recipients, 76.25 ± 4.79% carried donor cells in their gonads. Six months later, semen of three triploid male recipients was analyzed using PCR. One recipient was confirmed to have produced donor-derived sperm. Two diploid females produced round-shaped and elongated-shaped eggs. DNA analysis of the offsprings confirmed that round-shaped eggs were derived from Kyushu bitterling. Thus, we successfully established a germ cell transplantation system to produce gametes for an endangered bitterling species.

Intraperitoneal germ-cell transplantation technique in pelagic-egg spawning marine teleosts had been studied using sciaenid fish as models. We have previously demonstrated that the interspecific hybrid offspring obtained from a cross between blue drum Nibea mistukurii females and white croaker Pennahia argentata males possessed germ-cell deficient gonads and could be useful as surrogate broodstock. In the present study, we tested whether the hybrid recipients produced genetically distant xenogenic donor-derived gametes.

Testicular cells prepared from the immature testis of 1.5-year-old mulloway were intraperitoneally transplanted to the total of 430 hybrid recipient larvae at 14 day-post-hatch over the course of four independent experiments. In each trial, testes collected from 3-5 males were pooled, dissociated by enzymes, and labelled by PKH-26 fluorescent dye. Colonization of donor-derived PKH 26-labelled cells was observed in 3 of 20 (15 ± 5%) hybrid recipients. At 4 month-post-transplantation (mpt), gonads from the 25 recipients were tested for the presence of the donor-derived germ cells by RT-PCR. Both mulloway-specific vasa and dmc1 PCR fragments were amplified in 2 of 25 (8%) recipient gonads, indicating that xenogenic donor germ cells had been maintained and entered meiosis in the recipient gonads. Among 19 recipients survived at 11 mpt, 2 (10%) recipients produced motile sperm carrying mulloway-specific vasa DNA sequence. Progeny tests conducted by inseminating blue drum eggs with recipient-produced sperm, and the resulting hatched larvae were sampled for genotyping. As a result, all F1 offspring from two recipient males possessed mulloway-specific vasa DNA sequences, suggesting that these hybrid recipients produced mulloway sperm.

Because functionally sterile triploid blue drum recipients failed to produce donor-derived mulloway sperm (data not shown), we speculated that microenvironment of germ-cell deficient gonads of hybrid recipients supported the gametogenesis of xenogenic donor germ cells.

We have been attempting to establish a surrogate broodstock that can produce Pacific bluefin tuna (PBT) gametes by xenogeneic germ-cell transplantation. Previously, we revealed that only type A spermatogonia (ASG), among various germ cells, have the potency to colonize recipient gonads. Therefore, we aimed to visualize ASG specifically, which would facilitate sequential analyses of donor-cell behaviour, including colonization, proliferation, and differentiation in the recipient gonads. However, the introduction of reporter genes such as gfp is not suitable for food fish and seedlings used for releasing into natural water. To overcome these difficulties, we produced monoclonal antibodies that can recognize the cell surface antigen of ASG in PBT, aiming to visualize live ASG without any transgene. We first generated 1,152 antibodies by inoculating mice with 5 × 10⁶ PBT testicular cells containing ASG. Next, antibodies that could specifically recognize the cell membrane of ASG were screened by cell ELISA, immunocytochemistry, and flow cytometry (FCM); we successfully selected 20 out of 1,152 antibodies. Further, to make ASG visualization easier and simpler, we directly labeled one of the 20 antibodies (No152) with a fluorescent dye, Alexa 488. Immunocytochemistry with Alexa-labeled No152 could visualize ASG-like cells in a one-step procedure. Further, RT-PCR was performed against the FCM-sorted fluorescent cells to confirm whether Alexa-labeled No152 can surely visualize ASG. We found that the fluorescent cells fraction expressed the ASG markers vasa and dead end but not the somatic-cell marker gsdf, suggesting that ASG were specifically isolated with No152. Finally, the visualized ASG were transplanted into the peritoneal cavity of nibe croaker larvae to evaluate their migratory ability. As a result, the incorporated fluorescent cells were observed in the recipient gonads, suggesting that the visualized ASG possess migration and colonization ability. Thus, the visualization of ASG made PBT germ-cell-transplantation easier and simpler.

Among the silver crucian carp Carassius langsdorfii, both bisexually reproducing diploids (2n) and gynogenetically reproducing polyploids (triploids (3n) and tetraploids (4n)) are found in Japan. Triploids are all-female, but 2n and 4n have both sexes. All 4n females spawn genetically identical unreduced 4n eggs which spontaneously develop by gynogenesis. However, reproduction of 4n males has been scarcely studied due to their low frequencies in wild populations. Here, we report the presence of two types of spermatogenesis in 4n silver crucian carp. Among 12 tetraploid males collected from the Gunma Prefectural Fisheries Experiment Station, 11 males gave fertile 2n spermatozoa, which are presumably generated by regular meiosis. On the contrary, sperm of 12th 4n male gave wide distribution of flow-cytometrical histograms corresponding to the ploidy status between 0.5n and 4n with approximate mode ranging from 1.5n to 2n. Thus, this male formed various kinds of aneuploid spermatozoa. Similar aneuploid spermatozoa were also observed in nine 4n male progeny appeared from the experimental cross between 3n female and 2n goldfish male. Aneuploidies were also suggested by variation in sperm head volumes (25.63±10.13μm³), when compared with haploid sperm of the diploid goldfish male (14.75±2.46μm³) and diploid sperm of the other 4n male (28.39±4.22μm³). Spermatogenesis may depend on genomic constitution of tetraploid males: amphidiploidy may give rise to normal meiosis for the formation of diploid spermatozoa, while allotetraploidy including odd number of sub-genome set may induce meiotic configuration including univalents and multivalents and then may result in the formation of aneuploid spermatozoa.

This study was observation of parameter physiology and morphology of sperm, the determination of physiological solution, as well as the evaluation of the effects of voltage and number of electric shocks differently to fish sperm motility and viability of climbing perch. Observations indicate that the total length, head length, length of the middle section, and the length of the sperm tail climbing perch are respectively 35.54 ± 4.66; 1.83 ± 0.20; 0.93 ± 0.23; 33.05 ± 1.40 µm, and the sperm head width of 1.48 ± 0.21 µm. Climbing perch sperm motility in the water is over 116 ± 34 seconds. Osmolarity levels of body and testicular climbing perch fish ranged between 390-405 mOsmol kg-1, equivalent to a rate of 1.3% NaCl salinity (osmolarity: 407 mOsmol kg-1). Storage of sperm is diluted with physiological solution can only last a maximum of 60 minutes at room temperature (27^o C) and cold temperature (4^o C) using a dilution NaCl 1.3%, whereas without dilution can last for 360 minutes. Preservation of testicular fish for artificial spawning climbing perch is not different from the conventional method (semi-artificial breeding) (p> 0.05) related to the fertilization rate, and survival rate of larva up to 10 days after hatching. The process of electroporation in climbing perch fish sperm was diluent using 1.3% physiological solution successfully performed using a program with a long shock of 0.5 milliseconds, the interval shock 0.1 seconds, and the voltage 50, 125, 250, and 500 volts, and the amount of shock 1, 3, and 5 times. Increasing the voltage and the number of interacting shock was given lower levels of motility and viability of sperm (p<0.05), treatment voltage 50 volts and 1 time of shock does not provide a significant difference to the control treatment on parameters of sperm motility and viability.