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Genetics in Tuna Aquaculture
Chapter 13
Genetics in Tuna Aquaculture Yoshifumi Sawada and Yasuo Agawa Fisheries Laboratories, Kindai University, Kushimoto, Wakayama, Japan
13.1 INTRODUCTION Human beings have been breeding animals for more than 10,000 years (Shoda, 2010). During this period, new breeds and strains have been developed with desirable phenotypic traits such as rapid growth, higher tolerance to environmental conditions, enhanced disease resistance and improved meat quality. In aquaculture, selective breeding has a much shorter history but has been conducted for several species including Atlantic salmon (Salmo salar: Gjedrem, 1983), rainbow trout (Oncorhynchus mykiss: Donaldson and Olson, 1957), tilapia (Oreochromis niloticus: Eknath et al., 1993), and carp (Cyprinus carpio: Babouchkine, 1987; Gjedrem, 2005). Selective breeding programs for aquatic species provide better outcomes compared to terrestrial livestock. This higher response to selection of farmed aquatic species can be attributed to their high fecundity enabling higher selection intensity. In addition, large phenotypic and genetic variations exist in the genetically underdeveloped fish traits. However, in the tuna aquaculture industry, selection programs are not commonly used and production still relies heavily on catching wild fingerlings and broodstock. The completion of the life cycle of Pacific bluefin tuna (PBFT) has provided the potential to develop selective breeding programs for this species and this is now underway in Japan (Sawada et al., 2004). In this process, biotechnology will play an important role in the area of tuna reproduction, breeding, and harvested product quality management.
13.2 TARGET TRAITS IN TUNA BREED IMPROVEMENT In any tuna breeding program the following biological traits are important targets for selection, fast growth rate, high survival rate during early development, high disease resistance, superior traits in reproduction, and high meat quality. D.D. Benetti, G.J. Partridge, A. Buentello (Eds): Advances in Tuna Aquaculture. DOI: http://dx.doi.org/10.1016/B978-0-12-411459-3.00013-8 © 2016 Elsevier Inc. All rights reserved.
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FIGURE 13.1 Diagram of tuna individual selection for breed improvement. The procedure of individuals having superior genetic traits encompasses capture, DNA material sampling and analysis, and tagging where multiple times of juvenile handling is inevitable.
Although tuna have extremely high growth rate compared to other fishes (Miyashita, 2002), it is desirable to further increase the speed of growth and development by selective breeding in order to improve production efficiency. Developing breeds of tuna that are selectively optimized for commercial aquaculture is a dynamic and intensive process. Steps involve broodstock capture and maturation concurrent with genetic sampling and tagging. Records are kept to correlate larval success, growout metrics, and other significant biological traits to breeding individuals, and subsequent resampling for future brood fish. Figure 13.1 depicts an outline of the steps involved in the continuous improvement of tuna breeds. The technology for tuna fingerling production is still underdeveloped and the survival rate during the hatchery phase is 10% at most. Improvements to survival rate will be attained by the improvement of larval and juvenile rearing techniques but selection of breeders whose offspring show high survival during early development can also be a goal of a selective breeding program. In the larval rearing of tuna, low survival rate due to an unsuitable rearing environment, such as water surface condition and current in the rearing water is the largest obstacle for their mass production (Miyashita, 2002; Sawada et al., 2005; Kurata et al., 2011). Therefore, it is important to create strains which have tolerance to environmental stress. Domestication and selection of families with high survival will be attained naturally in the early stage of
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tuna aquaculture technology development, as poor larval survival inevitably leads to selection against such breeds. However, as a more positive step, selection of domesticated and high survival breeds for specific environments should be implemented in tuna aquaculture. PBFT do not tend to suffer from serious diseases from 1 year after hatching. However, PBFT juveniles experience viral, bacterial, and parasitic diseases that lead to high mortality (Munday et al., 2003), and development of disease-resistant strains is also important. Under aquaculture conditions, female adult tuna with high reproductive activity are scarce (Masuma et al., 2010) compared to males, the majority of which reach maturity at a younger age than females (Sawada et al., 2008). Therefore, prolific breeders are required for stable reproduction especially for female tuna in tuna aquaculture. One of the most important criteria in evaluating quality of the aquacultureproduced tuna is the muscle fat content. Fatty portions such as the belly flaps called “toro” are preferable as raw materials for the preparation of sushi and sashimi, also deriving a higher market value than other muscle sections. However, demand also exists for leaner meat called “akami”. Selective breeding programs can be directed at controlling muscle fat contents is in PBFT muscle.
13.3 GENETIC TECHNOLOGIES IN TUNA BREEDING 13.3.1 Genomic DNA Analysis Genomic information is applicable to the improvement of tuna aquaculture by using DNA marker-assisted selection. For this aim, it is necessary to establish a series of DNA microsatellite markers. Although tuna DNA microsatellite markers have been developed by various scientists (Takagi et al., 1999; McDowell et al., 2002; Nakadate and Chow 2008; Tagami et al., 2008; Morshima et al., 2009; Agawa et al., 2009; Clark et al., 2004; Takagi et al., 2001), the number of microsatellites is still insufficient to identify the desired traits or to make a high-density genetic map of tuna in the breeding program. In the analysis of genes, such as base sequence determination, it is inefficient and difficult to use genomic DNA. As a substitute, genome libraries, which are composed of cloning vectors (inserted fragments of targeted genome DNA), are ordinarily utilized. To make a genome library, genome DNA is fractionated by the use of restricted enzymes, sonication or shearing, then the fragments are inserted into genetically engineered vectors, such as plasmids, bacteriophages (such as phage λ), bacterial artificial chromosomes (BACs), or yeast artificial chromosomes (YACs). For PBFT genome, a BAC library has been established (Yagishita et al., 2006). Identification of sex-specific DNA markers of PBFT is a good example of genomic DNA analysis (Agawa et al., 2011). Under aquaculture conditions,
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FIGURE 13.2 The sex-specific DNA PCR products were confirmed by gel electrophoresis. M; molecular size markers. The sizes (base pairs) were indicated left of the fragments. Two females and two males PBFTs were used. n.; negative control means PCR were conducted without template DNA. Md6; male delta 6 locus, male positive PCR (Agawa et al., 2015). β-actin; beta actin gene PCRs were used for positive control.
it has been reported that adult females have a low percentage of participation in reproduction (Masuma et al., 2010), and to make a broodstock group with a high percentage of females is necessary for the stable reproduction and for securement of appropriate genetic diversity in the laboratory hatched and raised PBFT strains. Because adult PBFT are large in size, vulnerable, and difficult to handle, sex determination should be conducted during early developmental stages of juveniles. Therefore, for PBFT juveniles, whose sexes cannot be distinguished phenotypically, sex identification by genetics using small biotic samples such as fin tips is very useful. These biotic samples can then be screened using AFLP-selective DNA amplification including primer-specific secondary amplifications for increased fidelity and sequenced through high-throughput next-generation sequencing technologies (Agawa et al., 2015). Combined with common electrophoresis methodologies, male characteristic DNA fragments (Figure 13.2) can be observed and are attributable to specific breeding fish. As a more comprehensive result for PBFT, draft analysis of the genomic DNA was performed by the Fisheries Research Agency (FRA) cooperatively with the University of Tokyo and Kyusyu University (Saito, 2010; Fisheries Research Agency, 2010). In this project, they reportedly found approximately 86,000 microsatellite DNA markers. These results of genomic analysis will contribute largely to the PBFT breeding programs.
13.3.2 Analysis of Mitochondrial DNA Polymorphism The mitochondrial DNA D-loop region in tuna of the genus Thunnus is highly polymorphic as in other fishes (Bremer et al., 1998; Bremer et al., 1999; Chow et al., 2000), and this variation can be detected using conventional PCR-RFLP analysis in several tuna and billfish species (Chow et al., 1997; Chow et al., 2000; Chow and Takeyama, 2000; Niwa et al., 2003). The analysis of mitochondrial DNA polymorphism has been well used as the tool to identify tuna species (Chow and Kishino, 1995; Takeyama et al., 2001).
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This kind of genetic tool to identify tuna species is useful to prevent mislabeling of tuna fishery and aquaculture products. To sustain consumers’ confidence, such effort should be encouraged. Mitochondrial DNA polymorphism has also been used to analyze the genetic structure of natural populations of yellowfin tuna (YFT) (Scoles and Graves, 1993; Ward et al., 1997; Wu et al., 2010), bigeye (Chow et al., 2000; Grewe and Hampton, 1998; Grewe et al., 2000; Appleyard et al., 2002; Durand et al., 2005; Chiang et al., 2006; Chiang et al., 2008), albacore (Chow and Ushiyama, 1995; Vin˜as et al., 1999), and Atlantic bluefin tuna (ABFT) (Ely et al., 2002.; Vin˜as et al., 2003; Boustany et al., 2008; Carlsson et al., 2004). To raise the precision of these analyses, genome DNA microsatellite markers are often combined (Nakadate, 2005). Such combination can detect genetic foreignness among pedigrees and even individuals with high sensitivity. Therefore, this combined analysis of mitochondrial and genome DNA polymorphism can be used in genetic monitoring for spawning ecology of captive tuna (Niwa et al., 2003), and it has the possibility to elucidate how individual variation among broodstock, degree of maturity, and nutritional state influence the egg quality and larval vitality. In addition, it can be a very powerful tool to register newly developed tuna strains in aquaculture and to identify artificially hatched and released stocks to the open sea for natural population enhancement in the near future.
13.3.3 Other Genetic Technologies Chromosome manipulation in cultured fishes is effective to improve their performance. Inviable aneuploids such as triploids have been induced to improve growth (Arai et al., 1991; Hussain, 1996; Pandian and Koteeswaran, 1998; Arai 2001). Cloned fishes have been also produced in rainbow trout Oncorhynchus mykiss (Okada, 1985), common carp Cyprinus carpio (Komen et al., 1991), tilapia Oreochromis niloticus (Mu¨ller-Belecke and Ho¨rstgenSchwark, 1995; Mu¨ller-Belecke and Ho¨rstgen-Schwark, 2000), olive flounder “hirame” Paralichthys olivaceus (Yamamoto, 1999), and red sea bream Pagrus major (Kato et al., 2002). Cloned strains have a possibility of being advantageous in the mass production by their homogeneity in their superior phenotypic traits such as the digestion ability of nutrients, sensitivity to medicinal agents, and environmental stress resistance. The useful characteristic of these technologies is to create strains without breeding potential. It will contribute to more efficient mass production of cultured species. At present, there is no example of application of these technologies in tuna aquaculture industry. Genetic modifications should not be used by the tuna aquaculture industry until rigorous examination in terms of food safety is completed. In addition, genetically-modified tuna escapees could potentially interfere with the genetic material of wild tuna populations should they breach their enclosures in net pens. Although containment of other fin fish species is easily attained
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FIGURE 13.3 Sampling of DNA material and tagging of a PBFT juvenile. Tuna juveniles have low handling tolerance.
in land-based culture facilities, tuna species require significant space/water volume such as the one provided by massive net pens. Today, it is impractical to practice commercial tuna grow out in land-based facilities. Finally, in practical tuna breed improvement, it is necessary to develop a new technology for the treatment of live individuals. To create a new variety having superior traits for mating, it is necessary to make a brood stock group by the selection of live individuals having targeted phenotypic characteristics. In the DNA marker-assisted selection to obtain such varieties, collecting DNA samples from each individual is necessary. These individuals should be tagged and kept alive during the genetic examination. However, tuna are very difficult to handle, even for the juveniles that are smaller and easier to handle than huge adult tuna (Ottolenghi, 2008). Handling stress using current technologies during genetic sampling (Figure 13.3) results in accumulation in plasma lactate, cortisol, and glucose (Addis et al., 2012), yet is currently necessary for a breed development. For example, PBFT juveniles have a severe mortality by handling stress in the transfer from land-based rearing tanks to sea cages for further grow out culture. Therefore, it is necessary to develop the technologies for tagging of small-sized juveniles of 20 30 cm TL and for collection of DNA samples without injuring many small-sized juveniles. Therefore, it will be fundamental to elucidate the details of stress response in their handling which is unclear at present.
13.4 PROTECTION OF INTELLECTUAL PROPERTY OF TUNA AQUACULTURE PRODUCTS AND THEIR BREEDING METHODS When new tuna breeds are established or breeding methods are newly developed, it is very important to protect the intellectual property of such breeds
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and breeding methods from the view point of the tuna industry. Securement of intellectual property rights over the improved breeds guarantees inventors’ rights of preferential utilization both domestically and abroad (Fisheries Research Agency, 2010). Genetics and molecular biology provide scientific basis to protect intellectual property, for example, by pursuing patents of new breeds and breeding methods by providing complete traceability of the products and live stocks of fishes. However, care should be exercised in the necessary condition to protect intellectual property, for example, requirement of patentability. Generally, the nucleotide sequences of DNA fragments which are not eligible for patent are those which do not have the identified function or suggestion of usefulness of the synthesized protein (Japan Patent Office, 1999). For newly developed breeds of tuna, development of legal systems such as the reinforcement of the Plant Variety Protection and Seed Act in Japan will be very useful to protect their intellectual property. In addition, product branding of new breeds by trademark registration will be an important strategy to protect their intellectual property. These efforts to protect intellectual property should be exercised not only for the producers’ benefit but also for that of consumers.
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Bremer, A.J.R., Naseli, I., Ely, B., 1999. Heterogeneity of northern bluefin tuna populations. ICCAT Coll. Vol. Sci. Pap. XLIX, 127 129. Bremer, A.J.R., Stequert, B., Robertson, N.W., Ely, B., 1998. Genetic evidence for inter-oceanic subdivision of bigeye tuna (Thunnus obesus) populations. Mar. Biol. 132, 547 557. Carlsson, J., Jan, R., McDowell, L., Diaz-Jaimes, P., Carlsson, J.E.L., Boles, S.B., et al., 2004. Microsatellite and mitochondrial DNA analyses of Atlantic bluefin tuna (Thunnus thynnus thynnus) population structure in the Mediterranean Sea. Mol. Ecol. 13, 3345 3356. Chiang, H.C., Hsu, C.C., Lin, H.D., Ma, G.C., Chiang, T.Y., Yang, H.Y., 2006. Population structure of bigeye tuna (Thunnus obesus) in the South China Sea, Philippine Sea and western Pacific Ocean inferred from mitochondrial DNA. Fish. Res. 79, 219 225. Chiang, H.C., Hsu, C.C., Wu, G.C.C., Chang, S.K., Yang, H.Y., 2008. Population structure of bigeye tuna (Thunnus obesus) in the Indian Ocean inferred from mitochondrial DNA. Fish. Res. 90, 305 312. Chow, S., Kishino, H., 1995. Phylogenetic relationships between tuna species of the genus Thunnus (Scombridae: Teleostei): inconsistent implications from morphology, nuclear and mitochondrial genomes. J. Mol. Evol. 41, 741 748. Chow, S., Okamoto, H., Miyabe, N., Hiramatsu, K., Barut, N., 2000. Genetic divergence between Atlantic and Indo-Pacific stocks of bigeye tuna (Thunnus obesus) and admixture around South Africa. Mol. Ecol. 9, 221 227. Chow, S., Okamoto, H., Uozumi, Y., Takeuchi, Y., Takeyama, H., 1997. Genetic stock structure of the swordfish (Xiphias gladius) inferred by PCR-RFLP analysis of the mitochondrial control region. Mar. Biol. 127, 359 367. Chow, S., Takeyama, H., 2000. Nuclear and mitochondrial DNA analyses reveal four genetically separated breeding units of the swordfish (Xiphias gladius). J. Fish. Biol. 56, 1087 1098. Chow, S., Ushiyama, H., 1995. Global population structure of albacore (Thunnus alalunga) inferred by RFLP analysis of the mitochondrial ATPase gene. Mar. Biol. 123, 39 45. Clark, T.B., Ma, L., Saillant, E., Gold, J.R., 2004. Microsatellite DNA markers for populationgenetic studies of Atlantic bluefin tuna (Thunnus thynnus thynnus) and other species of genus Thunnus. Mol. Ecol. Notes 4, 70 73. Durand, J.-D., Collet, A., Chow, S., Guinand, B., Borsa, P., 2005. Nuclear and mitochondrial DNA markers indicate unidirectional gene flow of Indo-Pacific to Atlantic bigeye tuna (Thunnus obesus) populations, and their admixture off southern Africa. Mar. Biol. 147, 313 322. Eknath, A.E., Tayamen, M.M., Palada-de Vera, M.S., Danting, J.C., Reyes, R.A., Dionisio, E.E., et al., 1993. Genetic improvement of farmed tilapias: the growth performance of eight strains of Oreochromis niloticus tested in different farm environments. Aquaculture 111, 171 188. Ely, B., Stoner, D.S., Bremer, A.J.R., Dean, J.M., Addis, P., Cau, A., et al., 2002. Analyses of nuclear IdhA gene and mtDNA control region sequences of Atlantic northern bluefin tuna populations. Mar. Biotechnol. 4, 583 588. Fisheries Research Agency, 2010. Strategy for genomic research in fisheries science. http:// www.fra.affrc.go.jp/pressrelease/pr21/220331/0331besshi1.pdf. Gjedrem, T., 1983. Genetic variation in quantitative traits and selective breeding in fish and shellfish. Aquaculture 33, 51 72. Gjedrem, T., 2005. Status and scope of aquaculture. In: Gjedrem (Ed.), Selection and Breeding Programs in Aquaculture. Springer, Dordrecht, pp. 1 8. Grewe, P.M., Appleyard, S.A., Ward, R.D., 2000. Determining genetic stock structure of bigeye tuna in the Indian Ocean using mitochondrial DNA and DNA microsatellites. Fish. Res. Dev. Corp. Rep. 97/122, CSIRO Marine Research, Hobart.
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Grewe, P.M., Hampton, J., 1998. An assessment of Bigeye (Thunnus obesus) Population Structure in the Pacific Ocean, Based on Mitochondrial DNA and DNA Microsatellite Analysis. CSIRO Marine Research, Hobart. Hussain, M.G., 1996. Advances in chromosome engineering research in fish: review of methods, achievements and applications. Asian Fish. Sci. 9, 45 61. Japan Patent Office, 1999. Comparative study on the patentability of DNA fragments in tripartite patent offices (in Japanese). http://www.jpo.go.jp/torikumi/kokusai/kokusai3/tizai3.htm. Kato, K., Hayashi, R., Yuasa, D., Yamamoto, S., Miyashita, S., Murata, O., et al., 2002. Production of cloned red sea bream, Pagrus major, by chromosome manipulation. Aquaculture 207, 19 27. Komen, J., Bongers, A.B.J., Richter, C.J.J., Van Muiswinkel, W.B., Huisman, E.A., 1991. Gynogenesis in common carp (Cyprinus carpio L.): II. The production of homozygous clones and F1 hybrids. Aquaculture 92, 127 142. Kurata, M., Seoka, M., Nakagawa, Y., Ishibashi, Y., Kumai, H., Sawada, Y., 2011. Promotion of initial swimbladder inflation in Pacific bluefin tuna, Thunnus orientalis (Temminck and Schlegel), larvae. Aquaculture Res. 43, 1 10. Masuma, S., Takebe, T., Sakakura, Y., 2010. A review of the broodstock management and larviculture of the Pacific northern bluefin tuna in Japan. Aquaculture 315, 2 8. McDowell JR, Diaz-Jaimes, P., Graves, J.E., 2002. Isolation and characterization of seven tetranucleotide microsatellite loci from Atlantic northern bluefin tuna Thunnus thynnus thynnus. Mol. Ecol. Notes 2, 214 216. Miyashita, S., 2002. Studies on the seedling production of the Pacific bluefin tuna, Thunnus thynnus orientalis. Bull. Fish. Lab. Kinki Univ. 8, 1 171. Morshima, K., Yamamoto, H., Sawada, Y., Miyashita, S., Kato, K., 2009. Developing 23 new polymorphic microsatellite markers and simulating parentage assignment in the Pacific bluefin tuna, Thunnus orientalis. Mol. Ecol. Resour. 9, 790 792. Mu¨ller-Belecke, A., Ho¨rstgen-Schwark, G., 1995. Sex determination in tilapia (Oreochromis niloticus): sex ratios in homozygous gynogenetic progeny and their offspring. Aquaculture 137, 57 65. Mu¨ller-Belecke, A., Ho¨rstgen-Schwark, G., 2000. Performance testing of homozygous lines in Oreochromis niloticus. Aquaculture 184, 67 76. Munday, B.L., Sawada, Y., Cribb, T., Hayward, C.J., 2003. Diseases of tunas, Thunnus spp. J. Fish. Dis. 26, 187 206. Nakadate, M., 2005. Genetic isolation between Atlantic and Mediterranean albacore populations inferred from mitochondrial and nuclear DNA markers. J. Fish. Biol. 66, 1545 1557. Nakadate, M., Chow, S., 2008. Isolation and characterization of single copy nuclear DNA markers in Atlantic bluefin tuna Thunnus thynnus. Fish. Sci. 74, 1333 1335. Niwa, Y., Nakazawa, A., Margulies, D., Scholey, V.P., Wexler, J.B., Chow, S., 2003. Genetic monitoring for spawning ecology of captive yellowfin tuna (Thunnus albacares) using mitochondrial DNA variation. Aquaculture 218, 387 395. Okada, H., 1985. Studies on the artificial sex control in rainbow trout, Salmo gairdneri. Scientific Report of the Hokkaido Fish Hatchery 40, 1 49. (In Japanese with English summary.) Hokkaido National Fisheries Research Institute, Japan. Ottolenghi, F., 2008. Capture-based aquaculture of bluefin tuna. In: Lovatelli, A., Holthus P.F. (Eds.), Capture-based aquaculture. Global overview. FAO Fisheries Technical Paper. No. 508. Rome, FAO. pp. 169 182. Pandian, T.J., Koteeswaran, R., 1998. Ploidy induction and sex control in fish. Hydrobiologia 384, 167 243.
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Saito, K., 2010. A large-scale sequencing project of Pacific tuna genome (in Japanese). Nippon Suisan Gakkaishi 76, 1132 1134. Sawada, Y., Miyashita, S., Murata, O., Kumai, H., 2004. Seedling production and generation succession of the Pacific bluefin tuna, Thunnus orientalis. Mar. Biotechnol. 6, S327 S331. Sawada, Y., Okada, T., Miyashita, S., Murata, O., Kumai, H., 2005. Completion of the Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel) life cycle. Aquaculture Res. 36, 413 421. Sawada, Y., Seoka, M., Kato, K., Tamura, T., Nakatani, M., Hayashi, S., et al., 2008. Testes maturation of reared Pacific bluefin tuna, Thunnus orientalis Temminck and Schlegel, at two plus years old. Fish. Sci. 73, 1070 1077. Scoles, D.R., Graves, J.E., 1993. Population genetic structure of yellowfin tuna, Thunnus albacares, from the Pacific Ocean. Fish. Bull. 91, 690 698. Shoda, K., 2010. History of animal breeding and progress of genetics (in Japanese). In: Shoda, Y. (Ed.), World History of Breeding. Yushukan, Tokyo, pp. 1 22. Tagami, M., Arakawa, T., Sano, H., Fuji, K., Hasegawa, O., Sakamoto, T., et al., 2008. Ultrahigh throughput microsatellite marker development for linkage map construction by the next generation DNA sequencers. 5th World Fisheries Congress Program & Abstracts, 5th World Fisheries Congress Committee, Yokohama, Japan, pp. 373. Takagi, M., Okamura, T., Chow, S., Taniguchi, N., 2001. Preliminary study on genetic stock structure of albacore (Thunnus alalunga) inferred from microsatellite DNA analysis. Fish. Bull. 99, 697 701. Takagi, M., Okmura, T., Chow, S., Taniguchi, N., 1999. PCR primers for microsatellite loci in tuna species of the genus Thunnus and its application for population genetics study. Fish. Sci. 65, 571 576. Takeyama, H., Chow, S., Tsuzuki, H., Matsunaga, T., 2001. Mitochondrial DNA sequence variation within and between Thunnus tuna species and its application to species identification. J. Fish. Biol. 58, 1646 1657. Vin˜as, J., Pla, C., Tawil, M.Y., Hattour, A., Farrugia, A.F., de la Serna, J.M., 2003. Mitochondrial genetic characterization of Bluefin tuna (Thunnus thynnus) from three Mediterranean (Libya, Malta, Tunisia); and one Atlantic locations (Gulf of Cadiz). Col. Vol. Sci. Pap. ICCAT 55 (3), 1282 1288. Vin˜as, J., Santiago, J., Pla, C., 1999. Genetic characterization and Atlantic Mediterranean stock structure of albacore, Thunnus alalunga. ICCAT Coll. Vol. Sci. Pap. 49, 188 191. Ward, R.D., Elliott, N.G., Innes, B.H., Smolenski, A.J., Grewe, P.M., 1997. Global population structure of yellowfin tuna, Thunnus albacares, inferred from allozyme and mitochondrial DNA variation. Fish. Bull. 95, 566 575. Wu, G.C.-C., Chianga, H.-C., Choua, Y.-W., Wong, Z.-R., Hsuc, C.-C., Chend, C.-Y., et al., 2010. Phylogeography of yellowfin tuna (Thunnus albacares) in the Western Pacific and the Western Indian Oceans inferred from mitochondrial DNA. Fish. Res. 105, 248 253. Yagishita, N., Oohara, I., Kobayashi, T., 2006. Construction of a BAC Library for the Pacific Bluefin Tuna Thunnus orientalis. Ecology and Aquaculture of Bluefin Tuna 21st century COE program, Kinki University joint symposium on Bluefin Tuna, Kinki University, Japan, pp. 85 88. Yamamoto, S., 1999. Studies on sex-manipulation and production of cloned populations in hirame, Paralichthys olivaceus (Temminck et Schlegel). Aquaculture 173, 235 246.
Genetics in Tuna Aquaculture Yoshifumi Sawada and Yasuo Agawa Fisheries Laboratories, Kindai University, Kushimoto, Wakayama, Japan
13.1 INTRODUCTION Human beings have been breeding animals for more than 10,000 years (Shoda, 2010). During this period, new breeds and strains have been developed with desirable phenotypic traits such as rapid growth, higher tolerance to environmental conditions, enhanced disease resistance and improved meat quality. In aquaculture, selective breeding has a much shorter history but has been conducted for several species including Atlantic salmon (Salmo salar: Gjedrem, 1983), rainbow trout (Oncorhynchus mykiss: Donaldson and Olson, 1957), tilapia (Oreochromis niloticus: Eknath et al., 1993), and carp (Cyprinus carpio: Babouchkine, 1987; Gjedrem, 2005). Selective breeding programs for aquatic species provide better outcomes compared to terrestrial livestock. This higher response to selection of farmed aquatic species can be attributed to their high fecundity enabling higher selection intensity. In addition, large phenotypic and genetic variations exist in the genetically underdeveloped fish traits. However, in the tuna aquaculture industry, selection programs are not commonly used and production still relies heavily on catching wild fingerlings and broodstock. The completion of the life cycle of Pacific bluefin tuna (PBFT) has provided the potential to develop selective breeding programs for this species and this is now underway in Japan (Sawada et al., 2004). In this process, biotechnology will play an important role in the area of tuna reproduction, breeding, and harvested product quality management.
13.2 TARGET TRAITS IN TUNA BREED IMPROVEMENT In any tuna breeding program the following biological traits are important targets for selection, fast growth rate, high survival rate during early development, high disease resistance, superior traits in reproduction, and high meat quality. D.D. Benetti, G.J. Partridge, A. Buentello (Eds): Advances in Tuna Aquaculture. DOI: http://dx.doi.org/10.1016/B978-0-12-411459-3.00013-8 © 2016 Elsevier Inc. All rights reserved.
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FIGURE 13.1 Diagram of tuna individual selection for breed improvement. The procedure of individuals having superior genetic traits encompasses capture, DNA material sampling and analysis, and tagging where multiple times of juvenile handling is inevitable.
Although tuna have extremely high growth rate compared to other fishes (Miyashita, 2002), it is desirable to further increase the speed of growth and development by selective breeding in order to improve production efficiency. Developing breeds of tuna that are selectively optimized for commercial aquaculture is a dynamic and intensive process. Steps involve broodstock capture and maturation concurrent with genetic sampling and tagging. Records are kept to correlate larval success, growout metrics, and other significant biological traits to breeding individuals, and subsequent resampling for future brood fish. Figure 13.1 depicts an outline of the steps involved in the continuous improvement of tuna breeds. The technology for tuna fingerling production is still underdeveloped and the survival rate during the hatchery phase is 10% at most. Improvements to survival rate will be attained by the improvement of larval and juvenile rearing techniques but selection of breeders whose offspring show high survival during early development can also be a goal of a selective breeding program. In the larval rearing of tuna, low survival rate due to an unsuitable rearing environment, such as water surface condition and current in the rearing water is the largest obstacle for their mass production (Miyashita, 2002; Sawada et al., 2005; Kurata et al., 2011). Therefore, it is important to create strains which have tolerance to environmental stress. Domestication and selection of families with high survival will be attained naturally in the early stage of
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tuna aquaculture technology development, as poor larval survival inevitably leads to selection against such breeds. However, as a more positive step, selection of domesticated and high survival breeds for specific environments should be implemented in tuna aquaculture. PBFT do not tend to suffer from serious diseases from 1 year after hatching. However, PBFT juveniles experience viral, bacterial, and parasitic diseases that lead to high mortality (Munday et al., 2003), and development of disease-resistant strains is also important. Under aquaculture conditions, female adult tuna with high reproductive activity are scarce (Masuma et al., 2010) compared to males, the majority of which reach maturity at a younger age than females (Sawada et al., 2008). Therefore, prolific breeders are required for stable reproduction especially for female tuna in tuna aquaculture. One of the most important criteria in evaluating quality of the aquacultureproduced tuna is the muscle fat content. Fatty portions such as the belly flaps called “toro” are preferable as raw materials for the preparation of sushi and sashimi, also deriving a higher market value than other muscle sections. However, demand also exists for leaner meat called “akami”. Selective breeding programs can be directed at controlling muscle fat contents is in PBFT muscle.
13.3 GENETIC TECHNOLOGIES IN TUNA BREEDING 13.3.1 Genomic DNA Analysis Genomic information is applicable to the improvement of tuna aquaculture by using DNA marker-assisted selection. For this aim, it is necessary to establish a series of DNA microsatellite markers. Although tuna DNA microsatellite markers have been developed by various scientists (Takagi et al., 1999; McDowell et al., 2002; Nakadate and Chow 2008; Tagami et al., 2008; Morshima et al., 2009; Agawa et al., 2009; Clark et al., 2004; Takagi et al., 2001), the number of microsatellites is still insufficient to identify the desired traits or to make a high-density genetic map of tuna in the breeding program. In the analysis of genes, such as base sequence determination, it is inefficient and difficult to use genomic DNA. As a substitute, genome libraries, which are composed of cloning vectors (inserted fragments of targeted genome DNA), are ordinarily utilized. To make a genome library, genome DNA is fractionated by the use of restricted enzymes, sonication or shearing, then the fragments are inserted into genetically engineered vectors, such as plasmids, bacteriophages (such as phage λ), bacterial artificial chromosomes (BACs), or yeast artificial chromosomes (YACs). For PBFT genome, a BAC library has been established (Yagishita et al., 2006). Identification of sex-specific DNA markers of PBFT is a good example of genomic DNA analysis (Agawa et al., 2011). Under aquaculture conditions,
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FIGURE 13.2 The sex-specific DNA PCR products were confirmed by gel electrophoresis. M; molecular size markers. The sizes (base pairs) were indicated left of the fragments. Two females and two males PBFTs were used. n.; negative control means PCR were conducted without template DNA. Md6; male delta 6 locus, male positive PCR (Agawa et al., 2015). β-actin; beta actin gene PCRs were used for positive control.
it has been reported that adult females have a low percentage of participation in reproduction (Masuma et al., 2010), and to make a broodstock group with a high percentage of females is necessary for the stable reproduction and for securement of appropriate genetic diversity in the laboratory hatched and raised PBFT strains. Because adult PBFT are large in size, vulnerable, and difficult to handle, sex determination should be conducted during early developmental stages of juveniles. Therefore, for PBFT juveniles, whose sexes cannot be distinguished phenotypically, sex identification by genetics using small biotic samples such as fin tips is very useful. These biotic samples can then be screened using AFLP-selective DNA amplification including primer-specific secondary amplifications for increased fidelity and sequenced through high-throughput next-generation sequencing technologies (Agawa et al., 2015). Combined with common electrophoresis methodologies, male characteristic DNA fragments (Figure 13.2) can be observed and are attributable to specific breeding fish. As a more comprehensive result for PBFT, draft analysis of the genomic DNA was performed by the Fisheries Research Agency (FRA) cooperatively with the University of Tokyo and Kyusyu University (Saito, 2010; Fisheries Research Agency, 2010). In this project, they reportedly found approximately 86,000 microsatellite DNA markers. These results of genomic analysis will contribute largely to the PBFT breeding programs.
13.3.2 Analysis of Mitochondrial DNA Polymorphism The mitochondrial DNA D-loop region in tuna of the genus Thunnus is highly polymorphic as in other fishes (Bremer et al., 1998; Bremer et al., 1999; Chow et al., 2000), and this variation can be detected using conventional PCR-RFLP analysis in several tuna and billfish species (Chow et al., 1997; Chow et al., 2000; Chow and Takeyama, 2000; Niwa et al., 2003). The analysis of mitochondrial DNA polymorphism has been well used as the tool to identify tuna species (Chow and Kishino, 1995; Takeyama et al., 2001).
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This kind of genetic tool to identify tuna species is useful to prevent mislabeling of tuna fishery and aquaculture products. To sustain consumers’ confidence, such effort should be encouraged. Mitochondrial DNA polymorphism has also been used to analyze the genetic structure of natural populations of yellowfin tuna (YFT) (Scoles and Graves, 1993; Ward et al., 1997; Wu et al., 2010), bigeye (Chow et al., 2000; Grewe and Hampton, 1998; Grewe et al., 2000; Appleyard et al., 2002; Durand et al., 2005; Chiang et al., 2006; Chiang et al., 2008), albacore (Chow and Ushiyama, 1995; Vin˜as et al., 1999), and Atlantic bluefin tuna (ABFT) (Ely et al., 2002.; Vin˜as et al., 2003; Boustany et al., 2008; Carlsson et al., 2004). To raise the precision of these analyses, genome DNA microsatellite markers are often combined (Nakadate, 2005). Such combination can detect genetic foreignness among pedigrees and even individuals with high sensitivity. Therefore, this combined analysis of mitochondrial and genome DNA polymorphism can be used in genetic monitoring for spawning ecology of captive tuna (Niwa et al., 2003), and it has the possibility to elucidate how individual variation among broodstock, degree of maturity, and nutritional state influence the egg quality and larval vitality. In addition, it can be a very powerful tool to register newly developed tuna strains in aquaculture and to identify artificially hatched and released stocks to the open sea for natural population enhancement in the near future.
13.3.3 Other Genetic Technologies Chromosome manipulation in cultured fishes is effective to improve their performance. Inviable aneuploids such as triploids have been induced to improve growth (Arai et al., 1991; Hussain, 1996; Pandian and Koteeswaran, 1998; Arai 2001). Cloned fishes have been also produced in rainbow trout Oncorhynchus mykiss (Okada, 1985), common carp Cyprinus carpio (Komen et al., 1991), tilapia Oreochromis niloticus (Mu¨ller-Belecke and Ho¨rstgenSchwark, 1995; Mu¨ller-Belecke and Ho¨rstgen-Schwark, 2000), olive flounder “hirame” Paralichthys olivaceus (Yamamoto, 1999), and red sea bream Pagrus major (Kato et al., 2002). Cloned strains have a possibility of being advantageous in the mass production by their homogeneity in their superior phenotypic traits such as the digestion ability of nutrients, sensitivity to medicinal agents, and environmental stress resistance. The useful characteristic of these technologies is to create strains without breeding potential. It will contribute to more efficient mass production of cultured species. At present, there is no example of application of these technologies in tuna aquaculture industry. Genetic modifications should not be used by the tuna aquaculture industry until rigorous examination in terms of food safety is completed. In addition, genetically-modified tuna escapees could potentially interfere with the genetic material of wild tuna populations should they breach their enclosures in net pens. Although containment of other fin fish species is easily attained
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FIGURE 13.3 Sampling of DNA material and tagging of a PBFT juvenile. Tuna juveniles have low handling tolerance.
in land-based culture facilities, tuna species require significant space/water volume such as the one provided by massive net pens. Today, it is impractical to practice commercial tuna grow out in land-based facilities. Finally, in practical tuna breed improvement, it is necessary to develop a new technology for the treatment of live individuals. To create a new variety having superior traits for mating, it is necessary to make a brood stock group by the selection of live individuals having targeted phenotypic characteristics. In the DNA marker-assisted selection to obtain such varieties, collecting DNA samples from each individual is necessary. These individuals should be tagged and kept alive during the genetic examination. However, tuna are very difficult to handle, even for the juveniles that are smaller and easier to handle than huge adult tuna (Ottolenghi, 2008). Handling stress using current technologies during genetic sampling (Figure 13.3) results in accumulation in plasma lactate, cortisol, and glucose (Addis et al., 2012), yet is currently necessary for a breed development. For example, PBFT juveniles have a severe mortality by handling stress in the transfer from land-based rearing tanks to sea cages for further grow out culture. Therefore, it is necessary to develop the technologies for tagging of small-sized juveniles of 20 30 cm TL and for collection of DNA samples without injuring many small-sized juveniles. Therefore, it will be fundamental to elucidate the details of stress response in their handling which is unclear at present.
13.4 PROTECTION OF INTELLECTUAL PROPERTY OF TUNA AQUACULTURE PRODUCTS AND THEIR BREEDING METHODS When new tuna breeds are established or breeding methods are newly developed, it is very important to protect the intellectual property of such breeds
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and breeding methods from the view point of the tuna industry. Securement of intellectual property rights over the improved breeds guarantees inventors’ rights of preferential utilization both domestically and abroad (Fisheries Research Agency, 2010). Genetics and molecular biology provide scientific basis to protect intellectual property, for example, by pursuing patents of new breeds and breeding methods by providing complete traceability of the products and live stocks of fishes. However, care should be exercised in the necessary condition to protect intellectual property, for example, requirement of patentability. Generally, the nucleotide sequences of DNA fragments which are not eligible for patent are those which do not have the identified function or suggestion of usefulness of the synthesized protein (Japan Patent Office, 1999). For newly developed breeds of tuna, development of legal systems such as the reinforcement of the Plant Variety Protection and Seed Act in Japan will be very useful to protect their intellectual property. In addition, product branding of new breeds by trademark registration will be an important strategy to protect their intellectual property. These efforts to protect intellectual property should be exercised not only for the producers’ benefit but also for that of consumers.
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