Yasuo Agawa

Gonadogenesis and slow proliferation of germ cells in juveniles of cultured yellowfin tuna, Thunnus albacares.

To develop techniques for seedling production of yellowfin tuna, the behavior of primordial germ cells (PGCs) and gonadogenesis were examined at 1-30 days post hatching (dph) using morphometric analysis, histological examination, and in situ hybridization. Immediately after hatching, PGCs were located on the dorsal side of the posterior end of the rectum under the peritoneum of the larvae, and at 3 dph they came into contact with stromal cells. PGCs and stromal cells gradually moved forward from the anus prior to 5 dph. At 7-10 dph, germ cells were surrounded by stromal cells and the gonadal primordia were formed. In individuals collected at 12 dph, PGCs were detected by in situ hybridization using a vasa mRNA probe that is a germ-cell-specific detection marker. The proliferation of germ cells in the gonadal primordia began at 7-10 dph. We observed double the number of germ cells at 30 dph (22 ± 3.2 cells), compared to that at 1 dph (11 ± 2.1 cells). Therefore, based on our data and previous reports, the initial germ cell proliferation of yellowfin tuna is relatively slower than that of other fish species.

Molecular cloning and expression profiling of procollagen α1 (I) of cultured Pacific bluefin tuna

Type I collagen is widely distributed in most organs in teleosts. It plays a role not only in intercellular adhesion, but also in molecular signaling. In this study, Pacific bluefin tuna (PBT) procollagen α1 (I) cDNA was cloned and characterized. The nine fragments of a procollagen α1 (I) chain cDNA clone were prepared and spliced together to create the complete coding region. The resulting amino acid sequence was homologous with that of other teleosts. The mRNA expression profile of PBT procollagen α1 (I) in various tissues and the phylogenetic analysis with other vertebrate procollagen α1 (I) chains suggest that PBT procollagen α1 (I) could be a precursor form of the PBT type I collagen α1 chain. In addition, its level of expression in PBT larvae and early juveniles gradually increased with somatic growth. This increase was related to the standard length, wet body weight, and protein content of each individual fish. Therefore, the expression profile of procollagen α1 (I) may be a useful indicator for somatic growth in fish larvae and juveniles.

Development of a quantitative polymerase chain reaction assay for the detection of skin fluke Neobenedenia girellae larvae from environmental water

Skin fluke Neobenedenia girellae infection is a chronic problem in marine finfish aquaculture. Current control measures rely on bath treatments, which are labor intensive and cause significant stress to fish. Although there is an urgent need to develop a strategy to prevent this infection, little is known about the distribution of the larval stage of the skin fluke around culture cages. We aimed to develop a Neobenedenia-specific real-time quantitative polymerase chain reaction (qPCR) assay to detect and quantify N. girellae larvae in environmental water. New PCR primers targeting mitochondrial DNA were designed and showed specificity for N. girellae among five capsalid monogeneans known to occur in Japanese waters. A single N. girellae oncomiracidium is estimated to carry 2.2 million copies of mitochondrial DNA, and the qPCR assay reliably detects DNA equivalent to half of that. We used this qPCR to quantify N. girellae larvae from a fish culture site. Analyses of water samples collected inside shaded culture cages of Seriola dumerili revealed lower larval density than that in the unshaded cage. The results indicate the usefulness of this newly developed qPCR assay for monitoring skin fluke larval density and distribution in fish farms.

Isolation and characterization of 25 microsatellite loci from the Pacific bluefin tuna Thunnus orientalis (Perciformes, Scombridae)

The pacific bluefin tuna (PBT) Thunnus orientalis is one of the most important species for fisheries in the world. This species has been highly exploited in fisheries, resulting in dwindling of its natural resources. The artificial hatching of a third generation of fully cultured PBT was a success in 2007, suggesting the possibility of mass production of PBT for food and for seed release. To enhance stock structure studies and investigate genetic feature of seedlings, we isolated 25 polymorphic microsatellite loci (2–18 alleles/locus; expected heterozygosity, 0.042–0.941) from PBT.

Genetics in Tuna Aquaculture

Efforts to develop techniques for breed improvement in aquaculture have been conducted on numerous species, including salmonids, tilapias, and carps. Although attempts at breed improvement for tuna only recently began, rearing technologies used to close the Pacific bluefin tuna (PBFT) life cycle could potentially expedite the development of PBF breed improvement. The PBFT breeding program at Kinki University currently targets the following desirable traits: improved reproduction, increased survival rate during early larval development, faster growth rates, enhanced disease resistance, and improved meat quality. This technology emphasizes the use of marker-assisted breeding programs based on genomic and mitochondrial DNA analysis conducted on carefully selected fish held in bio-secure oceanic net pens. This approach prevents the potential “genetic pollution” of natural PBFT populations from encounters with escaped fish. Together with PBFT breed improvement, the simultaneous development of stress-free (reduced) handling technologies for activities such as juvenile tagging is also essential as tuna species have a low tolerance for handling stress. Finally, a comprehensive strategy for intellectual property protection including copyrights, patents, and trademarks should be implemented in any novel breeding program such that the hard-earned products and technologies developed can be protected from unfair competition.