United States-Japan
Natural Resources Panel on Aquaculture (UJNR)
43rd Scientific Symposium“Evaluation of the impact of breeding organisms on the ecosystem
and aquaculture industry”
November 10-11, 2015
Nagasaki

Program and Abstracts

Program
The 43rd Scientific Symposium of UJNR Aquaculture Panel

Evaluation of the impact of breeding organisms on the ecosystem andaquaculture industry
Date:
November 10

15:00-17:30

Poster Session

November 11

9:30-17:30

Oral Session, Poster Session during lunch break

Venue:
Lecture room, 2F General education and research building, Bunkyo Campus, Nagasaki
University

Aim of the Symposium
Breeding research will be of great help with the improvement of aquaculture technology. The
utilization of wild broodstock/seeds does not result in genetically improved stocks, and theimportance of genetics and breeding studies has increased to meet various needs.

Althoughgenetic improvement is common in agriculture and the resulting benefits unquestioned, there arefew se1ective1y bred commercially available strains in the aquaculture industry except in inlandaquaculture.
Moreover, we should pay attention to the ecological impacts of breeding technology. At the sametime selection may increase risks when selected animals escape and breed with wild stocks unlessproper safeguards are put into place. Integration of scientific knowledge of the benefits and risks
can help contribute to genetic improvement programs. Our UJNR activity should deepen
discussions on the issue of genetics/breeding studies, which is expected to contribute to thedevelopment of the aquaculture industry in both countries of Japan and United States.
This Symposium consists of oral session and poster session. In oral session, we will deal with thesubjects relating to ‘genetics and breeding’ such as: the impact of breeding organisms on theecosystem and aquaculture industry; selective breeding techniques; bioinformatics, etc. In poster
session, we will present and discuss broader topics relating to Aquaculture.

Tuesday, November 10th, 2015
Registration

14:00-17:30

Poster Session
Poster presentation

15:00-17:30

Wednesday, November 11th, 2015
Registration

9:00-12:00

Oral Session
Opening Session.
(Moderators: J. Higano & M. Rust)
Welcome to Nagasaki University
Atsushi Hagiwara (Dean of the Graduate School of Fisheries and Environmental Sciences,
Nagasaki University)
Aim of the Symposium
Fuminari Ito (Japan Chair, Fisheries Research Agency)

9:30- 9:35
9:35- 9:50

Session I. Breeding technique(Moderators: S.Watanabe & A. Fuller)
1. Appropriate conditions for the production of triploidy induced by cold shock in yellowtail
Seriola quinqueradiata
Yukinori Shimada (National Research Institute of Aquaculture, FRA)

9:50-10:15

2. Potential Application of Germplasm Preservation in Breeding Programs for Molluscan
Shellfish Aquaculture and Restoration
Huiping Yang (University of Florida)

10:15-10:35

3. Improving Aquaculture Production in Haliotis Species Through the Development of a
Genomic Toolkit
Catherine Purcell (NOAA Fisheries)

10:35-10:55

4. Culture Protocols and Production of Triploid Purple-Hinge Rock Scallops
Paul Olin (California SeaGrant)

10:55-11:15

Session II. Genetic improvement 1(Moderators: A. Ozaki & B. Bosworth)
5. Big Data in Agriculture and the USDA/ARS Initiative
Jeffrey Silverstein (USDA Agricultural Research Service)

11:15-11:35

6. Signature of artificial selection in a breed of coho salmon Oncorhynchus kisutch
Sho Hosoya (Fisheries Laboratory, University of Tokyo)

11:35-12:05

Group Photo

12:05-12:10

Lunch Break

12:10-13:10

7. Genetic Selection in Animals Using Pedigree, Phenotypic, and Genomic Information
Shogo Tsuruta (University of Georgia)

13:10-13:40

8. Exploring Transcriptomic Patterns in Slow- and Fast-Growing Seriola dorsalis Larvae
Catherine Purcell (NOAA Fisheries)

13:40-14:00

Session III. Risk evaluation of escaped fish
(Moderators: K. Ikuta & B. Iwamoto)
9. Modeling the Variable Effects of Using Wild and Cultured Broodstock on the Fitness Risk Due
to Escaped Farmed Fish
Kristen Gruenthal (NOAA Fisheries)

14:00-14:20

10. Did farmed Coho salmon Oncorhynchus kisutch that escaped during the earthquake andtsunami disaster of 2011 interbreed with native Masu salmon Oncorhynchus masou?
Kei Sasaki (Tohoku National Fisheries Research Institute, FRA)

14:20-14:45

11. Evaluation of the tsunami impact on the genetic diversity of the marbled flounder
Pseudopleuronectes yokohamae in Sendai Bay, Miyagi, Japan

14:45-15:10

Yuki Minegishi (Tohoku Ecosystem-Associated Marine Sciences, Tohoku University)
12. Competition between Atlantic salmon (Salmo salar) and Japan’s native salmonids.
Kazuo Araki (National Research Institute of Aquaculture, FRA)
Break

15:10-15:35
15:35-15:50

Session IV. Genetic improvement 2(Moderators: M. Ototake & H. Yang)
13. Hybrid Striped Bass National Breeding Program: Research Towards Genetic Improvement ofa Non-Model Species
Adam Fuller (USDA Agricultural Research Service)

15:50-16:10

14. Production of Benedenia-resistant Yellowtail (Seriola quinqueradiata) Families –A
Preliminary Approach to the Candidates–
Tsutomu Noda (Seikai National Fisheries Research Institute, FRA)
15. Coho Salmon Broodstock Development:

16:10-16:35

A Case Study of the Domsea Coho (1977 to 2015)

Bob Iwamoto (Spring Salmon LP)

16:35-16:55

16. Development of Improved Catfish Germplasm at the Warmwater Aquaculture Research Unit,
USDA-ARS
Brian Bosworth (USDA Agricultural Research Service)
Discussion

16:55-17:15
17:20-17:50(Moderators: F. Ito & M. Rust)
Closing remarks (Michael B. Rust, U.S.A. Panel Chair)

17:50-18:00

Abstracts with Annotated Bibliography of the 43rd
Scientific Symposium of UJNR Aquaculture PanelTitles of Oral Presentation
1. Appropriate conditions for the production of triploidy induced by cold shock in yellowtail Seriola
quinqueradiata (Yukinori Shimada, National Research Institute of Aquaculture, FRA)
2. Potential Application of Germplasm Preservation in Breeding Programs for Molluscan Shellfish
Aquaculture and Restoration (Huiping Yang, University of Florida)
3. Improving Aquaculture Production in Haliotis Species Through the Development of a Genomic
Toolkit (Catherine Purcell, NOAA Fisheries)
4. Culture Protocols and Production of Triploid Purple-Hinge Rock Scallops (Paul Olin, California
SeaGrant)
5. Big Data in Agriculture and the USDA/ARS Initiative (Jeffrey Silverstein, USDA Agricultural
Research Service)
6. Signature of artificial selection in a breed of coho salmon Oncorhynchus kisutch (Sho Hosoya,
Fisheries Laboratory, University of Tokyo)
7. Genetic Selection in Animals Using Pedigree, Phenotypic, and Genomic Information (Shogo
Tsuruta, University of Georgia)
8. Exploring Transcriptomic Patterns in Slow- and Fast-Growing Seriola dorsalis Larvae (Catherine
Purcell, NOAA Fisheries)
9. Modeling the Variable Effects of Using Wild and Cultured Broodstock on the Fitness Risk Due
to Escaped Farmed Fish (Kristen Gruenthal, NOAA Fisheries)
10. Did farmed Coho salmon Oncorhynchus kisutch that escaped during the earthquake and tsunami
disaster of 2011 interbreed with native Masu salmon Oncorhynchus masou? (Kei Sasaki,
Tohoku National Fisheries Research Institute, FRA)
11. Evaluation of the tsunami impact on the genetic diversity of the marbled flounder
Pseudopleuronectes yokohamae in Sendai Bay, Miyagi, Japan (Yuki Minegishi, Tohoku
Ecosystem-Associated Marine Sciences, Tohoku University)
12. Competition between Atlantic salmon (Salmo salar) and Japan’s native salmonids (Kazuo Araki,
National Research Institute of Aquaculture, FRA)
13. Hybrid Striped Bass National Breeding Program: Research Towards Genetic Improvement of a
Non-Model Species (Adam Fuller, USDA Agricultural Research Service)
14. Production of Benedenia-resistant Yellowtail (Seriola quinqueradiata) Families –A Preliminary
Approach to the Candidates– (Tsutomu Noda, Seikai National Fisheries Research Institute,
FRA)
15. Coho Salmon Broodstock Development: A Case Study of the Domsea Coho (1977 to 2015)(Bob Iwamoto, Spring Salmon LP)
16. Development of Improved Catfish Germplasm at the Warmwater Aquaculture Research Unit,
USDA-ARS (Brian Bosworth, USDA Agricultural Research Service)1

O-1
Appropriate conditions for the production of triploidy induced by cold shockin yellowtail Seriola quinqueradiata
Yukinori Shimada*1, Hiroyuki Nagoya1, Hiroyuki Okamoto1, Toshiya Yamaguchi1, Nariaki
Inoue1, Takachi Ishikawa1, Kazuhisa Hamada2, Kazuharu Nomura3,
Hironori Usuki1, Kazunori Yoshida41

National Research Institute of Aquaculture, FRA, Tsuiura, Kamiura, Saiki, Oita 879-2602, Japan,
National Research Institute of Aquaculture, FRA, Komame 330, Ootsuki, Kochi 788-0315,
3
National Research Institute of Aquaculture, FRA, Nakatsuhamaura 422-1, Minamiise, Mie 5160193, 4Seikai National Fisheries Research Institute, FRA, Nunoura 122-7, Tamanoura, Goto,
Nagasaki 853-0508
2*E-mail: yukinoris at affrc.go.jp
In yellowtail Seriola quinqueradiata, FRA aims to contribute to the aquaculture industry throughpromoting of the artificial seedling production of the strain having economic traits (e.g. resistanceto disease and rapid growth). However, in terms of preventing genetic contamination and breeding
strategy, the development of reliable infertility method is desired. We focused on the induction of
triploidy because triploidy is expected being infertile so far. In this study, we estimated a releasingtime of the second polar body, and then examined the appropriate condition induced triploidy bycold shock. A releasing time of the second polar body in fertilized eggs reared at 20°C wasinvestigated by histological sections using samples of fertilized eggs, fixed by Bouin solution at 5,
10 and 15 min after fertilization. The second polar body was observed to be released during 5–10
min after fertilization. Two factors (i.e. water temperature and duration of the cold shock) were
examined to maximize the triploidy rate. Ploidy was determined by DNA contents using flow
cytometry. The triploid rate was the highest (100%) in 10 min at 3°C. The hatching rate of thiscondition was 0.2–34.9% when the hatching rate of control (i.e. non-treatment fertilized eggs) was
13.3–45.2%. In the future, we verify the usefulness as an infertility method by investigating sex
ratio, fertility and growth performance of triploidy.
Keywords: yellowtail, Seriola quinqueradiata, triploid, cold shock

Annotated Bibliography of Key Works
W. R. Wolters, G. S. Libey, and C. Larry Chrisman (1982). Effect of Triploidy on Growth and
Gonad Development of Channel Catfish. Transactions of the American Fisheries Society 111:
102-105.
Diploid and triploid in channel catfish Ictalurus punctatus were reared in indoor tanks. Triploids
were significantly heavier than diploids at 8 months of age and older. Triploid female and male
had smaller gonads with altered histology. Triploids converted feed more efficiently, and may
provide greater profits in commercial culture than diploids.
2

S. K. Allen, Jr. and J. G. Stanley (1978). Reproductive Sterility in Polyploid Brook Trout,
Salvelinus fontinalis. Transactions of the American Fisheries Society 107 (3): 473-478.
Red blood cell size were measured in brook trout (Salvelinus fontinalis) from Phillips Hatchery in
Maine to investigate naturally occurring polyploid sterility. In eight brook trout in which gonads
were lacking or undeveloped, the red blood cells were large, suggesting polyploidy. The average
size of the red blood cells in other sterile fish fit into the normal range but all of the eight fish
appeared to have some red blood cells that were polyploid. All polyploids appeared to be mosaics,
containing diploid, triploid, or pentaploid cells. The cause of the polyploidy was not determinedbut may have been caused by the inadvertent exposure of the eggs to low temperatures after
fertilization.
T. J. Benfey (1999). The Physiology and Behavior of Triploid Fishes. Reviews in Fisheries
Science 7(1): 39-67.
Induced triploidy is widely accepted as the most effective method for producing sterile fish foraquaculture and fisheries management. Artificially produced triploids generally differ fromconspecific diploids in three fundamental ways: they are more heterozygous, they have larger but
fewer cells in most tissues and organs, and their gonadal development is disrupted to some extent.
Despite these basic biological differences, triploids are similar in most respects to diploids when
examined at the whole animal level. The only clear differences relate to the effects of impaired
gametogenesis on the reproductive physiology and behavior of triploids, especially in females.
Other apparent differences include reduced aggressiveness, occasional specific morphologicalabnormalities, and inferior performance when reared under suboptimal conditions. The causes ofthese latter two problems are poorly understood but must be addressed if triploids are to be used
more extensively.

3

O-2
Potential Application of Germplasm Preservation in Breeding Programs for
Molluscan Shellfish Aquaculture and Restoration
Huiping Yang
School of Forest Resources and Conservation, Institute of Food and Agricultural Sciences,
University of Florida, 7922 NW 71st Street, Gainesville, FL 32653,*E-mail: huipingyang.at.ufl.edu
Germplasm are the genetic materials of germ cells including gametes, embryos, or larvae.
Preservation of germplasm is usually achieved through cryopreservation. The technology ofcryopreservation has been applied for human artificial reproduction as a clinical treatment for
infertility, and for livestock as a tool for breeding programs worldwide. For fish and shellfish,
cryopreservation has been studied in more than 200 species for preservation of natural resources
and conservation of endangered species. For molluscan shellfish aquaculture, this technology canhave the following potential applications: 1) Preservation of specific lines or strains. Ongoingbreeding programs have yielded specific strains and lines, such as disease resistant oysters.
Cryopreservation can be used to preserve these valuable strains and provide gametes for assistanceof breeding programs. 2) Preservation of natural wild populations. The cryopreserved germplasm
of natural populations will act as a repository of genetic diversity and allow for the continued
adaptive genetic variation for aquaculture populations through infusion of new material from wild
populations. In addition, a germplasm repository of wild populations can provide easy access as
study materials for researchers. 3) Creation of self-fertilization inbred lines. Inbred lines are one of
the most valuable resources for breeding programs, but difficult to produce and require years of
repeated crossing of brothers and sisters or backcrossing. Most bivalves are protandrous, beginning
life as males and changing into females as they age. Therefore, with the techniques of non-lethal
sperm collection and cryopreservation, self-fertilized lines can be created by using cryopreserved
sperm and oocytes from the same individual after sex reversal; 4) Preservation of sperm fromtetraploid oysters. Triploid-tetraploid technology is probably the most promising in oyster
aquaculture because of the superior traits of triploids. Cryopreservation of sperm from tetraploids
can extend the commercialization of triploid-tetraploid technology by the sale of frozen sperm, andprovide cost savings and security for maintaining tetraploids. In addition, cryopreservation oftetraploids produced each year can offer benefit for maintenance of the tetraploid populations. 5)
Assistance for creation of mutant lines. Mutant breeding is an effective approach for creation of
new strains or lines, but seldom used in animal breeding programs because of its low efficiency.
Recently, a new approach called TILLING (Targeting Induced Local Lesions in Genomes) was
developed in plants for creation of mutant lines. To use this new technique for animal mutant
breeding, sperm cryopreservation is an absolutely required technique. So far this approach has been
applied to zebrafish and puffer fish. With the genome sequencing accomplished in oysters,
establishment of mutant lines by TILLING and sperm cryopreservation will benefit the aquaculture
and research on oyster functional genetics, and 6) Assistance of aquaculture hatchery practice forregular and hybrid seed production. The cryopreserved germplasm materials can function as a4reservoir to meet the need for regular and hybrid seed production practice. For example, the hardclam hybrid offspring of Mercenaria mercenaria with Mercenaria campechiensis showed fast
growth and higher survival.

Annotated Bibliography of Key Works
Polge, C., A. U. Smith, A. S. Parkes. 1949. Revival of spermatozoa after vitrification anddehydration at low temperatures. Vol 164:4172. Pp. 666.
This is the first paper to report the successful cryopreservation and cold-dry of sperm from fowl
by using glycerol as cryoprotectant. The finding in this one-page short report opened the door forhuman sperm cryopresrevation which is now a major clinical treatment for infertility, and bull
sperm cryopreservation which is now a huge industry for its breeding programs worldwide. For
fish, the first report was published after this report in 1953 in herring (Blaxter, J. H. S. 1953.
Sperm storage and cross-fertilization of spring and autumn spawning herring. Nature 172, 1189 –
1190), and this preliminary communication showed that it was possible to cross-fertilize the two
spawning ‘types’ of herring Clupea harengus found in the north-east Atlantic by using the
cryopreserved sperm.
Yang H., Y. Wang, X. Guo, T. R. Tiersch. 2015. Production of inbred larvae through selffertilization using oocytes and cryopreserved sperm from the same individuals after sex reversal ineastern oyster Crassostrea virginica. Aquaculture Research, Vol 46:9. Pp. 2153-2165.
The authors in this study reported the production of self-fertilization larvae in eastern oyster
Crassostrea virginica for the first time, and demonstrated the feasibility of creating self-fertilized
inbred lines by use of non-lethal sperm collection and cryopreservation. In this study, small (~1year old) and large (~2–3 years old) oysters were biopsied for sperm collection. Survival of thebiopsied oysters after 1 year was 50% for small oysters and 17% for large oysters, and sex reversal
was observed bidirectional (from female to male and also from male to female). Oocytes werecollected from sex-reversed females, and self-fertilized with cryopreserved sperm. Of the 24
cryopreserved samples, 14 individuals had ≤1% fertility when crossed with oocytes fromunrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10
individuals had a fertility of 39 ± 25% when crossed with oocytes from unrelated females (nonselfing), but showed a significantly lower success of self-fertilization (12 ± 16%) (P = 0.008),
while aliquots of the same oocytes had a fertilization of 83 ± 11% when crossing with fresh sperm.
Larvae were produced in the self-fertilized families (12–94% of the fertilized oocytes), andsurvived to eyed-larvae stage at days 11–14. Genotyping with 9 microsatellite markers confirmedthat the larvae resulted from self-fertilization in four families.
Yang H., E. Hu, R. Cuevas-Uribe, J. Supan, X. Guo, T. R. Tiersch. 2012. High-throughput sperm
Crassostrea virginica through systematic evaluation of the factors in process of cryopreservation.
Aquaculture, Vol 344-349. Pp. 223–230.
This study provided a reliable protocol for sperm cryopreservation in the eastern oyster
Crassostrea virginica with potential for high-throughput processing. In this study, DMSO yielded
the highest post-thaw motility at a cooling rate of 20 °C/min when thawed at 30 or 40 °C among
the three tested cryoprotectants. Further evaluation of cooling rates of 10, 15, 20, 25 and 30 °C/min showed that 20 or 25 °C /min yielded the highest post-thaw motility (34 ± 5%) and fertility5

(77 ± 12%) for French straws and CBS straws (28 ± 3% and 69 ± 14%). Equilibration times of 10
to 60 min did not cause significant differences in post-thaw motility when freezing with 10%
DMSO at a cooling rate of 25 degrees C/min. Also, sperm concentrations ranging from 1×108 to
1×109 cells/ml at freezing did not cause significant differences in post-thaw motility. Finally, afterthawing, sperm cryopreserved from 16 males with this protocol showed 58 ± 24% fertility (from18 to 86%) for French straws, and 54 ± 21% fertility for CBS straws (from 18 to 95%). Overall,this research provided an outline template for developing a basic protocol for sperm
cryopreservation for any other molluscan shellfish species.
Fuller B, Lane N, Benson E. 2004. Life in the Frozen State, CRC Press, New York. Pp 672.
Cryobiology is a research field encompassing disciplines including mathematics, biophysics, cellbiology, molecular biology, and metabolism physiology. This book brings together the knowledges
about cryobiology from these aspects into one platform for readers to understand this research,and is probably the first major textbook on cryobiology. It includes four themes with a total of 23
chapters, and the four themes are Fundamental Aspects, Life and Death at Low Temperatures,Freezing and Banking of Living Resrouces, and Medical Applications.
Tiersch T. R. and C. C. Green. 2011. Cryopreservation in Aquatic Species, World Aquaculture
Society, Baton Rouge, LA. Pp. 1034.
This is the 2nd version of the book published electronically by the World Aquaculture Society(www.was.org). The first version is the volume 7 in the series of publication “Advances in World
Aquaculure” published in 2000. The revised version is designed as a comprehensive single
compendium of informatio on cryopreservation in aquatic species, and provide a broad overview
of the princles, procedure and perspectives which are necessary for development and applicationof the cryopreservation technology. It includes 101 chapters (compared to the 55 in the firstedition) organized into 11 sections. This book can assist with teaching, research and program
development, and is available for readers to purchase through the World Aquaculture Society witha very affordable price for students and researchers.

6

O-3
Improving Aquaculture Production in Haliotis Species Through theDevelopment of a Genomic Toolkit.
Catherine Purcell*1, Andrew Severin2, Paula Sylvia1, Nick Wegner1, Luis Rodriguez1, and John
Hyde1
1

Southwest Fisheries Science Center, NOAA Fisheries, 8901 La Jolla Shores Dr., La Jolla, CA
92037, Genome Informatics Facility, 206 Science I, Iowa State University, Ames, Iowa 50011*E-mail: Catherine.Purcell.at.noaa.gov

Commercial abalone aquaculture has greatly expanded over the past decade, becoming a thriving
global industry valued at over $100 million USD. Abalone is one of the few species where culture
production dominates the global market as a result of increasing demand and declining natural
stocks from overexploitation and disease. U.S. abalone production is also growing due to highmarket value and demand. Most farms operating in California utilize three native west coast species:
red abalone (Haliotis rufescens), green abalone (H. fulgens), and pink abalone (H. corrugata). These
species differ in commercially important traits that are key to culture expansion in California andimproved production efficiency (e.g., growth rate, disease resistance, thermal tolerance). Next
generation sequencing has opened the door for extensive and explorative genetic research on
abalone, and several studies were recently published examining transcriptomic data for several
species including the domestically important red abalone, H. rufescens (Franchini et al. 2011;
Bester-Van der Merwe et al. 2013; De Wit and Palumbi 2013). Recently, researchers working with
the SWFSC used restriction site associated DNA sequencing (RAD-Seq) methods to identify
genome-wide SNP markers in H. fulgens and to examine population structure in wild populations(Gruenthal et al. 2014). However, transcriptomic and RAD-mapping analyses were also limitedby the paucity of genomic information available for abalone; without knowledge of the genomic
structure it is very difficult to ascertain coverage depth in these studies (Franchini et al. 2011; Araiand Okamura 2013). We are working to create the first de novo abalone genome assembly using
sexed H. rufescens samples, generate tissue specific transcriptomes for H. rufescens, and conductcomparative genomic analyses with other commercially important California abalone. Comparative
analyses will include H. fulgens, H. corrugata, and endangered white (H. sorenseni) and black (H.
cracherodii) abalone. Genomic, transcriptomic, and comparative analyses will improve ourunderstanding of sex-determination, thermal/environmental preference, disease resistance,
hybridization outcomes, and local adaptation, especially for commercially important California
abalone. This will enable identification of candidate genes of interest and marker development formarker assisted selection to improve aquaculture practices in the U.S. abalone industry andelsewhere. This research will also help guide restoration and wild stock enhancement along the west
coast for these species, including for the endangered white and black abalone.

7

Annotated Bibliography of Key Works
Gruental, K., Witting, D., Ford, T., Neuman, M., Williams, J., Pondella, D., Bird, A., Caruso, N.,
Hyde, J., Seeb, L., and Larson, W. (2014). Development and application of genomic tools to therestoration of green abalone in southern California. Conservation Genetics, 15: 109-121.
The authors describe the use of next generation sequencing technology to develop an extensive setof markers to test population structure and effective population size in green abalone in Southern
California. In this study, RADSeq (restriction site associated DNA sequencing) was used togenerate millions of short sequences, from which, many thousands of SNPs may be identified thatspan a greater proportion of the genome compared to previous types of marker development. A
total of 1209 polymorphic SNPs were developed from this sequencing. While the extensive set ofmarkers did not detect population structure in green abalone in the range that was sampled, they
were able to estimate an effective population size (Ne) of 1,100-3,600 individuals. Importantly,
this work generated valuable genomic resources that can be used to further build the set of tools
available to study Haliotis species.
Franchini, P., van der Merwe, M., and Roodt-Wilding, R. (2011) Transcriptome characterization of
the South African abalone Haliotis midae using sequencing-by-synthesis. BMC Research Notes,
4: 59.
This study describes the use of applying next generation sequencing technology to develop
molecular tools for a South African abalone species, Haliotis midae. They use the Illumina
Genome Analyzer II to generate 25 million sequences. Using the transcriptome sequences, they
did a de novo assembly that resulted in 27,761 contigs with an average length of 260 bp.
Importantly, although abalone have a relatively poor representation in genome databases (likely
due to their large genome size), a good number of the contigs had BLAST matches to known
annotated genes in Genbank; with a stringent e-value set, 16.8% of the contigs had a homologous
BLAST match against Genbank. These sequences were assigned to functional categories using
GO and KOG databases. The authors were also able to use this data to identify thousands of SNPs,
and out of those, they developed 420 primer sets.
De Wit, P., and Palumbi, S. (2013) Transcriptome-wide polymorphisms of red abalone (Haliotis
rufescens) reveal patterns of gene flow and local adaptation. Molecular Ecology, 22: 2884-2897.
The authors of this study tested whether signals of environmental selection could be detected insamples of red abalone (Haliotis rufescens) collected from three locations in California: Monterey
Bay, Sonoma, and north of Cape Mendocino. These particular areas are especially distinct in termsof their temperature, aragonite saturation, exposure to hypoxia stress, and disease pressure; assuch, the authors hypothesized that genes related to shell biomineralization, resistance to hypoxia,
temperature tolerance, and resistance to pathogens would show the strongest signals of local
adaptation. The authors tested this by conducting RNA-Seq analyses on mantle tissue of 39 red
abalone individuals from the above locations. A total of 21,579 SNPs were genotyped for eachindividual, and out of these 691 showed significant differentiation. From this set of 691, 163 locicould be identified through BLAST annotation; many of these genes had functions related tobiomineralization, energy metabolism, heat-, and disease- or hypoxia-tolerance. These genes are
now candidates for further studies to look for signals of local adaption.

8

O-4
Culture Protocols and Production of Triploid Purple-Hinge Rock Scallops
Paul G. Olin
California Sea Grant, UCSD / Scripps Institution of Oceanography, 133 Aviation Blvd., Suite 109,
Santa Rosa CA 95403*E-mail: polin.at.ucsd.edu
The goal of this ongoing research and outreach is to expand the West Coast shellfish industry
through creation of triploid seed and demonstration of efficient culture methods for the native
purple-hinge rock scallop (Crassadoma gigantea). Shellfish aquaculture is a low trophic levelmeans of seafood production that provides many benefits to coastal communities and theenvironment, while at the same time increasing the supply of locally produced safe and nutritious
seafood. There is a strong desire to develop native species for aquaculture development to diversify
the shellfish industry and help to avoid concerns often voiced today about the use of non-native
species. While new native species of shellfish for aquaculture are highly sought after, there arealso genetic concerns associated with rearing native species for aquaculture using hatchery-reared
seed that may have undergone significant domestication selection or been produced from distantbroodstock populations. This may occur as a normal consequence of rearing in the hatcheryenvironment or through highly directed selection, crossbreeding, or other means to geneticallychange the production characteristics of the organism. These risks are significant and must be
addressed to realize the potential for growth of the U.S. west coast shellfish industry. Issues
associated with potential genetic risk to wild rock scallop populations could be resolved through thecreation of tetraploid scallop stocks which could be mated to diploids producing 100% triploidoffspring, or by using chemical means. Scallops were successfully spawned and cultured at the
Taylor Shellfish hatchery in Washington State and at the Bodega Marine Laboratory of UC Davisin Bodega Bay, California. Growth and survival of larvae was highly variable among batches andpopulations. Despite broodstock maintained in common conditions and similar larval dietary rations.
Causes for this variability, and generally low larval survival are being investigated. Initial efforts toculture scallop larvae relied mainly on C-Isochrysis sp. These efforts produced weak larvae withlow survival and nutrition was identified as a limiting factor early on. Subsequent efforts relied ona mixed diet of C-Isochrysis sp., Nannochloropsis sp., Pavlova sp., Chaetoceros sp., andThallasiosira sp. The optimal timing for production of 3N scallops by inhibition of second polar
body extrusion using 6-DMAP has been determined to be a 20 min treatment, 55-60 min post
fertilization at 17° C. The optimal dosage of 6-DMAP for production of 3N scallops by inhibitionof second polar body extrusion has been determined to be 425 uM 6-DMAP.

9

Annotated Bibliography of Key Works
Bourne, N. C. A Hodgson and J. N. C. White. 1989 A manual for Scallop Culture in British
Columbia. Canadian Technical Report of Fisheries and Aquatic Sciences. No. 1694 Department of
Fisheries and oceans, Biological Sciences Branch, Pacific Biological Station, Nanaimo, BC
A comprehensive manual for hatchery production of scallops.
The hatchery culture of bivalves: a practical manual. FAO
http://www.fao.org/docrep/007/y5720e/y5720e09.htm
A comprehensive manual for hatchery production of bivalve shellfish
Mortalities in a hatchery of the great scallop, Pecten maximus, . University of Bergen, Departmentof Biology Bergen Norway, 2004.
A bacteriological study was carried out in a scallop hatchery in western Norway. The hatchery bad
suffered severe mortalities during the larval stages, and prophylactic use of antibacterial agentswas necessary to produce larvae. A number of bacterial strains were isolated from the hatchery. A
challenge test was performed with the isolates. Six of the strains caused mortalities not statisticallydifferent from Vibrio pectenicida, a known pathogen froze scallop larvae in France. From 16s
rDNA analysis on these strains, the phylogenetic tree indicated two groups of apparent pathogens;one strain that resembles the Alteromonas/Pseudoalteromonas group, and a cluster of strains that
resemble the species Vibrio splendidus. Since the antibacterial agent used in the hatchery waschloramphenicol, which is now banned in Norway and Europe, application of alternative
antibacterial agents were investigated. In this study the minimum inhibitory concentration (MIC)
values of chloramphenicol, florfenicol, fluemequine, the combination trimethoprim/sulfadiazine,
oxytetracycline, oxolinic acid and Pyceze on bacterial strains isolated from scallop larvae were
investigated. Based on these MIC values, procedures for the treatment of scallop larvae with
antibacterial agents were evaluated. Since the therapeutic procedures will be used in a marine
environment, the antagonizing effect of seawater on some of the antibacterial agents wasmeasured. For fluemequine and trimethoprim/sulfadiazine the average MIC values increased
significantly when using seawater with salinity of 25ppt compared to 2% NaCl.
Caines, J; Crocker, K. 1999.Hatchery production of sea scallop spat (Placopecten magellanicus)
in Newfoundland, Canada
A commercial hatchery for production of sea scallop spat was commissioned in 1995 at
Belleoram, Newfoundland, by the provincial Department of Fisheries and Aquaculture. Fourannual production seasonal cycles have been completed to date, where spat have been transferred
to grow-out at shellfish farms. The annual yield from the hatchery has been 2-25% of the plannedtarget of 20 million spat per year. This presentation will report our experiences in 1998 regarding
algal culture, broodstock conditioning, larval growth, spat settlement and growth, andbacteriological monitoring of water quality. Algal culture: Algae (flagellates and diatoms) weregrown in a continuous culture system purchased from Seasalter Shellfisheries in England. The
intent was to provide scallop larvae and spat with a diet of mixed species of varied lipid andcarbohydrate content. The following flagellates and diatoms were cultured in 500-L polyethylenebags under continuous light at 22 degree C with the addition of CO sub(2): Isochrysis galbana,
T.Iso, Pavlova lutheri, Tetraselmis suecica, Chaetoceros calcitrans, C. ceratosporum, C. muelleri,

10Thalassiosira pseudonana, T. weissflogii, and Chroomonas salina. The growth rate wasapproximately 0.30 and 0.35 divisions/day for the flagellates and diatoms respectively.
Meng, Qinglei; Bao, Zhenmin; Wang, Zhaoping; Wang, Shi; Hu, Jingjie. 2012. Growth andReproductive Performance of Triploid Yesso Scallops (Patinopecten yessoensis) Induced byHypotonic Shock. Journal of Shellfish Research 31.4 (Dec 2012): 1113-1122.
The successful induction of triploid embryos or larvae has been performed in Patinopecten
yessoensis during the past 2 decades. However, no research has been reported about theperformance of triploid P. yessoensis cultured in the field. This study induced triploidy in P.
yessoensis by hypotonic shock and compared the growth and reproductive performance of
triploids and diploids reared under commercial conditions for up to 24 mo. The main results ofthis study are as follows: Triploid scallops were smaller in size and weight compared with diploids
and had a retarded absolute growth rate (AGR). After 24 mo. of cultivation, the mean shell height,
shell length, shell width, and body weight of triploids were 9%, 10%, 9%, and 25% less than
diploids, respectively (P < 0.01). Although normal in sex ratio, the reproductive potential of
triploids was significantly reduced. Only 87% of the triploids exhibited sex-discernible gonads
during the breeding season. None of the male triploids spawned, and the percentage of female
spawners among the triploid population was only 27% of that for the diploid population. The
relative fecundity of triploid females was only 4% of diploid females. Triploid eggs produced
mostly aneuploid larvae and had an extremely small chance of generating viable offspring when
fertilized by sperm from diploid males. Most aneuploid larvae died before the D-shaped stage, andno survival exceeded 7 days. The potential to yield viable offspring from the triploid population
was estimated to be only 4% 10-6 of that of the diploid population. Despite the growth
disadvantage of triploids, this study may support, in part, the energy reallocation hypothesis,
because triploid AGR was similar to diploid AGR (2% variance) during the sexual maturationseason. Our results also indicate that there would be no growth advantage, but instead adisadvantage, for triploid P. yessoensis growing at the experiment site. In addition, this research
provides the first evidence that viable triploid molluscs can be induced by hypotonic shock, ofwhich the practical and evolutionary implications are also discussed.
Cogswell, A. T., Roach, S. E.; MacDonald, B. W. 2006. Triploid bay scallops (Argopecten
irradians): induction methodology, early gonadic development and growth. Department of
Fisheries and Oceans, Dartmouth, NS (Canada). Science Branch. Canadian technical report offisheries and aquatic sciences/Rapport technique canadien des sciences halieutiques et aquatiques
2635. (2006).
Triploid (3N) Pacific oysters (Crassostrea gigas) account for more than 50% of total oyster
production in the US. The success of this species has created an interest in producing triploids ofother commercial shellfish species, including scallops, clams and mussels. Here, we report on 3N
induction trials with the bay scallop, Argopecten irradians. The most commonly employed 3N
induction technique involves exposing early embryos to chemicals (e.g., Cytochalasin B (CB)).
CB inhibits the release of the second polar body immediately following fertilization, causing
retention of both pairs of female chromosomes in addition to the male chromosome set. It does
this by disrupting actin polymerization. Four concentrations of CB were evaluated for ability to
produce 3N larvae. The efficacy of current and proposed 3N induction techniques (i.e., 4N x 2N
crosses) and the commercial potential of 3N bay scallops are discussed.
11

Ruiz-Verdugo, CA; Allen, S K, and ; Ibarra, A M. 2001. Family differences in success of triploid
induction and effects of triploidy on fecundity of catarina scallop (Argopecten ventricosus).
Aquaculture 201.1-2 (Sep 14, 2001): 19-33.
Mass induction of triploidy in the catarina scallop (Argopecten ventricosus) results in low successin the percentage of triploids produced. To understand whether this is a treatment effect affecting
all eggs equally, families were individually induced to triploidy with cytochalasin-B (CB),comparing their survival from egg to D-larvae and spat, and the percent of triploidy withinfamilies. Differences in percent triploidy success were evident between families, obtaining somewith no triploids, and some with high triploidy. Among the possible causes for these differences
are quality of eggs, different developmental rates, and differences in susceptibility to the treatment(CB or DMSO) itself. Regardless of those differences, overall triploidy production was increased
by inducing individually eggs of each scallop rather than in mixed egg batches. In the firstexperiment, it was improved by 17%, and in the second experiment by 42%, as indicated by theweighted mean of triploids among the families, and when compared with previous results withthis same species, where triploidy success was 58%.In a second experiment with three different
families, the growth and fecundity of triploid and diploid catarina scallop were evaluated. The
growth superiority of triploids was confirmed. The results indicated that triploid catarina scallop
had a significantly reduced fecundity when compared with diploid scallop. The reduced fecundity
appears to be mostly of a random nature, possibly associated with a reduced capability to produce
balanced gametes. Whereas the successful production of tetraploid catarina scallop from fertile
triploid scallop is in principle possible, the low number of eggs shed by triploid catarina scallop
could diminish that success rate, even more if single triploid females are required to optimize
tetraploid induction.

12

O-5
Big Data in Agriculture and the USDA/ARS Initiative
Jeffrey SilversteinSoutheast Area, US Department of Agriculture, Agricultural Research Service, P.O. Box 225,
Stoneville MS 38776*E-mail: jeff.silverstein.at.ars.usda.gov
In recent years, technological achievements have permitted the relatively inexpensive and rapid
production of vast amounts of data. The large and often complex datasets produced in the scientificsphere demand new approaches to gain value and to turn data into information. Management of thegrowth in the volume, variety, and velocity of data is often referred to as the ‘Big Data problem’.
The Agricultural Research Service of the U.S. Department of Agriculture (USDA/ARS) has long
been a strong, science-based, problem solving agency. In the past, our computational infrastructure
has primarily been based on meeting administrative needs and security requirements, whereas
computational, analytical, and sharing of scientific data were regarded as secondary priorities. The
Big Data problem has required a reassessment of scientific computing needs. In February 2013,
we held a workshop led by ARS scientists to assess scientific data needs, and this resulted in a $25
million initiative to develop the USDA/ARS capacity to collect, share, and analyze Big Data. Our
Big Data initiative contains three elements. First, we have developed a dedicated scientific research
network (SCInet) which will leverage Internet2 to facilitate large scale transfer of research data at
high-speeds with low latency. Second, we have constructed a high-performance computing (HPC)
system with high memory, high processing capacity and the potential to burst computational
workloads to commercial resources when necessary. SCInet and the HPC have been largely
constructed and connected in the first 18 months of the initiative. Finally, we are developing a virtualresearch support core of individuals who will provide scientific computational and informatics
support. These individuals will work with agency scientists to facilitate specific projects and will
also develop standardized solutions and training for common challenges.
Key steps of this initiative, 1) determining the needs of the agency, 2) developing a plan to connect
more than 90 locations in the agency, 3) building the technical capacity in our labs, and 4)
controlling costs due to restrictive budgets are challenges that will be shared and discussed.

13

O-6
Signature of artificial selection in a breed of coho salmon Oncorhynchus
kisutchSho Hosoya1*, Kiyoshi Kikuchi1, Hiroshi Nagashima2, Junichi Onodera2, Kouichi Sugimoto2, Kou
Satoh2, Keisuke Matsuzaki2, Masaki Yasugi3, Atsushi J. Nagano3,4, Akira Kumagayi2, Kenichi
Ueda2, Tadahide Kurokawa5
1

Fisheries Laboratory, University of Tokyo, 2Miyagi Prefecture Fisheries Technology Center,3
Center for Ecological Research, Kyoto University, 4JST PRESTO, 5Tohoku National Fisheries
Research Institute*E-mail: ahosoya.at.mail.ecc.u-tokyo.ac.jp
Artificial selection has often left signatures within the genome of the breeding population. Suchsignatures can be detected by means of a population genomics approach. Identification of thesefootprints of selection can pave the way to find genes and genetic markers that may facilitate further
genetic improvement. In this study, we analyzed two divergent populations of coho salmon(Oncorhynchus kisutch), one of which was obtained by selection for increased growth during thepast 15 years and the other with no intentional selection, in order to identify loci affected in the earlystages of a breeding program. The two populations originated from the hatchery at Lower Kalama,
WA, USA. Eyed eggs were introduced to Japanese National Research Institute of Aquaculture in
1978. Subpopulation was transferred to Inland Fisheries Experimental Station, Miyagi Prefecture
Fisheries Technology Center in 1980. Two selected lines were established in 2000 from two
different year classes: six and ten selected females were crossed to three unselected males each. The
second and the third selection were done in 2003 and 2006, respectively. In 2009, individuals fromthe selected population were randomly crossed within the population (F3). The forth selection was
done in 2013 (F4). Genetic analysis was done using the F3 and the size comparison using the F4.
We compared fork length between the unselected and the F4 populations reared in a communal pond
and found that the average fork length of the selected population was significantly longer than thatof the unselected population. From the F3 selected and the unselected population, 100 individuals
were randomly chosen and a genome-wide set of SNPs was collected by ddRAD-seq. Output reads
were first quality trimmed and survived reads at both paired ends were mapped on the rainbow trout
genome individually, and analyzed variations using Stacks (ver 1.35). Close to 166,000 SNP sites
were detected, out of which approximately 1,100 were considered reliable, and were used for
subsequent analysis. Multidimensional scaling analysis based on SNPs showed that the two
populations are clearly separated from one another, indicating that two populations have geneticallydifferentiated after several generations of captive breeding. We compared allele frequencies
between populations at each locus, and found significant divergence at 57 sites. We also performed
GWAS and detected 19 loci where P-values were less than 0.01. A subset of these sites are likely tobe linked to regions associated with the growth phenotype, and will be available as genetic markers
for the improvement of the breed. As a next step, we are planning to implement genomic selection
into the breeding program. This work was supported by AFFRC, Japan.

14

Key words: coho salmon, Oncorhynchus kisutch, artificial selection, body size, ddRAD-seq,
marker assisted s

15

O-7
Genetic Selection in Animals Using Pedigree, Phenotypic, and Genomic
Information
Shogo Tsuruta1*, Daniela, A. L. Lourenco1, Yutaka Masuda1, Breno Fragomeni1, Ignacio Aguilar2,
Andres Legarra3, Ignacy Misztal1
1

Department of Animal and Dairy Science, The University of Georgia, Athens, GA, 30602, USA,
Instituto Nacional de Investigacion Agropecuaria, Canelones, 90200, Uruguay, 3INRA, UMR1388
GenePhySE, Castanet Tolosan, 31326, France*E-mail: shogo.at.uga.edu
2Genetic selection of quantitative traits in animals has been performed for over a century in the
United States. In the beginning, artificial selection was conducted using only phenotypes. Later,
pedigree information was added, from parent-offspring relationships, siblings, and families to all
available information among related animals. Around the same time, advanced statistical methods
were developed (e.g., regression analysis, least square analysis, and BLUP) to estimate genetic
parameters and to predict breeding values for economically important traits. As a result, animal
productions have dramatically increased in the last half-century. In the last two decades, we has
contributed to various livestock industries, including dairy cattle, beef cattle, swine, chicken, goats,
sheep, horses, bees, and fish. We have analyzed genetic components in animal production,
reproduction, and disease traits using our own computer programs in collaborations with USDA,
breed associations, breeding companies, and other institutes all over the world. Those programs
have been used by breed associations and breeding companies in routine genetic evaluations.
Recently, we have developed a method called single-step genomic BLUP (ssGBLUP) to predictgenomic breeding values by combining pedigree, phenotypes, and genotypes (SNP markers). With
ssGBLUP, we found that we could increase the accuracy of genomic breeding values in dairy andbeef cattle, pigs, and chickens by 10-30% compared with traditional breeding values. Thismethodology has been expanded to use a large number of genotyped animals (> 1 million). The
application programs are available on our website at http://nce.ads.uga.edu/projects/programs/~.
Genomic selection has a greater advantage when more genotyped animals are available, because
predictions are more accurate and more complex models can be applied. Also, if animals are
genotyped in the earlier stage of their life, genetic progress can be accelerated and genetic gains canbe maximized. These methodologies are applicable to other organisms, such as farming fish,shellfish, anadromous fish (salmon), and even plants.

Annotated Bibliography of Key Works
Misztal, I., S. Tsuruta, T. Strabel, B. Auvray, T. Druet, and D. Lee. 2002. BLUPF90 and related
programs (BGF90). Proc. 7th WCGALP, Montpellier, France.
Aguilar, I., I. Misztal, D. L. Johnson, A. Legarra, S. Tsuruta, and T. J. Lawlor. 2010. A unified
approach to utilize phenotypic, full pedigree, and genomic information for genetic evaluation of
Holstein final score. J. Dairy Sci. 93:743-752.
16

Tsuruta, S., I. Misztal., I. Aguilar, and T. J. Lawlor. 2011. Multiple-trait genomic evaluation of linear
type traits using genomic and phenotypic data in Holsteins. J. Dairy Sci. 94:4198-4204.
Lourenco, D. A. L., S. Tsuruta, B. O. Fragomeni, Y. Masuda, I. Aguilar, A. Legarra, J. K. Bertrand,
D. Moser, and I. Misztal. 2015. Genomic evaluation using single-step genomic BLUP in American
Angus. J. Anim. Sci. 93:2653-2662.
Fragomeni, B. O., D. A. L. Lourenco, S. Tsuruta, Y. Masuda, I. Aguilar, A. Legarra, T. J. Lawlor,and I. Misztal. 2015. Use of genomic recursions in single-step genomic BLUP with a large numberof genotypes. J. Dairy Sci. 98: 4090-4094.
The authors have been working on estimation of genetic parameters and prediction of breedingvalues for economically important traits in animals. They have recently developed a method called
single-step genomic BLUP (ssGBLUP) to predict genomic breeding values by combining pedigree,phenotypic, and genomic information and have implemented the method to their existing computer(BLUPF90) programs (e.g., BLUP, REML, and Gibbs Sampler) to use SNP marker information.
Recently, they published several studies of ssGBLUP to maximize accuracy and minimize bias ingenomic breeding values, applying this methodology to dairy cattle, beef cattle, pigs, and chickens.
Currently, they have been working on how to include a large number of genotyped animals (> 1
million) in genomic evaluation. Breed associations and breeding companies that have been using
their software are now planning to introduce ssGBLUP to routine evaluations. The same or similar
methodology can be applied to fish breeding. The BLUPF90 family of programs and the manual
are available at http://nce.ads.uga.edu/~.

17

O-8
Exploring Transcriptomic Patterns in Slow- and Fast-Growing Seriola dorsalis
Larvae.
Catherine Purcell*1, Andrew Severin2, Vince Buonaccorsi3, Mark Drawbridge4, Kevin Stuart4, andJohn Hyde1
1

NRC Postdoctoral Fellow, Southwest Fisheries Science Center, NOAA Fisheries, 8901 La Jolla
Shores Dr., La Jolla, CA 92037, 2Genome Informatics Facility, 206 Science I, Iowa State
University, Ames, Iowa 50011, 3Juniata College, 1700 Moore St. Huntingdon PA 16652, 4Hubbs
Seaworld Research Institute, 2595 Ingraham Street, San Diego, CA 92109*E-mail: Catherine.Purcell.at.noaa.gov
Seriola species have traditionally been a major component of global commercial and recreational
fisheries, and in recent years, aquaculture value of these fish has grown into a ~$1.3 billion industry.
The California yellowtail (Seriola dorsalis) is a strong candidate for development of offshore
commercial aquaculture in southern California and neighboring Baja California. Althoughproduction from broodstock populations has been successful, it has not yet reached a sustainablelevel where it can satisfy the aquaculture demand, largely due to difficulties from highly variablesurvival and growth rates through the larval stages. Given the extremely fast growth and the major
physical changes that occur during the earliest life stages, one way to examine variability in survivaland growth is at the gene and molecular levels across those early-developmental periods. To
improve our understanding of the molecular processes underlying development, we examined
RNA-Seq profiles for several early life stages of yellowtail, categorized as either slow- or fastgrowing. Gene expression was measured in three replicates of pooled larval samples at 2, 7, and 17days post hatch for these two growth categories. Using the Illumina platform, an average of sixty
million reads was obtained per replicate; genes of related function were sorted into clusters, andthose found at high frequency in the differential gene expression set were identified. Differences inmolecular pathways, biological processes, and gene regulating patterns between the two fitness
groups were examined. There were many differentially expressed genes across developmental
stages and between the fitness groups, for example, genes involved in oxidative phosphorylation
pathways revealed interesting patterns both across developmental stages and between slow- andfast-growing larvae. Results of these analyses will be presented.

Annotated Bibliography of Key Works
Kocmarek, A., Ferguson, M., and Danzmann, R. (2014) Differential gene expression in small andlarge rainbow trout derived from two seasonal spawning groups. BMC Genomics, 15: 57.
In this study, the authors wanted to identify genes that showed similar expression patterns between
large and small rainbow trout from different spawning seasons in two types of tissue: liver andwhite muscle. The goal in identifying these genes was to be able to develop growth-related
markers for use in breeding programs. They hypothesized that 1) genes related to carbohydrate
and lipid metabolism, energy production, insulin, and growth factors would be down-regulated inboth tissue types in the small fish, 2) that genes involved in cytoskeletal structuring would be18

down-regulated in small fish, while myostatin will be up-regulated, and 3) that liver-specific lipid
binding, cytoplasmic components, signaling, and transcription would be up-regulated in small
fish. They observed that genes related to immune function were up regulated in large fish;suggesting that enhanced growth is associated with enhanced immune function. They also foundthat genes related to transcription, translation, and protein production were up regulated in small
fish (from Sept.) in white muscle, which supports patterns previously detected in liver tissue. This
indicates that protein production in small fish may not be translating effectively into finishedproteins. This study was able to identify patterns of differential gene expression in small and large
rainbow trout; this will enable future studies to delve deeper into the genes related to differencesin growth.
Meyer, E., and Manahan, D. (2010) Gene expression profiling of genetically determined growthvariation in bivalve larvae (Crassostrea gigas). The Journal of Experimental Biology, 213: 749758.
The authors compared gene expression patterns in larvae of the Pacific oyster (Crassostrea gigas)
that exhibited slow- and fast-growth (these larvae were produced frome xperimental crosses).
Based on a previous transcriptome-wide analysis, a set of 181 candidate genes for growthheterogeneity were analyzed with the goal of understanding the biological processes underlying
the differential growth rates. Of the genes identified by GenBank, ribosomal proteins were themost abundant, comprising 50% of the total genes with 17 different ribosomal protein genes. The
genes included nine components of the large ribosomal subunit, and eight components of the small
ribosomal subunit. Some of these genes were up-regulated in the fast-growing larvae (n = 6),
while others were up-regulated in the slow-growing larvae (n = 11). Since ribosome biogenesis
is a significant metabolic cost in cell proliferation, any changes in this pathway would likely havea large effect on the overall energy metabolism. The authors hypothesize that in the slow-growing
larvae there may be a high metabolic cost to synthesizing and degrading excess ribosomal protein
copies resulting from the higher expression of those genes.
Salem, M., Silverstein, J., Rexroad III, C., and Yao, J. (2007) Effect of starvation on global gene
expression and proteolysis in rainbow trout (Oncorhynchus mykiss). BMC Genomics, 8: 328.
The authors of this study used microarrays to identify genes and pathways involved in the
starvation response and protein turnover in rainbow trout, and to identify metabolic adaptations
that occur in the liver during these starvation periods. This study was of interest because, as theauthors point out, examining changes in metabolism during starvation is an effective way toidentify relationships between metabolic pathways and body processes. The experiments showed
down-regulated expression of genes involved in protein biosynthesis in the starved fish, but nosignificant changes in protein catabolic pathways, and a slight increase in 20S proteasome activity.
Responses in the liver to starvation included an overall decline in gene expression associated withdecreasing tissue metabolism, a reduction in protein synthetic capacity, an impairment of ATPbiosynthesis, and lower expression in hepatic lipid and fatty acid transport.
Moran, D. (2007) Size heterogeneity, growth potential and aggression in juvenile yellowtail
kingfish (Seriola lalandi Valenciennes). Aquaculture Research, 38: 1254-1264.
In this study, the authors are describing the occurrence of size heterogeneity and aggressive
behaviors in cultures of Seriola lalandi; this was done to examine the effectiveness of size-grading19

in reducing aggression and mortality, and increasing growth rates. To do this, graded and ungradedjuveniles were compared for various measures of aggression and growth, and a RNA/DNA ratio
was used as a measure of growth rate. The authors found that size heterogeneity became more
pronounced at 12 days post hatch (dph) when Artemia are offered as a food source. While thelarge and aggressive juveniles only accounted for 8% of the population, the small grade juveniles
that received the aggression accounted for 42% of the population. In the ungraded treatment, thisaggression was associated with mortality for most small fish. However, even without aggression,
the small-grade juveniles did not gain weight or increase their RNA/DNA ratio after 12 days. The
authors believe that these small fish appear to be on a degenerative developmental strategy
without any influence of the larger aggressive fish.

20

O-9
Modeling the Variable Effects of Using Wild and Cultured Broodstock on the
Fitness Risk Due to Escaped Farmed Fish
Kristen M. Gruenthal*1, Jason D. Volk2, Gregory R. Blair2, Gavin Key3 and Michael B. Rust1
1

NOAA Fisheries Office of Aquaculture, Northwest Fisheries Science Center, 2725 Montlake
Blvd E, Seattle WA, 98112, USA, 2ICF International, 710 Second Avenue Suite 550, Seattle, WA
98104, USA, 3Kampachi Farms, LLC, 1 Keahole Point Road, Kailua Kona, HI 96740*E-mail: kristen.gruenthal.at.noaa.gov

The rapid development of offshore marine aquaculture worldwide has raised concerns due in partto the potential for various negative ecological and genetic impacts if and when farmed fish escapeand mix with wild conspecifics. Impacts due to interbreeding between wild fish and escapees may
result in reduced genetic diversity within and among populations and a loss of fitness. Loss ofgenetic diversity and fitness compromise the adaptive potential of a mixed (wild plus cultured)
population, making it potentially less able to respond to stochastic changes or long-term trends (e.g.
climate change) in environmental conditions. Risk to wild population fitness is a function of 1)
the number of escapees relative to wild census size, 2) the genetic difference between escapees andwild fish, and 3) the fitness of escapees relative to wild fish. There is little scientific data, however,that reliably assigns the risk to population fitness due to escapes. The Offshore Mariculture
Escapes Genetics Assessment (OMEGA) model simulates the risk posed by escaped farmed fish towild population fitness. OMEGA is intended to provide insight into the variables affecting risk,
help identify information gaps and research priorities, inform policy and management decisions,and explore options for design or modification of culture programs, such that the aquacultureindustry itself can and will drive improvements in culture techniques and sustainability. Current
work continues to be directed at making OMEGA fully operational and includes fostering external
collaborations to develop model scenarios, evaluate model parameters, and validate the model withdata from current and planned domestic and international aquaculture operations. To illustrate
OMEGA’s application, we present results from evaluations of a theoretical sablefish (Anoplopoma
fimbria) aquaculture program and a practical pilot project for Almaco jack (Seriola rivoliana) in
Hawaii. Sablefish are a highly-prized groundfish with white flesh found along the North PacificRim. Commercial culture of the species currently occurs in Washington State, USA, and British
Columbia, Canada. The OMEGA scenarios for sablefish were originally developed for model
verification and include using a domesticated broodstock to produce up to 10,000 mt of fish stocked
among 50 offshore cage sites. Kampachi Farms is a leader in sustainable US marine aquaculture
systems development and hatchery methods. Recently, the group has engaged in iterative openocean pilot projects directed at expanding offshore farming of almaco jack to a commercial scalenear Kailua Kona, HI, USA, and elsewhere. The OMEGA scenario for Kampachi uses wild
broodstock and stocks F1 juveniles in tethered cages located approximately 11 km offshore of Kona,
with production of about 7 mt per year for two years. Escape scenarios for both species varied
from low to high base leak rates, cage failure probabilities, and catastrophic cage failure
probabilities. Whereas the high escapes scenario for sablefish resulted in a significant impact towild population fitness, even a total loss of almaco jack had a negligible effect. Key differences21

in the simulated fitness risk associated with each program included broodstock source (domesticatedor wild), offshore location, and the scale (size and longevity) of the operation.

Annotated Bibliography of Key Works
Baskett, M.L., Burgess, S.C., Waples, R.S., 2013. Assessing strategies to minimize unintended
fitness consequences of aquaculture on wild populations. Evolutionary Applications 6:1090-1108.
Baskett et al. (2013) address various factors associated with the management of cultured
populations and model their potential fitness impacts should farmed fish escape into the wild.
Factors assessed include the origin and level of domestication in the cultured stock (i.e. degree ofmaladaptation), induced sterility in the cultured stock, and the magnitude and frequency ofescapes (e.g. continuous low level leakage versus rare catastrophic events). Results indicate that,up to a point, the magnitude of the fitness impact rises as the maladaptation of escaped cultured
fish increases; an extremely maladapted, non-local origin cultured population may actually haveeffects similar to a weakly diverged stock. Second, sterilization reduces unintended fitness
consequences rapidly. Finally, it is more effective to reduce low-level leakage than guard against
sporadic large-scale escape events.
Ford, M.J. 2002. Selection in captivity during supportive breeding may reduce fitness in the wild.
Conservation Biology 16:815-825.
Escaped cultured fish may cause a potential loss of fitness in the wild, if breeding occurs between
escapees and wild conspecifics. This drop in fitness is associated with a difference in the
optimum trait values for hatchery and natural environments. The single-trait phenotypic fitness
model in Ford (2002) describes how mean phenotype values of the mixed population (captive plus
wild fish) may shift relative to the optimum values for each environment, based on thepresence/absence and amount of gene flow (interbreeding) between the cultured escaped and wild
fish. The overall fitness effect depends on the magnitude of the difference in optimum trait
value, trait heritability, and selection pressure against domestication in the wild, as well as habitat
capacity, magnitude and frequency of escape events, wild and captive population demographics,
and the potential for interaction between wild fish and escapees. Ford (2202) was used mostnotably in Pacific Northwest salmonids, where the All-H Analyzer, or AHA, model helped the
Hatchery Scientific Review Group explore the potential fitness consequences of supplementing
wild populations and of cultured fish straying into wild populations.
Hindar, K., Ryman, N., Utter, F., 1991. Genetic effects of cultured fish on natural fish population.
Canadian Journal of Fisheries and Aquatic Sciences 48:945-957.
Hindar et al. (1991) represents one of the original manuscripts reviewing the genetic impact
escaped farmed fish may have on wild populations and is one of the most often cited. The
authors recommend several strategies for protecting the genetic integrity of wild populations,many of which remain the focus of aquaculture programs today. These strategies include
improved containment technology and recovery, sterilization of the culture stock, and better
breeding practices, coupled with monitoring the genetic contribution of escaped fish to the mixed
population.

22

Waples, R.S., Hindar, K., Hard, J.J., 2012. Genetic risks associated with marine aquaculture.
NOAA Technical Memorandum NMFS-NWFSC-119. US Department of Commerce NOAANMFS, Washington, D.C.
Waples et al. (2012) is a comprehensive overview of the potential genetic impacts to wild
populations associated with marine aquaculture. As such, the authors “provide managers with abetter understanding of the genetic effects of marine aquaculture on natural populations,” with theintent of informing policy and management decision-making. The document synthesizes
relevant information and provides key references, identifies research priorities, provides a risk
assessment framework, and gives recommendations for monitoring and evaluation towardsustainable marine aquaculture development in the US. Waples et al. (2012) focuses oncommercial aquaculture of marine finfish but presents it in light of decades of research on salmon
hatcheries and marine stock enhancement.

23

O-10
Did farmed Coho salmon Oncorhynchus kisutch that escaped during theearthquake and tsunami disaster of 2011 interbreed with native Masu salmon
Oncorhynchus masou?
Kei Sasaki*, Hideki Nikaido, Daisuke Muraoka, Yoshiro Okada, Tadahide Kurokawa
Miyako Station, Tohoku National Fisheries Research Institute, FRA*E-mail: keis.at.affrc.go.jp
Sea farming of coho salmon (Oncorhynchus kisutch), a species that migrates to waters off Hokkaido
for feeding but does not naturally spawn in Japan has been conducted around the “Sanriku” coast inJapan since 1975. In recent years, production has remained at over 10,000 tons per year. Due tothe effects of the Great East Japan Earthquake and tsunami of March 11, 2011, several million
farmed coho salmon escaped into the Northeast Pacific Ocean off Tohoku, northeastern Japan. In
the fall of that year, sexually mature coho salmon migrated up rivers in this area. Farmed coho
salmon that migrated up rivers to breed may have affected the genetic material of native salmon
species and result in weakened populations. Especially, there is strong concern that coho salmon
may cross with the native masu salmon (Oncorhynchus masou); it is known that hybrids of these
species are able to survive (while the ability to survive is low for crosses with chum salmon).
Assuming that hybrids are present, it is unclear how many years they will need to mature. However,based on the maturation age of both species, the possibility that hybrids will return in spring 2014
is considered to be high. We conducted visual checks of masu and coho salmon landed at a fish
market during May to September 2014 (approx. 2,000 individuals). We selected 30 masu, and 5
coho salmon (all individuals were sampled during the survey) judged from appearance for analysis.
Furthermore another sample of individuals (n=9) that were caught at the same place and wereconsidered to potentially be crosses were also examined in the laboratory. For the morphologicalcomparison, we compared the number of rays of each fin, and the number and length of the gillrakers. We conducted sequence analysis of the intron C of growth hormone 1 (GH-1). PCR
amplified product of masu salmon (256bp) is 34bp less than that of coho salmon (290bp) using aprimer set with 17bp adapter sequence for the fluorescent label. Therefore, it is possible todistinguish both species. The amplicon is seen in both if there is a hybrid. As a result of themorphological comparison and DNA fragment analysis, hybrids were not confirmed, and all theindividuals that were initially considered to be potential hybrids were judged to be masu salmon.
Therefore based on the current survey, the impact on the genetic resources of masu salmon isconsidered to be low. However, three of five of coho salmon were of the 2011 brood and their gonads
were developed (although there is no conclusive evidence that these coho salmon individuals were
derived from the escaped coho salmon during the 2011 earthquake). Therefore it is necessary tocarefully monitor the occurrence of hybrids in the short-term future.
Key words: farmed coho salmon, Oncorhynchus kisutch, escaped, masu salmon, Oncorhynchus
masou, hybrids, DNA fragment analysis

24

O-11
Evaluation of the tsunami impact on the genetic diversity of the marbled
flounder Pseudopleuronectes yokohamae in Sendai Bay, Miyagi, Japan
Yuki Minegishi1*, Minoru Ikeda1, Yutaka Kurita2, Hiroyuki Togashi2, Yukinori Nakane2, andAkihiro Kijima1
1

Tohoku Ecosystem-Associated Marine Sciences, Graduate School of Agricultural Science,
Tohoku University, 2Tohoku National Fisheries Research Institute, FRA*E-mail: y.minegishi at m.tohoku.ac.jp
Evaluating genetic diversity is a fundamental step for appropriate stock management. The
information on genetic diversity such as population structure, migration rate and population size
helps identify management units and translocation ranges, and monitoring those alterations provides
the insights into the population vulnerability and viability. At the same time, genetic diversity is
influenced by environmental and demographic changes. Direct effects involve population
bottleneck and varied migration between populations, and indirect ones cause changes in habitat
structure and community composition. Therefore, genetic diversity should be carefully investigated,
especially where dramatic disturbances have occurred. Marbled flounder Pseudopleuronectes
yokohamae is a commercially valued fish overall Japan. Due to the benthic lifestyle including low
dispersal ability, this species possibly consists of multiple management units. The population in
Sendai Bay is considered to form a single management unit, and its spawning ground has beenprotected for stock conservation. However, this population is expected to have been strongly
affected by the great tsunami occurred along the Pacific coast in North Japan in 2011, andconsequently the genetic diversity could be changed. Thus, the temporal genetic variation of the
Sendai Bay population needs to be investigated and the geographic range of the management unit
should be determined based on the genetic population structure inferred together with other Pacific
Ocean populations. To develop a genomic resource, a 200-bp library was prepared using the
genomic DNA of a single specimen of the marbled flounder collected in Sendai Bay. IonTorrent
PGM sequencing was performed up to 35.4 × coverage (23.7 gigabases), and de novo assembly
generated a total of 525,502 contigs (N50 = 1994) and 10,732,070 unassembled reads. We thendesigned a total of 331,368 primer pairs for 86,732 unique microsatellite sequences detected in theunassembled reads. Among the arbitrarily selected 96 pairs that were experimentally tested, sixteen
pairs were characterized as novel microsatellite loci applicable for population genetic analyses ofthis species. Temporal change in genetic diversity after the tsunami impact was investigated using
thirteen populations collected in Sendai Bay from April 2012 to February 2014 (N = 807). Nine
populations from the Pacific Ocean coasts (N = 596) were also analyzed for comparisons.
Genotyping was performed at the newly developed sixteen microsatellite loci and population
genetic analyses were conducted. Significant genetic divergence was found between the east andwest sides of Boso peninsula, in which populations further diverged (pairwise FST = 0.02305 –0.19784). At the east side of Boso peninsula, the Mutsu Bay population was differentiated (FST =0.03829, P < 0.001) and gene flow was observed over the wide area from Onagawa Bay to Choshi
including Sendai Bay. The genetic variability of the Sendai Bay population did not show any
dramatic changes in time (HE = 0.5061 – 0.5463) and was relatively higher compared to those of25

the western populations of Boso Peninsula (HE = 0.3817 – 0.4557). It is thus suggested that thetsunami impact on the Sendai Bay population of the marbled flounder is considered to be minor but
a long-term monitoring may be needed.
Key words: Marbled flounder, Pseudopleuronectes yokohamae, genetic diversity, management
units, population structure
Annotated Bibliography of Key Works
Banks, S.C., G.J. Cary, A.L. Smith, I.D. Davies, D.A. Driscoll, A.M. Gill, D.B. Lindenmayer, R.
Peakall. 2013. How does ecological disturbance influence genetic diversity? Trends Ecol. Evol.,28, 670-679.
Environmental disturbance underpins the dynamics and diversity of many of the ecosystems of theworld, yet its influence on the patterns and distribution of genetic diversity is poorly appreciated.
We argue here that disturbance history may be the major driver that shapes patterns of genetic
diversity in many natural populations. We outline how disturbance influences genetic diversity
through changes in both selective processes and demographically driven, selectively neutral
processes. Our review highlights the opportunities and challenges presented by genetic
approaches, such as landscape genomics, for better understanding and predicting the demographic
and evolutionary responses of natural populations to disturbance. Developing this understanding is
now critical because disturbance regimes are changing rapidly in a human-modified world.
Waples, R.S., A.E. Punt, J.M. Cope. 2008. Integrating genetic data into management of marine
resources: how can we do it better? Fish Fish., 9, 423-449.
Molecular genetic data have found widespread application in the identification of population andconservation units for aquatic species. However, integration of genetic information into actual
management has been slow, and explicit and quantitative inclusion of genetic data into fisheriesmodels is rare. In part, this reflects the inherent difficulty in using genetic markers to draw
inferences about demographic independence, which is generally the information of the greatest
short-term interest to fishery managers. However, practical management constraints, institutional
structures and communication issues have also contributed to the lack of integration. This paper
identifies some of the organizational, conceptual and technical barriers that have hampered fulluse of genetics data in stock assessment and hence fishery management and outlines how such use
could be enhanced.

26

O-12
Competition between Atlantic salmon (Salmo salar) and Japan’s native
salmonids.
Kazuo Araki*, Tetuji Masaoka, Hiroyuki Okamoto, Hiroyuki Nagoya, Hidefumi
Nakamura, ,Kouji Muto and Toshiya Suzuki
National Research Institute of Aquaculture, FRA
224-1, Hiruda, Tamaki-cho, Watarai-gun, Mie, 516-0423, Japan*E-mail: arakin.at.affrc.go.jp
AquaAdvantage Salmon are a strain of Atlantic salmon that have been genetically modified by the
integration of Chinook salmon growth hormone gene and an anti-freezing protein promoter derived
from ocean pout. This strain grows more rapidly (> 2×) than other farmed Atlantic salmon. Issuanceof a draft Environmental Assessment (EA) and preliminary Findings Of No Significant Impact(FONSI) represent the first step in the U.S. Food and Drug Administration (FDA) evaluation of
AquaAdvantage Salmon. The FDA’s preliminary finding is that an approval of this application,
under the specific conditions proposed in the application, would not have a significant impact on
the US environment. The National Environmental Policy Act of 1969 (NEPA) requires the FDA andother federal agencies to perform such assessment whenever a major federal action is taken. If the
US government approves the commercial application of AquaAdvantage Salmon, there is apossibility that genetically modified (GM) salmon will be imported to be farmed in Japan. To date,Atlantic salmon have been farmed at a single case in Japan, in Aomori Prefecture (1983～1988).
As a result, there is little information describing the biology and ecology of Atlantic salmon in Japan.
To address this, we documented the maturation of Atlantic salmon in Japan, and evaluated thepotential for hybridization and competition with Japan’s native salmonids. We demonstrated thatseasonal water temperature is very important for Atlantic salmon maturation in Japan. Additionally,
we artificially crossed Atlantic salmon and native salmonids. Almost all hybrid embryos stop
development at the mesoderm induction stage. However, low numbers of Atlantic salmon and native
char (Salvelinus leucomaenis) hybrids, confirmed by RFLP, have survived for 2 years. We
cultured different lifestages of Atlantic salmon and several native salmonids in the same aquariumand evaluated the occurrence of competition for food and space, and changes in weight. Competitiondiffered depending on the combination of salmonid species and life stage. Among juveniles, allnative salmonids attacked by Atlantic salmon. During the adult stage, Amago (Oncorhynchus
masou ishikawae) and Biwa trout (Oncorhynchus masou rhodurus) divided habitat and did notcompete with Atlantic salmon. Among all life-stages, Japanese char and masu salmon(Oncorhynchus masou) attacked Atlantic salmon, and Atlantic salmon decreased in weight. Ourdata suggest that Atlantic salmon may not pose a competitive threat to Japan’s native salmonids.
Keywords: Atlantic salmon (Salmo salar), Japan, native salmonids, competition27

Annotated Bibliography of Key Works
Du SJ, Gong ZY, Fletcher GL, Shears MA, King MJ, Idler DR, Hew CL. 1992
Growthenhancement in transgenic Atlantic salmon by the use of an "all fish" chimeric growth hormone
gene construct. Biotechnology (N Y). 10(2):176-81.
We have developed an "all fish" growth hormone (GH) chimeric gene construct by using anantifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA
clone. After microinjection into fertilized, nonactivated Atlantic salmon eggs via the micropyle,transgenic Atlantic salmon were generated. The presence of the transgene was detected bypolymerase chain reaction (PCR) using specific oligonucleotide primers. A number of thesetransgenic fish showed dramatic increases in their growth rate. At one year old, the average increaseof the transgenic fish was 2 to 6 fold and the largest transgenic fish was 13 times that of the averagenon-transgenic control.
E. J. Deitch, G. L. Fletcher, L. H. Petersen, I. A. S. F. Costa, M. A. Shears, W. R. Driedzic and A.
K. Gamper 2006. Cardiorespiratory modifications, and limitations, in post-smolt growth hormone
transgenic Atlantic salmon Salmo salar. Journal of Experimental Biology 209, 1310-1325
In recent years, there has been a great deal of interest in how growth hormone (GH) transgenesis
affects fish physiology. However, the results of these studies are often difficult to interpretbecause the transgenic and non-transgenic fish had very different environmental/rearinghistories.
This study used a stable line of size-matched GH Atlantic salmon (Salmo salar) that were reared
in a shared tank with controls (at 10°C, for ~9·months) to perform a comprehensive examinationof the cardiorespiratory physiology of GH transgenic salmon, and serves as a novel test of thetheory of symmorphosis. The GH transgenic salmon had a 3.6 faster growth rate, and 21 and 25%
higher values for mass-specific routine and standard oxygen consumption (MO2), respectively.
However, there was no concurrent increase in their maximum MO2, which resulted in themhaving an 18% lower metabolic scope and a 9% reduction in critical swimming speed. Thisdecreased metabolic capacity/performance was surprising given that the transgenics had a 29%
larger heart with an 18% greater mass-specific maximum in situ cardiac output, a 14% greater
post-stress blood haemoglobin concentration, 5–10% higher red muscle and heart aerobic enzyme(citratesynthase or cytochrome oxidase) activities, and twofold
higher resting and 1.7 higher post-stress, catecholamine levels. However, gill surface area was the
only cardiorespiratory parameter that was not enhanced, and our data suggest that gill oxygen
transfer may have been limiting. Overall, this research: (1) shows that there are significant
metabolic costs associated with GH transgenesis in this line of Atlantic salmon; (2) provides thefirst direct evidence that cardiac function is enhanced by GH transgenesis; (3) shows that auniversal upregulation of post-smolt (adult) GH transgenic salmon cardiorespiratory physiology,
as suggested by symmorphosis, does not occur; and (4) supports the idea that whereas differencesin arterial oxygen transport (i.e.cardiac output and blood oxygen carrying capacity) are important
determinants of inter-specific differences in aerobicity, diffusion-limited processes must be
enhanced to achieve substantial intra-specific improvements in metabolic and swimming
performance.
John P. Volpe, Bradley R. Anholt, and Barry W. Glickman 2001 Competition among juvenile
Atlantic salmon (Salmo salar) and steelhead (Oncorhynchus mykiss): relevance to invasion
potential in British Columbia Can. J. Fish. Aquat. Sci. 58: 197–207
28

Atlantic salmon (Salmo salar) are routinely captured in both freshwater and marine environmentsof coastal British Columbia (Canada). Recent evidence suggests that this species is now naturally
reproducing in Vancouver Island Rivers. Our objective was to quantify the performance of eachspecies in intra- and inter-specific competition by assessing the competitive ability of Atlantic
salmon sympatric with native niche equivalent steelhead – rainbow trout (Oncorhynchus mykiss).
Significant behavioural differences, particularly with respect to agonism, were observed between
species; however, the status of an individual as resident or challenger was the best predictor of
performance. Resident fish always outperformed challengers, regardless of species. Thus, wesuggest that Atlantic salmon may be capable of colonizing and persisting in coastal British
Columbia river systems that are underutilized by native species, such as the steelhead.

29

O-13
Hybrid Striped Bass National Breeding Program: Research Towards Genetic
Improvement of a Non-Model Species
S. Adam Fuller
Harry K. Dupree Stuttgart National Aquaculture Research Center, US Department of Agriculture,
Agricultural Research Service, 2955 HWY 130 EAST, STUTTGART, AR, 72160*E-mail: adam.fuller.at.ars.usda.gov
The hybrid striped bass (HSB) farming industry at present relies almost totally on wild broodstock
for annual production of larvae and fingerlings, and industry efforts to domesticate the parent
species of the HSB (white bass WB Morone chrysops and striped bass SB M. saxatilis) have been
fairly limited in scope. At the USDA-ARS HKD Stuttgart National Aquaculture Research Center(SNARC), multiple areas of research are being pursued, with the end result being to provide HSB
producers with a better performing line of broodfish. Among the areas of research that are currently
being pursued at SNARC include: 1) the development of genomic resources for WB and SB; 2) the
molecular and physiological consequences of alternative production diets on HSB; 3) the molecularand physiological consequences of exposure to different production environments (hypoxic
environments, etc); 4) research to evaluate differential susceptibility of HSB and WB families tocolumnaris disease; 5) the effects of diet/parental contribution/kisspeptin injection on gonad
maturation, lipid composition, and fry and fingerling performance. An overview of these findings
will be discussed.

Annotated Bibliography of Key Works
Li C, Beck BH, Fuller SA, Peatman E (2014) Transcriptome annotation and SNP discovery in whitebass (Morone chrysops) and striped bass (Morone saxatilis). Animal Genetics 45: 885-887.
The authors present the first ever multi-tissue reference transcriptomes for striped bass (Morone
saxatilis) and white bass (Morone chrysops) which are the parental species of
the hybrid striped bass, a major U.S. aquaculture species. Being non-model species, this was ofcritical importance, as prior to this there only existed a medium-density genetic linkage map and awell-annotated ovarian transcriptome. The assembled Moronid reference transcriptomes andidentified simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) should
advance ongoing studies of reproduction, physiology, and immunology in these species andprovide markers for broodstock management and selection.
Childress CJ, Fuller SA, Rawles SD, Beck BH, Gaylord TG, Barrows FT, McEntire ME (2015)
Lysine supplementation of commercial fishmeal-free diet in hybrid striped bass Morone chrysops
x M. saxatilis affects expression of growth-related genes. Aquaculture Nutrition DOI:
10.1111/anu.12300
The authors present a follow-up study to a study (Rawles et al. 2013) where ideal protein theory
accurately predicted first-limiting amino acids and optimum lysine level for a fishmeal-free,commercial-grade diet for hybrid striped bass (HSB). In the current study, authors sought to30

determine how dietary lysine supplementation of these same diets influences the expression of two
genes, myostatin and myogenin, controlling myogenesis in differentially growing groups of HSB.
Real-time rt-PCR results in HSB suggest that the levels of lysine added to the diet has an impact
on myogenin relative to the unsupplemented diet, but no effect on myostatin. Moreover, presented
data also suggests that the amount of dietary lysine supplementation influenced the ratio of
myostatin/myogenin expression in HSB and that this pattern mimicked that of most of the growth,composition of growth and nutrient retention data from the authors’ previous study and may
therefore be a useful marker for selecting fish for improved growth performance.
Fuller SA, Farmer BD, Beck BH (2014) White bass Morone chrysops is less susceptible than its
hybrid to experimental infection with Flavobacterium columnare. Diseases of Aquatic Organisms
109:15-22.
The authors present research regarding Flavobacterium columnare, the causative agent ofcolumnaris disease, susceptibility differences between hybrid striped bass (HSB) and white bass
(WB) in a series of 3 fundamental studies. In the first experiment, the authors sought todetermine whether columnaris disease could be developed using a low-water flow experimental
challenge in HSB using 3 levels of F. columnare (60-, 30-, 10- ml). Each of these treatment groups
exhibited significantly different survival rates: 0, 3.3, and 13.3%, with higher survival occurring intreatment groups exposed to less bacteria. In the second experiment (30ml), both HSB and WB
had a 0% survival rate, but the WB took significantly longer to reach 100% mortality. Finally inExpt 3 (10ml), no HSB survived, whereas 33% of WB survived. Compared to controls, theauthors observed extensive gill damage in HSB treated with 10 ml after 24 h, which they
hypothesized could have contributed to the higher mortality observed in HSB; an observation notseen on the WB gills. From these series of experiments, it is clear that HSB are more sensitive to
F. columnare, having lower survival and more extensive histological damage compared to WB
following the bacterial challenge.
Beck BH, Fuller SA, Peatman E, McEntire ME, Darwish AM, Freeman DW (2012) Chronic
exogenous kisspeptin administration accelerates gonadal development in basses of the genus
Morone. Comparative Biochemistry and Physiology, Part A: Molecular & Integrative Physiology
162: 265-273.
The authors present the effects of chronic administration of kisspeptins to immature and mature
white bass (WB), striped bass (SB), and hybrid striped bass (HSB) to determine its effects on
gonadal development in these species. The authors determined that bi-weekly injections (over 7
weeks) differentially accelerate puberty, as evidenced by increases in the prevalence of
spermatozoa in the testes of juvenile fish. Also, in sexually mature fish, kisspeptin treatment led toincreased gonad weight, gonadosomatic index, and spermatocrit in some white and striped bass.
Additionally, mature white bass treated with kisspeptins showed an advancement in oocyte
development as determined by histological examination. Importantly, the gonadal changes
occurred in the absence of any photothermal manipulation or hormone injections. This description
was the first report of kisspeptin-mediated pubertal initiation in fish, and the first evidence thatkisspeptins could modulate gonad maturation.

31

O-14
Production of Benedenia-resistant Yellowtail (Seriola quinqueradiata) Families–
A Preliminary Approach to the Candidates–
Tsutomu Noda*1, Kazunori Yoshida1, Takurou Hotta1, Hirotaka Mizuochi1, Taro Chujo1,
Hideaki Aono2, Akiyuki Ozaki3, Kazuo Araki4, Mitsuru Yoshimoto5, Hiroki Ogawa6,
Yasuhiro Shima7 and Yuichiro Fujinami1
Goto Laboratory, Seikai National Fisheries Research Institute, FRA, Nunoura 122-7, Tamanoura,
Goto, Nagasaki 853-0508, Japan, 2Seikai National Fisheries Research Institute, FRA, 3National
Research Institute of Aquaculture, FRA, , 4Tamaki Laboratory, National Research Institute of
Aquaculture, FRA, 5Aquafarm, Maruha Nichiro Corporation, 6Shimaura Aquafarm Branch, Maruha
Nichiro Corporation, 7Minami-Izu Laboratory, National Research Institute of Aquaculture, FRA*E-mail: ttmnoda at affrc.go.jp
1

The skin fluke Benedenia seriolae is a parasite specific to Seriola species. Its feeding on theepidermal tissues of yellowtail (Seriola quinqueradiata) causes external injuries that render the fishsusceptible to bacterial and viral infection. Infection with this parasite is a serious problem foryellowtail aquaculture because it also inhibits fish growth. A previous study has reported theexistence of an inherited Benedenia disease–resistance factor in yellowtail. Yellowtail families
resistant to Benedenia disease have not yet been produced, although the production of such families
for aquaculture would help to reduce not only rates of infectious disease but also the costs and labor
required to eradicate Benedenia on the fish’s body surface. Here, we examined the relationshipbetween susceptibility and genetic variation that was described in the previous study in order toselect candidate broodstock for breeding Benedenia-resistant yellowtail. In September 2014, weselected 961 fingerlings among 10,000 wild-caught 0-age yellowtail at an aquaculture farm on thebasis of their parasite burdens (3 or fewer Benedenia on each selected fish: first selection treatment);
the number of parasites on each of the 10,000 fish ranged from 1 to 48. These 961 selected fish werethen cultured in a net cage and the number of parasites on each was counted five times between
November 2014 and July 2015. The average number of parasites per fish had a wide range from 0.2
to 39.4 over this period and the overall mean was 8.9. The 160 fish with the lowest parasite burdens
were selected as broodstock candidates (second selection treatment). We are now using thesebroodstock candidates to produce Benedenia-resistant F1 yellowtail families by using DNA
marker–assisted-selection breeding methods.
Keywords: Yellowtail, Seriola quinqueradiata, Benedenia disease, Breeding

Annotated Bibliography of Key Works
Akiyuki Ozaki, Kazunori Yoshida, Kanako Fuji, Satoshi Kubota, Wataru Kai, Jun-ya Aoki, Yumi
Kawabata, Junpei Suzuki, Kazuki Akita, Takashi Koyama, Masahiro Nakagawa, Takurou Hotta,
Tatsuo Tsuzaki, Nobuaki Okamoto, Kazuo Araki, Takashi Sakamoto. 2013. Quantitative Trait Loci
(QTL) Associated with Resistance to a Monogenean Parasite (Benedenia seriolae) in Yellowtail(Seriola quinqueradiata) through Genome Wide Analysis. PLoS ONE 8(6): e64987.
32doi:10.1371/journal.pone.0064987
Benedenia infections caused by the monogenean fluke ectoparasite Benedenia seriolae seriously
impact marine finfish aquaculture. Genetic variation in host has been inferred to play a significant
role in determining the susceptibility to this parasitic disease. To evaluate the genetic basis of
Benedenia disease resistance in yellowtail (Seriola quinqueradiata), a genome-wide andchromosome-wide linkage analyses were initiated using F1 yellowtail families (n = 90 per family)
based on a high density linkage map with 860 microsatellite and 142 single nucleotide
polymorphism (SNP) markers. Two major quantitative trait loci (QTL) regions on linkage groups
Squ2 (BDR-1) and Squ20 (BDR-2) were identified. These QTL regions explained 32.9–35.5% of
the phenotypic variance. On the other hand, the relationship between QTL for susceptibility to B.
seriolae and QTL for fish body size were investigated. The QTL related to growth was found on
another linkage group (Squ7). As a result, the authors present first genetic evidence that
contributes to detailing phenotypic resistance to Benedenia disease, and the results will help
resolve the mechanism of resistance to this important parasitic infection of yellowtail.
Kazunori Yoshida, Akiyuki Ozaki, Masahiro Nakagawa, Takurou Hotta, Jun-ya Aoki, Takashi
Koyama, Kazuo Araki, Nobuaki Okamoto, Takashi Sakamoto and Tatsuo Tsuzaki. 2011.
Characteristic Evaluation Method Relating to Benedenia Disease of Yellowtail (Seriola
quinqueradiata). Proceedings of the forty U.S.-Japan aquaculture panel symposium (eds M. Rust,
P. Olin, A. Bagwill and M. Fujitani): Pp96-99.
Benedenia is a parasitic disease caused in Seriola species by Benedenia seriolae. This parasite cancause growth reduction and external injuries in yellowtail, increasing the risk of secondary viral orbacterial infection. The main method of parasite removal is to soak the fish in a freshwater bath.
However, this method requires a great deal of time, cost, and effort. We have been studying
DNA Marker-Assisted Selection (MAS) breeding, to select for resistance to Benedenia disease.
Three components ("Reproduction technology", "Character evaluation", and "DNA analysis") are
critically important to promote MAS breeding success. We focus on one of the key components,"Characteristic evaluation method” relating to Benedenia disease in yellowtail.
Mushiake, K., H. Yamazaki, and H. Fujimoto. 2005. Current Situation of Technical Developments
in Seed Production of Yellowtail (Seriola quinqueradiata) in Japan. Proceedings of the thirty-fourth
U.S.-Japan aquaculture panel symposium (eds R. Stickney, R. Iwamoto, and M. Rust): Pp1-4.
The National Center for Stock Enhancement (NCSE, formerly Japan sea-farming Association), of
the FRA, introduced the stock enhancement program for yellowtail (Seriola quinqueradiata andSeriola lalandi) in 1977. Technical developments in induced spawning as well as larval andjuvenile rearing techniques have increased the population of this species to 1 million juveniles per
year at NCSE. This project faced three major drawbacks: high mortality of larvae, cannibalism,
and the smaller size of released juveniles in comparison with their wild counterparts. The highmortality of larvae was overcome by utilizing strong aeration during the early larval stage, whilecannibalism was controlled by grading juveniles by size selection. The two-month delay in the
spawning season of reared broodstock (the usual spawning season is late April to early May),
which caused the smaller size of released juveniles, was solved by developments in advanced
spawning techniques. Photoperiod and water temperature manipulations were used to produce
eggs in February, thus producing yellowtail juveniles that can be released into the wild at a size
similar to that of the wild stock.
33

O-15
Coho Salmon Broodstock Development: A Case Study of the Domsea Coho
(1977 to 2015)
Robert N. Iwamoto1, Richard Towner2, James Myers3, and Greg Hudson1
1

Spring Salmon LP, Rochester, WA, 2 Genentech Consultants, Payette, ID., 3Northwest Fisheries
Science Center, Seattle, WA 98112*E-mail:
There are few long-term studies on Pacific salmon that inform on the potential for gains bysystematic selective breeding and the potential for inbreeding losses because of constrained
population sizes and matings of closely related individuals. In 1977, Domsea Farms Inc., theUniversity of Washington, and the Washington Sea Grant Program initiated a genetic selection
program for coho salmon for Domsea’s marine net-pen operations. Because little was known atthat time regarding the genetic potential for genetic improvement in coho salmon, the program wasinitially designed with two central goals: 1) collect basic information (heritabilities, genetic andphenotypic correlations) on the potential for genetic improvement for such economically important
traits such as smoltification, growth rate to harvest, flesh color, and reproductive fitness; and 2)
using that information, develop selection and mating protocols that would maximize selection gains
but minimize inbreeding. Despite significant changes in ownership, rearing environment andoperations, the program has remained remarkably consistent over the past 38 years or 19 generationsof selection. We have demonstrated that selection for improved growth to the smolt stage (7.113.1% per generation) and adult phases (43-53 g per generation) can be achieved. Overall, thegrowth rate of the Domsea coho salmon has improved between 3% and 8% per generation while
reproductive traits such as female weight, egg weight, and survival to ponding have remained
unaffected by inbreeding. The program has also demonstrated the importance of considering thepotential for genotype-environment interactions when designing selection programs for specificrearing applications. While traditional genetic approaches have been demonstrably successful forthis program, it is anticipated that further consideration and application of molecular approaches
will help further characterize and advance this broodstock program for coho salmon.

Annotated Bibliography of Key Works
1. a. Iwamoto, R.N., Saxton, A.M., Hershberger, W.K. 1982. Genetic estimates for length andweight of coho salmon (Oncorhynchus kisutch) during freshwater rearing. J. Hered. 73: 187-191.
b. Saxton, A.M., Hershberger, W.K., Iwamoto, R.N. 1984. Smoltification in the net-pen
culture of coho salmon: quantitative genetic analysis. Trans. Am. Fish. Soc. 113: 339-347.
c. Hershberger, W.K., Myers, J.M., Iwamoto, R.N., McAuley, W.C., Saxton, A.M. 1990.
Genetic changes in the growth of coho salmon (Oncorhynchus kisutch) in marine net-pens,produced by ten years of selection. Aquaculture 85: 187-197.
d. Iwamoto, R.N., Myers, J.M., and Hershberger, W.K. 1990. Heritability and genetic
correlations for flesh coloration in pen-reared coho salmon. Aquaculture 86: 181-190.

34

The papers cited above provide much of the early quantitative genetic parameters for coho salmon
traits. Genetic estimates for freshwater growth, smoltification and initial saltwater survival,
seawater growth, and flesh coloration for the Domsea coho salmon were derived from full- andhalf-sib analyses. In almost every case, the magnitude of the derived heritabilities and genetic
correlations indicated that a systematic selection program would be successful.

2. Myers, J.M., Iwamoto, R.N., Teel, D., Van Doornink, D., and Hershberger, W.K. 1999.
Coho salmon broodstock developmet---1977-1998. Ten generations of systematic selective
breeding. Bull. Natl. Res. Inst. Aquacult Suppl. 1, 63-70.
The authors summarize the concept of the Domsea coho salmon broodstock program from theselection sheme to the circular mating design. They also present allozyme comparisons of thefounder strain and the broodstock after 10 generations of selection and indicate that significant
genetic changes had occurred in the broodstock as a result of genetic selection and drift.
3. Myers, J.M., Heggelund, P.O., Hudson, G. and Iwamoto, R.N. 2001. Genetics andbroodstock management of coho salmon. In: Lee, Cheng-Sheng and Donaldson, E.M. (Eds.)
Reproductive biotechnology in finfish aquaculture, Aquaculture 197:43-62.
This paper summarized the information collected on the Domsea coho salmon broodstock
program after 13 generations of implementation. It discusses the results of sib trials in marine
net pens and two freshwater environments and consequently the importance of considering
genotype-environmnent interactions in broodstock programs for specific applications. It tracked
theoretical (pedigree data) and genetic changes (allozyme data and individual traits) in thebroodstock population.

35

O-16
Development of Improved Catfish Germplasm at the Warmwater Aquaculture
Research Unit, USDA-ARS.
Brian Bosworth* and Geoff Waldbieser
Warmwater Aquaculture Research Unit, USDA-ARS, P.O. Box 38, Stonville, MS 38776.
*E-mail: brian.bosworth.at.ars.usda.gov
Production of farm-raised catfish is the largest aquaculture enterprise in the U.S. in terms of bothvolume and value. The Warmwater Aquaculture Research Unit (WARU), USDA-ARS mission is
to improve production efficiency of catfish and other warmwater aquatic organisms, and includes
development and release of catfish germplasm improved for economically important traits.
Traditionally, the channel catfish comprised nearly all U.S. farm-raised catfish production, but inthe last 10 years production of the F1 hybrid between the channel and blue catfish has increased toabout 50% of total production. Therefore, the WARU breeding program is focused on genetic
improvement of purebred channel catfish and F1 hybrid catfish performance. The channel catfish
improvement program has used a traditional selection on BLUP breeding value estimates to improve
growth rate and carcass yield. We started with a diverse population of channel catfish derived
from 10 commercial farms in 2006, and have evaluated over 15,000 animals from 630 full-sib
families produced by 334 sires and 516 dams between 2010 and 2013. Heritabilities for growthand carcass yield are 0.33 and 0.36, respectively; and fish are selected based on an index that puts
equal weight of individual breeding values for growth and family average breeding values forcarcass yield. Improvements in growth have been 8-10% per generation and are in agreement withexpected improvements. Increases in carcass yield, although positive, have not been as dramatic,
due at least in part to the use of less accurate family average breeding values instead of individual
breeding values. The hybrid improvement program has focused on evaluation, identification andselection of blue catfish sires that produce superior performing hybrid progeny. Past work on
genetic improvement of blue catfish is very limited; therefore, we initiated a program to collectseveral blue catfish populations and evaluate performance of their purebred and hybrid progeny.
Initial evaluations suggest the majority of genetic variance for growth and carcass yield in hybrid
progeny is additive in nature, and that populations and individual sires that produce purebred
progeny with superior growth and carcass yield also produce hybrid progeny with superior
performance. We have ongoing experiments to estimate the genetic correlation between traits inthe purebred and hybrid state, which will give us direction in developing purebred blue catfish which
produce superior hybrid progeny. We currently use DNA markers to identify parentage in ourpedigreed populations and are developing a SNP chip to be used for genomic selection. Use of
genomic breeding values should improve our breeding value accuracy and rate of improvement,
particularly for carcass yield. We are evaluating other traits for inclusion in our selection index,
and we are collecting and cryopreserving sperm from superior channel and blue catfish sires for
future use. This combination of traditional animal breeding, genomic selection and cryopreservation
will result in improved catfish germplasm, improved production efficiency and greater profitability
for catfish farmers.

36

Annotated Bibliography of Key Works
Lush, J.L. 1947. Family Merit and Individual Merit as Bases for Selection. Part I. The American
Naturalist 81:241-261 and 81:362-379.
No abstract available. These 2 papers detail Lush’s development of the use of information on
individual performance and performance of relatives (family merit) to improve accuracy ofbreeding value estimates and form the basis for development of selection index theory anddevelopment of BLUP based breeding programs.
Henderson, CR. 1975. Use of all relatives in intraherd prediction of breeding values and producing
abilities. Journal of Dairy Science 58:1910–1916.
Commonly used cow evaluation methods apply principles of the selection index to herdmate
deviations on the cow and close relatives. In contrast, best linear unbiased prediction adjusts
records by best linear unbiased estimates of all fixed effects in the model and simultaneously
weights those adjusted records by selection index principles. It would be advantageous to utilize
all known relationships among animals in the herd in the latter method, but computations havebeen too laborious, requiring the inverse of the numerator relationship matrix. By a method of
writing this inverse rapidly without computing the relationship matrix itself all relationships can
now be used in intraherd cow evaluation. Further, tests of progeny by artificial insemination onsires used in the herd can be incorporated.
Wei, M., van der Werf, J.H.J., 1994. Maximizing genetic response in crossbreds using
purebred and crossbred information. Animal Production 59:401-413.
A combined crossbred and purebred selection (CCPS) method, i.e. using crossbred and purebred
information, was proposed to achieve genetic response in crossbred animals. Selection index
theory was applied to establish a CCPS index. The CCPS was compared with pure-line selection(PLS) and crossbred selection (CS) methods. The genetic correlation between purebred andcrossbred performance (rpc) and crossbred heritability (hc2) are crucial factors in the comparison.
The CCPS is always better than PLS or CS when a fixed number of purebred progeny is tested.
With a fixed total number of purebred and crossbred tested progeny, CCPS is only worse than PLS
for very high values of rpc (>0·8). Superiority of CCPS over PLS increases and over CS decreases
with decreasing rpc. The larger hc2 is, relative to purebred heritability (hc2 the more response CS
and CCPS will achieve. The robustness of CCPS against inappropriate assumptions on rpc andhc2 values was investigated. The expected response is always an overestimate, and the actual
response is smaller than the optimal response when rpc is assumed one but the true rpcis smaller.
The difference between actual and optimal response increases as rpc decreases but it is small forlarge rpc values (e.g. <3% for rpc >0·7). The expected response is smaller than the actual responsewhen rpc is large and hc2> hp2 Finally, the actual response to CCPS is larger than the optimal
response to PLS for positive values for rpc. The main conclusions are: (1) CCPS method is optimalfor obtaining genetic response in crossbreds; and (2) CCPS with inappropriate assumptions on
rpc and hc2 values (e.g. recognizing crossbreds as purebreds) achieves more genetic response than
PLS for common values of rpc and crossbred heritability.
Meuwissen, T.H.E., Hayes, B.J., Goddard, M.E. 2001. Prediction of total genetic value using
genome-wide dense marker maps. Genetics 157:1819-1829.

37

Recent advances in molecular genetic techniques will make dense marker maps available andgenotyping many individuals for these markers feasible. Here we attempted to estimate the effectsof z50,000 marker haplotypes simultaneously from a limited number of phenotypic records. A
genome of 1000 cM was simulated with a marker spacing of 1 cM. The markers surrounding
every 1-cM region were combined into marker haplotypes. Due to finite population size (Ne 5100), the marker haplotypes were in linkage disequilibrium with the QTL located between themarkers. Using least squares, all haplotype effects could not be estimated simultaneously. When
only the biggest effects were included, they were overestimated and the accuracy of predicting
genetic values of the offspring of the recorded animals was only 0.32. Best linear unbiased
prediction of haplotype effects assumed equal variances associated to each 1-cM chromosomal
segment, which yielded an accuracy of 0.73, although this assumption was far from true. Bayesian
methods that assumed a prior distribution of the variance associated with each chromosome
segment increased this accuracy to 0.85, even when the prior was not correct. It was concluded
that selection on genetic values predicted from markers could substantially increase the rate ofgenetic gain in animals and plants, especially if combined with reproductive techniques to shorten
the generation interval.
Legarra, A. O.F. Christensen, O.F., Aguilar, I. Misztal, I. 2014. Single Step, a general approach for
genomic selection. Livestock Production Science 166:54–65.
Genomic evaluation methods assume that the reference population is genotyped and phenotyped.
This is most often false and the generation of pseudo-phenotypes is uncertain and inaccurate.
However, markers obey transmission rules and therefore the covariances of marker genotypes
across individuals can be modelled using pedigree relationships. Based on this, an extension of the
genomic relationship matrix can be constructed in which genomic relationships are propagated to
all individuals, resulting in a combined relationship matrix, which can be used in a BLUP
procedure called the Single Step Genomic BLUP. This procedure provides so far the mostcomprehensive option for genomic evaluation. Several extensions, options and details aredescribed: compatibility of genomic and pedigree relationships, Bayesian regressions, multiple
trait models, computational aspects, etc. Many details scattered through a series of papers are put
together into this paper.

38

Titles of Poster Presentation
1. Whole genome re-sequencing of fugu populations (Sho Hosoya, Fisheries Laboratory, University ofTokyo)
2. Incipient transition of a sex-determining gene among closely related species of fugu.(Kiyoshi Kikuchi,

Fisheries Laboratory, University of Tokyo)
3. Estimation of breeding value in model fish, guppy (Poecilia reticulata) and its application for selective
breeding in aquaculture. (Masamichi Nakajima, Tohoku University)
4. The life table demography and population growth of the rotifer Brachionus angularis Gosse, from Kenya;
the influence of temperature and food density. (Erick Ochieng Ogello, Nagasaki University)
5. Effect of dissolved organic matter on electrochemical removal of ammonia in recirculating aquaculture
systems. (Satoshi Tada, Nagasaki University)
6. Effect of tetrodotoxin-containing diet on feeding, digestion and growth of tiger puffer, Takifugu rubripes
juveniles. (Kogen Okita, Nagasaki University)
7. Temperature tolerance in two clonal strains of mangrove killifish Kryptolebias marmoratus. (Marina

Yamada, Nagasaki University)
8. Effects of protozoa Euplotes sp. coexistence on the population growth of minute monogonont rotifer
Proales similis. (Naoshi Wakimura, Nagasaki University)
9. Distribution of larval and juvenile greater amberjack (Seriola dumerili) around the Penghu islands, Taiwan.
(Takamasa Hasegawa, Nagasaki University)
10. Effect of starvation on mixis induction in offspring and its genetic mechanism of the monogonont rotifer
Brachionus manjavacas. (Shohei Kamizono, Nagasaki University)
11. Body siza, Culture and fish larval ingestion on a minute rotifer, Colurella cf. adriatica. (Stenly Wullur,

Faculty of Fisheries and Marine Science, Sam Ratulangi University, Manado – Indonesia)
12. Production of myostatin gene-knockout Japanese anchovies (Eugraulis Japonicus) using TALEN-based
genome editing. (Keishi Sakaguchi, Fisheries Research Institute of Karatsu, Department of Joint

Research, Faculity of Agriculture, Kyushu University)
13. Isolation and Screening of Novel Probiotic Lactic Acid Bacteria for Aquaculture. (Nguyen Thi Hue

Linh, University of Miyazaki)
14. Effect of Protease Addition to EP diet on the Growth of amberjack, Seriola dumerili. (Yousuke Taoka,

University of Miyazaki)
15. Amino Acid Profile of Thraustochytrids Cells and Potential of Application to Aquafeed. (Kenya Horii,

University of Miyazaki)
16. Diurnal changes in frequency of the burst swimming behavior of adult Pacific bluefin tuna (Thunnus
orientalis) in a land-based tank. (Akiko Tsujita, Seikai National Fisheries Research Institute, FRA)
17. Spawning frequency of Pacific bluefin tuna Thunnus orientalis in a land-based tank. (Ayako Suzuki,

Seikai National Fisheries Research Institute, FRA)
18. Effect of timing of restricted feeding on sexual maturation in the yellowtail, Seriola quinqueradiata.

(Kentaro Higuchi, Seikai National Fisheries Research Institute, FRA)
19. Nitrogen excretion in Pacific bluefin tuna. (Toshinori Takashi, Seikai National Fisheries ResearchInstitute, FRA)
20. A high density genetic linkage map for yellowtail (Seriola quinqueradiata) containing 6,275 ESTbased SNPs. (Akiyuki Ozaki, National Research Institute of Aquaculture, FRA)

39

P-1
Whole genome re-sequencing of fugu populations
Sho Hosoya*, Satoshi Tasumi, Kiyoshi Kikuchi
Fisheries Laboratory, University of Tokyo, Hamamatsu, Bentenjim 2971-4 Japan*E-mail: ahosoya.at.mail.ecc.u-tokyo.ac.jp
Historically, the prevalence of intraspecific diversity, both neutral and adaptive, in many marine
species has been unclear. However, the recent advent of new technologies, in particular, highthroughput DNA sequencing is making it feasible to evaluate genetic variability at the wholegenome level. Fugu (tiger pufferfish Takifugu rubripes) is one of the important cultured marine
fish species in East Asia. The yield of cultured fugu accounted for approximately 5 % of total marine
fish aquaculture in Japan, and ranked 3rd-4th by value. In addition, this species is becoming animportant target for stock enhancement because of severe declines in the wild populations. In recent
years, approximately two million hatchery-born seedlings have been released each year. Tagging
and releasing experiments have suggested that fugu shows homing behavior to the natal site forspawning following long-range migration, and by extension, that distinct local populations may
exist. As expected, recent study using 21 microsatellite loci revealed a shallow divergence between
two wild populations from Japan Sea and Pacific Ocean. Nonetheless, the detailed degrees of
population and adaptive structuring remain unclear. Moreover there is virtually no information
regarding the extent and magnitude of linkage disequilibrium (LD) that play essential roles inchoosing marker loci for the management of brood stocks and conservation of wild populations.
In this study, we resequenced wild individuals of fugu from Wakasa Bay (Japan Sea) and Mikawa
Bay (Pacific Ocean) to compare the genetic diversity within and between populations. Two libraries
each containing ten individuals from either of the populations were constructed for paired-end
sequencing (2 x 101bp) on the Illumina Hiseq2000. We obtained 43.2M reads per sample yielding
coverage of 11.4 per genome, on average. We mapped these reads on the fugu reference genome(Fugu v.5) and called single-nucleotide polymorphisms (SNPs) using BWA, Samtools and GATK
software. The number of SNPs detected per individual was about 700 thousand and the SNP
frequency was about 480bp per SNP. Missing SNPs because of the shallower depth were estimatedas 2 to 2.5 per cent of the total SNPs for each sample. Multidimensional scaling plot clearly
separated the two populations, and individuals from Ise Bay were genetically closer than those from
Wakasa Bay. However, the global Fst (= 0.0057) was small and no outlier locus was detected by
BayeScan software. These results suggest that the genetic divergence between the two populations
is shallow. Linkage disequilibrium analysis was done using PLINK software. The two populations
were similar in the LD state. We detected putative 3,000 LD blocks from each population but 90 per
cent of them were smaller than 1kb. The mean r2 value was 0.48 between two SNPs at 100bp
distance whilst that was less than 0.20 at 1kb distance. This rapid LD decay indicates these
populations have maintained at relatively healthy states until recently. The expected SNP size forthe implementation of genomic selection program for fugu breeding was 300k.
Key words: fugu, Takifugu rubripes, conservation, genetic diversity, single nucleotide
polymorphism, Fst, linkage disequilibrium, genomic selection40

Annotated Bibliography of Key Works
Katamachi D., Ikeda M., Sato T., Suzuki S., Kikuchi K and Ojima D. 2014. Development of amultiplex PCR assay for population genetic analysis of the tiger puffer Takifugu rubripes using 16
microsatellite DNA loci. Aquaculture Sci., 62, 55－63
The tiger puffer Takifugu rubripes is a marine fish species economically important to East Asia,
particularly Japan. To evaluate the genetic variability and population structure of the tiger puffer indetail, we generated a multiplex PCR assay of microsatellite DNA loci, a fast and cost-effective
technique that allows high-throughput genotyping. In this study, we report the development offour multiplex PCR assays for this species using 16 microsatellite DNA loci located on
independent chromosomes. We ensured quality control throughout all steps of the multiplex PCR
assay development, i.e., exclusion of loci detected with stuttering, allele dropouts, or null alleles.
We evaluated this set of microsatellite DNA loci for polymorphisms using 113 fishes collectedfrom three different locations in the sea around Japan. This combination of loci will prove usefulfor future investigations of the fine-scale population genetic structure of this species.

41

P-2
Incipient transition of a sex-determining gene among closely related species of
fugu
Risa Ieda1, Sho Hosoya1, Satoshi Tasumi1, Shigenori Suzuki2, Kiyoshi Kikuchi1*
1Fisheries Laboratory, University of Tokyo, Hamamatsu, Bentenjim 2971-4 Japan
Minami-Izu Laboratory, National Research Institute of Aquaculture, FRA, Shizuoka, Minami-izu
415-0156 Japan*E-mail: akikuchi.at.mail.ecc.u-tokyo.ac.jp
2

Sex determination in teleosts fish is often genetic, where segregation of sex-determining loci assign
the phenotypic sex. In these species, sex-linked polymorphic markers can be used for sex
identification, potentially providing important information regarding the management of cultured
species and conservation of wild species. However, the different master sex-determining (SD) genes
in different fish lineages appear to have evolved independently and have been frequently replacedby new genes. Therefore, the sex-linked markers are often specific to a few stock population of the
focal species, greatly limiting use of such markers for the management of aquaculture species. To
gain more insights into the transition between SD systems in teleosts, we study closely-related
species of fugu belonging to genus Takifugu. This genus has undergone an adaptive radiation in thelast 2-5 million years, resulting in about 20 extant species including fugu (tiger pufferfish). Fugu is
one of the most economically important food fish in Japan and also is the first fish with a fully
sequenced genome. Previously, we have shown that sex in fugu appears to be determined by amissense single nucleotide polymorphism (SNP) in the Amhr2 gene. In this study, we have taken
advantage of this finding and the rich genomic resources of fugu to explore the genetic basis of sex
determination in closely-related species of fugu. We found that while sex in the majority of Takifugu
species is likely determined by the SNP in the Amhr2 gene, it is clearly not the case in a few species.
To confirm this, we performed genome-wide linkage analysis and identified novel SD loci distinct
from the Amhr2 locus in these species. Interestingly, the transition of the SD system appears to be
in progress at least in one species, as a small percentage of males still retains the “sex-determining
SNP” on the Amhr2 gene. These results indicate that fugu and its closely-related species can be an
excellent model group for investigating the transitions between alternative master SD genes.

42

P-3
Estimation of breeding value in model fish, guppy (Poecilia reticulata) and its
application for selective breeding in aquaculture
Masamichi Nakajima1*, Toyoko Nakajima1, Syunsuke Obinata1
1

Laboratory of Marin Life Science and Genetics, Graduate School of Agricultural Science,
Tohoku University, Sendai, Miyagi 981-8555, Japan*E-mail: mnkjm at bios.tohoku.ac.jp
Selective breeding is one of the most important methods for the genetic improvement in not only
livestock animals, but also aquatic organisms.
Many varieties and strains were produced inaquatic organisms; however the most succeeded example of selective breeding in aquatic organisms
is ornamental fish. In the case of quantitative traits, the example of succeeded selective breedingis scare. In the case of the selective breeding based on phenotypic value, many cases showed
inconsistent result. This is due to its inability to remove environmental and dominance effect.
Therefore the method to evaluate the accurate abilities of parent is necessary. The breeding value
was contrived from such circumstances. Though the breeding value possesses such importance,
the applications of the breeding value in the aquaculture are not so much. The reasons why theapplication in aquaculture is little are 1) the large number of offspring can obtain from small numberof parental fish; 2) short life cycle of target species caused difficulties to estimate breeding value inparental fish and their offspring. Although, it is hard to obtain the breeding value, the role ofbreeding value is very important for the efficient selection. From the above mentioned reason, it
is necessary to identify the breeding value for the effective selective breeding in aquaculture. In
this study, the breeding values were estimated from selection experiment for body size in the guppy,
Poecilia reticulata, and examined the efficiency of the selection between used phenotype andbreeding value. Comparison of breeding value in parental and offspring indicated significantly
positive correlation. Positive correlation also observed between breeding values of parental andbody size of their offspring. Selection based on breeding value showed 5% larger in body size
compared with selection using phenotype. Selection based on breeding value showed lesser
increment in coefficient of inbreeding compared to the selection based on phenotype. These
results suggest that the breeding values are effective to the evaluating parental fish and useful for
selective breeding.
It is expected that the application of breeding value in the industrially
important fish, such as Bluefin tuna and Japanese eel.
Keywords: Guppy（Poecilia reticulata）; Selective breeding ; Body size ; Breeding value

43

P-4
The life table demography and population growth of the rotifer Brachionus
angularis Gosse, from Kenya; the influence of temperature and food density
Erick Ochieng Ogello*1, 2, Hee-Jin Kim1, Koushirou Suga1 and Atsushi Hagiwara1
1

Graduate School of Fisheries & Environmental Sciences, Nagasaki University, 1-14 Bunkyo,
Nagasaki 852-8521, Japan, 2Kenya Marine & Fisheries Research Institute (KMFRI), Kegati
Aquaculture Research Station, P.O. Box 3259 - 4200 Kisii, Kenya*E-mail: bb53414801.at.cc.nagasaki-u.ac.jp
The nature of reproduction of the Kenyan strain of Brachionus angularis was investigated using
individual and small batch culture approaches. First, the Kenyan rotifer was identified usingmorphological and molecular techniques. The life-table demography and the population growthstudies were conducted at three temperatures (i.e. 20, 25 and 30°C) using Chlorella vulgaris at threedensities (i.e. 2.5x105, 2.5x106 and 2.5x107 cells mL-1). The lorica length (85.6 ± 3.1 µm) and width(75.4 ± 3.6 µm) of the Kenyan sample were smaller than those of similar species cited in theliterature. The phylogenetic tree grouped the Kenyan sample together with Brachionus caudatus
and Brachionus angularis. However, with additional morphological data e.g. presence of two
median occipital spines with either reduced or lacking sub-median spines, Brachionus angularis
was identified as the most likely match for the Kenyan sample. The rotifers were most fecund (2.11± 0.07 offspring female- 1 day -1) and reproductive (8.43 ± 0.24 offspring female -1) at 25oC with
2.5×106 cells mL-1 of C. vulgaris. The highest intrinsic rate of natural increase (0.74 ± 0.02 d-1),
specific population growth rate (0.49 ± 0.01), longest life expectancy at hatching (12.41 ± 0.28 d)and shortest generation time (2.87 ± 0.03 d) were also observed at 25oC with 2.5×106 cells mL-1 of
C. vulgaris. However, the duration of hatching to first egg spawning was shortest (2.86 ± 0.21 h) at
30oC with 2.5×107 and longest (8.83 ± 0.39 h) at 20oC with 2.5×105 cells mL-1 of C. vulgaris. In thebatch cultures, the highest population density (255.7 ± 12.6 ind mL–1) and lowest (122.0 ± 3.6 indmL–1) were realized at 25oC with 2.5×106 and at 20oC with 2.5×105 cells mL-1 of C. vulgaris on day
8 respectively. There was earlier population density peaks at higher food densities (2.5×107 cells
mL-1 of C. vulgaris) regardless of temperature. In conclusion, the Kenyan strain of B. angularis
seems to have favorable morphological and reproductive features making them suitable foraquaculture activities. The life table demography of this strain is optimal at 25oC with 2.5×106 cells
mL-1 of C. vulgaris. The results of this study are relevant for improvement of the freshwateraquaculture activities. Further studies on the population growth of the rotifer are recommended
using other different food types.
Key words: Rotifera, alga, life table parameters, fecundity, Brachionus angularis.

44

P-5
Effect of dissolved organic matter on electrochemical removal of ammonia inrecirculating aquaculture systems
Satoshi Tada* and Shigenobu Takeda
Graduate School of Fisheries and Environmental Sciences, Nagasaki University*E-mail: tdsts.goopy.at.gmail.com
Improvement of water treatment systems in closed recirculating aquaculture systems (RAS) is
necessary for reducing total volume of culture water, which determine the size of the whole system
as well as running costs. New RAS using electrochemical oxidation for removal of ammonia inculture seawater are being developed by Nagasaki Prefectural Institute of Fisheries. In this system,
hypochlorous acid, which produced by electrolysis of seawater, oxidizes the bromide ions, yielding
hypobromous acid. Both hypochlorous and hypobromous acids react with ammonium ion andoxidize it to nitrogen gas. Since these free chlorine and free bromine also react with dissolved
organic matter (DOM), efficiency of electrochemical ammonia removal could be affected byquantity and reactivity of DOM in culture seawater. In this study, removal of total ammonia
nitrogen (TAN) and DOM by additions of hypochlorous acid was investigated using culture
seawater of two RAS for tiger puffer Takifugu rubripes (RAS-1) and kelp grouper Epinephelus
bruneus (RAS-2). The RAS-1 comprised of a culture tank (20 m3), a solid settler, a circulation pump,a foam fractionator, an electrolysis unit, a reaction tank (for ammonia removal), and activatedcharcoal tank (for removal of residual chlorine). The RAS-2 has a biofiltration tank in addition to
the units provided in the RAS-1. The culture seawater was filtered through Whatman GF/F filterand dispensed into seventeen replicate 250-ml amber glass bottles. Additions of chlorine (Sodium
hypochlorite solution) were conducted to achieve 17 steps chlorine doses of 0~16 (or 70) mg/L as
Cl2. After 20 min contact period at 25ºC, free available residual chlorine and combined available
residual chlorine in the sample were analyzed by the DPD method. TAN was measured using an
autoanalyzer. DOM was determined as humic-like and protein-like fluorophores based on the threedimensional excitation emission matrix spectroscopy. Concentration of TAN in the culture
seawater consistently decreased along with the increase in chlorine dose and then disappeared (whatis called breakpoint). In the bottles with higher levels of chlorine dose, free available residual
chlorine was detected according to the excess amount of chlorine dose. Concentrations of combined
available residual chlorine were very low but small increase was observed around the breakpoint.
Humic-like and protein-like fluorophores showed large decrease by the addition of 1 mg/L Cl2 (thelowest chlorine dose) and then kept relatively constant concentrations until the breakpoint. In thebottled added with excess amount of chlorine (higher than the breakpoint), further decreases inhumic-like and protein-like fluorophores were observed. These results suggest that DOM consistsof highly reactive fraction and semi-labile fraction, and the former could reduce efficiency ofelectrochemical ammonia removal in the culture seawater. Compare to the chlorine demand toachieve the breakpoint in DOM-free artificial seawater, the culture seawater from RAS-2 required
17~42% more chlorine to oxidize TAN, but the difference between the artificial seawater and RAS1 culture seawater was not clear. Skin mucus of kelp grouper seems to be one of the sources ofhighly reactive DOM in RAS-2.
45

Key words: Recirculating aquaculture system, electrochemical oxidation, ammonia, dissolved
organic matter, seawater
Annotated Bibliography of Key Works
Ohwaki H., S. Yamamoto, A. Okamoto and Y. Kurokawa. 2011. Development of a large-scaleclosed recirculating aquaculture system for saltwater fish. Report of Industrial Technology
Center of Nagasaki, 40, 52-55 (in Japanese)
For the constraction of a large-scale closed recirculating system for aquaculture of saltwater fishon land, we have been developing a new seawater treatment unit using hypochlorous acidproduced by electrolysis of seawater with platinum modified titanium electrode, and a closed
recirculating aquaculture system with the electrochemical water treatment. Acidic water, which is
a byproduct of electrolysis of seawater, can be used for the removal of dissolved CO2 that
accumulated in the culture water. Our computer simulation model could reproduce the flow
pattern in the electrolysis unit and the estimated pH value of the out-flow water agreed well withthe observation. We found that dissolved CO2 in the culture water can be easily removed bybubbling of the acidic water.

46

P-6
Effect of tetrodotoxin-containing diet on feeding, digestion andgrowth of tiger puffer Takifugu ruburipes juveniles
Kogen Okita1*, Tomohiro Takatani1, Osamu Arakawa1,
Atsushi Hagiwara1, Yoshitaka Sakakura1
1

Graduate School of Fisheries and Environmental Sciences, Nagasaki University*E-mail: jj20140049 at cc.nagasaki-u.ac.jp
Marine pufferfish contain tetrodotoxin (TTX), an extremely potent neurotoxin. We recently
clarified that a tiger puffer Takifugu rubripes juvenile detects TTX by olfactory organ, and actively
ingests and accumulates TTX not only in liver and skin but also in central nervous system. We
further revealed that gene expression of some appetite peptides in the brain of hatchery-reared nontoxic fish changed by TTX-sensing and TTX-administration. In the present study, feeding trial
using non-toxic and toxic diets was conducted with hatchery-reared T. rubripes juveniles in order
to examine the relation between the appetite and TTX ingestion. A total of 120 non-toxic cultured
juveniles (mean body weight of 3.6 g) were randomly divided into two groups where one group was
fed with non-toxic commercial diets and the other was fed with TTX-containing diets (5.3 MU/gfeed). Fish were maintained in 3 tanks (20 fish/200 l) for each group with flow through system (2l/min) and vigorous aeration. The fish were fed with non-toxic and toxic diets to apparent satiation
three times a day at 08:00, 13:00 and 17:00 hours for 28 days. All fish survived until the last day
of feeding trial. There were no significant differences in the growth performance between fish fed
non-toxic diets and toxic diets: total length (85.8±1.2 vs 85.1±2.7 mm), standard length (81.1±1.0
vs 80.1±1.5 mm), degree of loss of caudal fin (77.7±0.6 vs 75.5±5.5 %), body weight (18.4±1.7 vs17.6±1.1 g), feed intake (11.7±0.6 vs 11.3±0.8 g), assimilation rate (91.1±1.6 vs 86.3±3.0 %),weight gain (14.9±1.6 vs 14.0±1.1 g), feed efficiency (127.3±7.1 vs 123.7±5.3 %). Total amountof administered and accumulated TTX in the fish fed toxic diets are 59.9±4.1 MU/fish and 33.6±2.0
MU/fish (56.4±7.1% of administered), respectively. These results indicate that TTX at this highdose may not have function as feeding stimulant for T. rubripes juveniles, but that feeding activity
and growth of the juveniles are not inhibited by potent neurotoxin. We will further perform
quantitative analysis and investigate the immunohistochemical localization of appetite peptides inthe brain of TTX-sensed and TTX-administered hatchery-reared T. rubripes juveniles.
Key words: Tiger puffer, Takifugu rubripes, tetrodotoxin, feeding trial, appetite

47

P-7
Temperature tolerance in two clonal strains of mangrove killifish,
Kryptolebias marmoratus
Marina Yamada1*, Yoshitaka Sakakura1
1

Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Japan.
*E-mail: bb53115032.at.cc.nagasaki-u.ac.jp

Mangrove killifish Kryptolebias marmoratus is the only known self-fertilizing vertebrate. This
unique species broadly distributes in coastal mangrove habitats from southern Brazil through the
Caribbean Islands and Central America to North Florida. They are capable of synchronous selffertilization, producing homozygous clones as a consequence. Our laboratory has two clonal strains,
PAN-RS and DAN, which were originally collected from near Bocas del Toro, Republic of Panama
and Dangriga, Belize, respectively. PDHy strain which is the hybrid of PAN-RS and DAN was
produced by artificial insemination (Nakamura et al. 2008). The descendants of PDHy are divided
into 4 strains, PDHyI, PDHyII, PDHyIII and PDHyIV according to the growth rate. Since Panama
and Belize show different climate, we hypothesized that PAN-RS, PDHy and DAN show different
temperature tolerance.
We used two clonal strains (PAN-RS and DAN) and hybrid strains (PDHyI, II, III, and IV). Fishwere kept in plastic containers filled with 60 mL of 17 ppt artificial brackish water under 25 ˚C andphotoperiod of 14L:10D. “Upper and lower thermal acclimation limits” were quantified for eachstrain using chronic thermal tolerance methodology (Fangue et al. 2006). All fish were kept under
25 ˚C for one week, and 20 fish from each strain were subjected to either increasing or decreasingwater temperatures of 0.5 ˚C per day. This experiment was continued until all fish died. The
respective chronic thermal maximum or minimum value was taken as the high or low temperature
at which 50 % morbidity was observed. The respective chronic thermal maximum wassignificantly higher in DAN (36.6 ˚C) than PAN-RS (35.7 ˚C), (Log-rank test, p<0.01). The
respective chronic thermal maximum was 30.7 ˚C, 33.6 ˚C, 33.7 ˚C and 32.5 ˚C for PDHyI, PDHyII,PDHyIII and PDHyIV, respectively. PDHyI showed the lowest respective chronic thermal
maximum among all strains. Respective chronic thermal maximum of each PDHy strain was lower
than their parents. The respective chronic thermal minimum was 9.4 ˚C for PAN-RS and 9.6 ˚C for
DAN, with no significant difference (Log-rank test, p=0.59).
Key words: Mangrove killifish, Kryptolebias marmoratus, thermal tolerance, hybrid

48

P-8
Effects of coexistence of protozoa Euplotes sp. coexistence on the population
growth of minute monogonont rotifer Proales similis
Naoshi Wakimura1*, Yoshitaka Sakakura1, and Atsushi Hagiwara1
1

Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Japan.
*E-mail: bb53115036.at.cc.nagasaki-u.ac.jp

The rotifer Brachionus rotundiformis (SS-type) is commonly used as starter food for rearing smallmouthed marine fish larvae. However, several tropical marine fishes have much smaller mouth gap,
hence cannot feed on the B. rotundiformis. To solve this problem, the minute monogonont rotifer
Proales similis is preferred due to its smaller size than B. rotundiformis. However, the culture of P.
similis is easy to be collapsed due to inability to withstand the handling stresses and environmental
changes in the culture medium. In this study, we investigated the interaction between P. similis andthe protozoa Euplotes sp. in the rotifer culture water. We independently cultured P. similis, Euplotes
sp. and a combination of P. similis and Euplotes sp. in 144 ml glass jar containing 50 ml of seawater(22 ppt) at an initial density of 1 ind./ml at 28°C in darkness for 14 days. All the treatments weretriplicated and daily fed with Nannochloropsis oculata at 8.0 x 10⁵ cells/ml without water exchange.
A similar experiment was conducted separately with Chlorella vulgaris as food at 2.9 x 10⁵ cells/ml
daily without water exchange. When fed with N. oculata, population density increased in Euplotes
sp. in the monoculture and mixed-cultures. However, the population growth of P. similis in themixed-culture decreased after 8 days and P. similis disappeared after 14 days of culture. There wasa significant difference in the population density of either P. similis or Euplotes sp. between the
monoculture and mixed-culture on day 14. When cultured with C. vulgaris, population density of
P. similis decreased from day 6, while that of Euplotes sp. reached its highest peak on the same day.
P. similis dissapeared completely on day 14. These results suggest that the presence of Euplotes
sp. in the P. similis cultures significantly suppressed the population growth of P. similis presumably
due to stressful interactions. Furthermore, the competition between P. similis and Euplotes sp. forbacteria may also have existed. Even though we did not observe a behavioral interaction of Euplotes
sp. and P. similis, the swimming speed of the Euplotes sp. was higher than the P. similis.
Key words: Proales similis, Euplotes sp., monoculture, mixed-culture

49

P-9
Distribution of larval and juvenile greater amberjack (Seriola dumerili) aroundthe Penghu Islands, Taiwan
Takamasa Hasegawa1*, Hsin-Ming Yeh2, June-Ru Chen3, Ryo Kawabe4, Yoshitaka Sakakura1
Graduate School of Fisheries and Environmental Science, Nagasaki University, Japan 2Coastal
and Offshore Resources Research Center, Fisheries Research Institute, Council of Agriculture,
Taiwan 3Fishery Research Institute, Council of Agriculture, Taiwan 4Institute for East China Sea
Research, Nagasaki University, Japan*E-mail: bb53512002.at.cc.nagasaki-u.ac.jp
1

The greater amberjack Seriola dumerili (family Carangidae) widely distributes around the world.
Because of its commercial importance, rapid growth and good adaptation to captivity, S. dumerili isa very important species for aquaculture in Japan. Juveniles of S. dumerili associate with floating
objects such as drifting seaweeds. However, there is very limited knowledge about larval and earlyjuvenile stages of this species in the wild. In order to investigate the early life history of S. dumerili,
firstly we validated the otolith daily increments using artificially-raised fish (11-51 days after
hatching). Then, field surveys were made by R/V Hai-an, Fishery Research Institute, Council ofAgriculture, Taiwan around the Penghu Islands, Taiwan, from May to August 2015. Frontal zone
and drifting seaweeds were visually observed during survey, and drifting seaweeds were scooped
together with associated fishes by a hand net (Φ45 cm, 3 mm mesh). Surface tows of plankton net(Φ1.3 m, 0.33 mm mesh) were conducted for 10 minutes with towing speed of 2 knots in frontal
zones and other areas. At each sampling station, vertical profile of water temperature and salinity
were measured by a CTD (SBE-19 plus, Sea-Bird Electronics Inc.). Fish species were identified
and zooplankton abundance (mg DW·m-3) and species composition (%) were calculated. We alsomeasured four S. dumerili samples caught in 2014 deposited at Penghu Marin Biology ResearchCenter, Fishery Research Institute, Council of Agriculture, Taiwan. Increments of sagittal otolithof reared fish showed the same number as their age in days after hatching (ANCOVA, df=1, p=0.32).
Relationships between otolith diameter (ydia, μm) and age (xday, days), and between otolith diameter(ydia, μm) and TL (xTL, mm), were described as following equations: ydia=0.0003· . (r=0.991),
and ydia=0.1937·xTL-0.1018 (r=0.981), respectively. We caught a total of four larval and juvenile S.
dumerili by surface towing, but not from drifting seaweeds. Total length of S. dumerili ranged from
7.4 to 42.5 mm, and age ranged from 18 to 56 days after hatching. All S. dumerili were caught inopen water and in frontal zone, however we could not detect significant differences in zooplankton
abundance between frontal zone and other station (t-test, p=0.87). Thecostraca was significantly
abundant in the stations away from the frontal zones (t-test, p<0.01). In 2014, larval and juvenile S.
dumerili were caught in January and May, and body length ranged from 5.8 mm in NL to 54.2 mm
in TL. Our study indicates that S. dumerili spawns from April to June in 2015, and frontal zone inopen water is nursery ground for S. dumerili around the Penghu Islands, Taiwan.
Key wards: Greater amberjack, Seriola dumerili, early life, spawning ground, otolith

50

P-10
Effect of starvation on mixis induction in offspring andgenetic mechanism of the monogonont rotifer Brachionus manjavacas
Shohei Kamizono, Koushirou Suga, Yoshitaka Sakakura, Atsushi Hagiwara
Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Bunkyo 1-14,
Nagasaki 852-8521, Japan．
*E-mail: bb53415001.at.cc.nagasaki-u.ac.jp
Short time starvation of neonates hatched from rotifer resting eggs induces active mixis up to the10th generation. However, this phenomenon remains unexplained whether it can last for further
generations. Also, the mode of heredity and the acquired parental characteristics inherited by thesubsequent generations are not clear. In this study, we used the rotifer Brachionus manjavacas andBrachionus plicatilis to investigate the heredity of mixis induction in rotifers up to the 80th
generation and the inheritance of the acquired traits by methylation of DNA. The maternal rotifers
hatched from resting eggs were starved for 12 hours before determination of the acquired
characteristics. The control group was not starved. A set of 8 individual rotifers were randomly
selected from starved and control group. Rotifers were individually cultured in 0.2 mL of sea water(22 ppt.) in 8 well microplate at 1 individual per well untill the 80th generation. The rotifers weredaily provided with Nannochloropsis oculata suspension at 6.0 × 106 cells/mL. The mixis inductionin each generation was determined. We determine the genetic factors that changed the mixis
induction rate of the parent and future generations. First, we designed the primer using partial basesequence of the methyltransferase, which was the DNA methylase which EST analysis of B.
plicatilis performed PCR as template cDNA of B. manjavacas. The mixis induction of the offspringfrom starved parents increased until 38th generation. In addition, mixis induction during
accumulated generations peaked at the 17th generation. We observed repetitions of increase anddecrease of beyond the 17th generation. The amplification of the DNA fragment of the predictedsize was confirmed through PCR analysis using the primer designed from the DNA
methyltranferase (DNMT) gene of B. plicatilis. This phenomenon could be explained by the
epigenetic inheritance involving methylation of the DNA. It was estimated that the DNMT genefragment by BLAST analysis. We have been investigating this by comparing the DNMT gene
expression level among generations.

51

P-11
Body Size, Culture and Fish Larval Ingestion on a Minute Rotifer Colurella cf.
adriatica
Stenly Wullur1, Petrus Letsoin2, Rina P. Astuti3, Atsushi Hagiwara4
1

Faculty of Fisheries and Marine Science, Sam Ratulangi University, Manado - Indonesia.
State Polytechnic of Fisheries. Tual, Southeast Maluku – Indonesia, 3Gondol Research Institue
for Mariculture, Bali – Indonesia, 4Graduate School of Fisheries Science and Environmental
Studies, Nagasaki University, Japan*E-mail: miracle_given.at.yahoo.com
2

Current procedure of rearing small mouthed marine fish larvae is using the Super Small (SS) typeof rotifer Brachionus rotundiformis as starter food during first days of larval first feeding. The B.
rotundiformis, however, is ineffective or even unsuitable for larvae of several marine tropical fishwith even smaller mouth size including, Napoleon fish (Cheilinus undulatus, groupers (genus
Epinephelus), Angelfishes (family Pomachantidae). In present study, we examined the feasibilityof a minute rotifer Colurella cf. adriatica as live food by measuring its body size, analyzing
population growth and fish larval ingestion on the rotifer.
Rotifer Colurella cf. adriaticawas isolated using a plankton net (45 μm mesh size) from an estuary
in Mangket, North Minahasa, North Sulawesi, Indonesia. Water temperature and salinity at thetime of sampling were 28±1 0C and 30±1 ppt, respectively. Sixty adults of the rotifer were
measured for its body length and width. As comparison, body length and width of a local strain
Brachionus rotundiformis were also measured. Population growth of Colurella cf. adriatica wasassessed by culturing the rotifer under four densities (3, 6, 9, 12 x 106 cells/mL) of Nanochloropsis
oculata as food source. The rotifer was cultured at salinity of 20 ppt and placed in a controlled room
temperature at 25±1 0C. Water volume of the culture was 4 mL (using 3x4, multiwell plate) andthe initial density of the rotifer was 1 ind./mL. Observation was made daily by counting thenumbers of rotifer in each well until the density decline. Larval ingestion on the rotifer wasinvestigated in Gondol Research Institute for Mariculture, Bali-Indonesia. Approximately 10 ind./L
eggs of humpback grouper (Cromileptes altivelis) were transferred to four 200-L larval rearing tank.
The first two tanks were fed with 10 ind./mL of rotifer Colurella cf. adriatica from day 2 till day 5
after hatching, while the other two tanks were left without any addition of food. All surviving
larvae were harvested on day 5 and the numbers of remaining larvae were counted. Gut content
of the surviving larvae was analyzed to see the presence of rotifer. Body length and width of
Colurella cf. adriatica were distributed from 82.8-103.2 and 46.8-61.7 μm, respectively. The
mean body length (95.9±3.8μm; mean± standard deviation) and width (46.8-61.7 μm) of Colurella
cf. adriatica were significantly smaller/narrower than B. rotundiformis (175.2±9.2 and 123.5±7.7μm, respectively) (t-test, p<0.05). Rotifer Colurella cf. adriatica grew well in all N. oculata
treatments. The rotifer attained highest population densities on day 16 (774±167 ind./mL) and 18(656±139 ind./mL) at N. oculata densities of 6 and 9x106 cells/mL, while it was reached on day 26(646±85 ind./mL) and 34 (560±58 ind./mL) at N. oculata densities of 3 and 12x106 cells/mL,
respectively. Humpback grouper larvae show higher survival (1.5%) on Colurella cf. adriatica
treatment than control (0.3%) (t-test, p<0.05). By analyzing gut content of the remaining larvae,52

it was found that individual of rotifer Colurella cf. adriatica presence in gut of the larvae indicatingthat larvae of humpback grouper ingested the rotifer.
Key words: Minute rotifer, Colurella cf. adriatica, body size, population growth, fish larvae,
ingestion

53

P-12
Production of myostatin gene-knockout Japanese anchovies (Engraulisjaponicus) using TALEN-based genome editing
Keishi Sakaguchi1*, Kanako Nakashima2, Hajime Kitano1, Naoki Nagano1, and Michiya
Matsuyama2
1

Fisheries Research Institute of Karatsu, Department of Joint Research, Faculty of Agriculture,
Kyushu University, 2Laboratory of Marine Biology, Faculty of Agriculture, Kyushu University*E-mail: keishi_s.at.agr.kyushu-u.ac.jp

Genome editing techniques such as zinc finger nucleases (ZFNs), transcription activator-like
effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats(CRISPR) have attracted considerable attention in recent years since these technologies can mediatetargeted and efficient genetic modifications (knockout, knock-in, and gene modification) in variousorganisms. Thus, targeted genome editing, which enables researchers to tailor genomic loci ofinterest, is one of the most promising approaches for plant, animal, and fish breeding.
Nevertheless, the application of the techniques to teleost fishes have been limited almost exclusively
to popular experimental fish models, such as zebrafish (Danio rerio) and Japanese medaka (Oryzias
latipes). There are no literature reports on the application of these techniques to marine fish, which
includes many important species for the fisheries industry. In this context, we regard the Japanese
anchovy (Engraulis japonicus) as a most suitable candidate for genome-editing experiments inmarine fish, because they have the following advantages: 1) easy rearing and breeding in a smallscale fish tank. 2) year-round and multi-year spawning under photoperiod and temperature control.
3) quick growth into mature individuals, which produce another generation of eggs, about threemonths. Myostatin (MSTN), previously referred to as growth differentiation factor 8 (GDF8), isa negative regulator of skeletal muscle growth. In mammals, MSTN-deficient animals resulted in
an increase of skeletal muscle mass with both hyperplasia and hypertrophy. Likewise, recent
studies revealed that the MSTN gene inhibits skeletal muscle growth even in fish. Thus, toproduce a fish breeding model generated by genome editing, we performed targeted gene disruptionof the MSTN gene in Japanese anchovies using TALEN technology. We constructed three TALEN
pairs targeting the first intron of the MSTN gene and the in vitro transcribed RNAs of the pairs were
injected into the yolk of embryos at the one-cell stage. As a result, mutant F0 embryos wereobtained with a very high insertion and/or deletion (indel) mutation rate (~96.9%), and thus the F0
founders were mated with each other to produce MSTN-knockout anchovies at the F1 generation.
To our knowledge, this is the most advanced study for genome editing in marine fishes. The
rearing of F1-individuals and their genotyping is now in progress.
Key words: Japanese anchovy, Engraulis japonicus, myostatin, knockout, TALEN, genome
editing54

P-13
Isolation and screening of novel probiotic lactic acid bacteria for aquaculture
Nguyen Thi Hue Linh1*, Masato Otani2, Yousuke Taoka3
1

Interdisciplinary Graduate School of Agriculture and Engineering, University ofMiyazaki,2Graduate School of Agriculture, University of Miyazaki,3Department of Marine
Biology and Environmental Sciences, Faculty of Agriculture, University of Miyazaki*E-mail: nb14005.at.student.miyazaki-u.ac.jp
Most of probiotics used in aquaculture are considered as alternative therapies for the use ofantibiotics to prevent diseases in aquatic animal. Among of probiotic bacteria, lactic acid bacteria
are one of potential candidate due to several strains have been isolated from fish gut as beneficial
microflora and acted antagonistic against Gram-negative fish pathogens. Therefore, in this study weconducted to isolate lactic acid bacteria from fermented food as candidate strains which may be
used to reduce the antibiotics using for sustainable aquaculture. The probiotic properties of isolates
were surveyed. Lactic acid bacteria were isolated from samples such as fermented foods by an agar
plating method using GYP and MRS media. The antagonistic activity test against fish pathogens
Lactococcus, Streptococcus and Edwardsiella was carried out according to the method of the double
layer agar method. The tolerant ability of isolates on NaCl (0%, 3%, 5%, 10%), pH (from 2 to 9),
artificial gastric juice (at pH range 2-4) with pepsin and intestine juice (at pH 8) with gall powder
were evaluated. Isolates were identified based on the sequences of 16 S rRNA gene (~700bp).
Totally 55 strains of lactic acid bacteria were isolated from rice bran and several kinds of fermented
vegetables. In antagonistic test, three isolates showed positive results against three strains of
Edwardsiella tarda, three strains of Streptococcus disgnactie, three strains of S. iniae and threestrains of Lactococcus garvie. These three strains of GYP 31, GYP 69 and GYP 4-20 were identified
as Lactobacillus sp.. The relative growth of GYP 31 strain at pH range 5-8 was from 100% to 80%,
at pH 2-4 and pH 9 were below 20%; while GYP 69 strain and GYP 4-20 strain only grew well at
pH 5, pH 6 with 100% of the relative growth. Three strains grew well at NaCl concentration from
0-5%, 3-5% and 0-3% with the relative growth from 80% to 100%, respectively. In tolerance teston acid and artificial gastrointestinal juices, GYP 31 strain expressed the sustained ability andsurvival itself better than GYP 69 strain and GYP 4-20 strain. GYP 31 strain showed the highest
viable count as 1x108, 1.4x108, 5.6x107cfu/ml in acid solution (at pH 3,5-4), artificial gastric juiceat (pH 4) and intestine juice (at pH 8), respectively, although the viable count was lower than thosein the control group (2.2x109cfu/ml at pH 7). From these results, Lactobacillus sp.. GYP 31 strain
is considered as a potential probiotic candidate in aquaculture due to its ability competition withpathogens and high tolerance in the gastrointestinal tract of fish.
Key words: probiotics, lactic acid bacteria, Lactobacillus sp., pathogen, antagonistic activity,
tolerance in gastrointestinal juice

55

P-14
Effect of protease addition to EP diet on the growth of amberjack, Seriola
dumerili
Yousuke Taoka1*, Takayuki Minami2, Nguyen Thi Hue Linh3, and Terumi Koga4
1

Department of Marine Biology and Environmental Sciences, Faculty of Agriculture, Universityof Miyazaki, 2 Miyazaki Prefectural Fisheries Research Institute, 3Interdisciplinary Graduate
School of Agriculture and Engineering, University of Miyazaki, 4TOA Pharmaceutical CO., Ltd.
*E-mail: yousuketao.at.cc.miyazaki-u.ac.jp
In aquaculture, the late growth of the cultured fish in winter season is serious problem foreconomical aquaculture. The extruder pellet (EP) has been used as an composed feed for the culture
fish because EP can be produced efficiently. However, the EP is very hard to decompose it bydigestive enzymes as compared with raw diet. The activities of digestive enzymes in thegastrointestinal tracts of the cultured fish significantly decrease due to the low temperature during
winter season. Therefore, it is very difficult that the digestive enzymes with low activities
decompose the EP diet efficiently. To dissolve this problem, in this study, the application ofproteolytic enzymes from microorganisms was examined to accelerate the digestion of the EP dietfor the cultured fish. Acid and alkaline proteases from microorganisms was used. The effect ofpH and temperature on enzyme reaction was also investigated. Alkaline and acid protease wereadded to the EP diets in a 0.1M Tris-HCl buffer and 0.1M glycine-HCl buffer, respectively. The
reaction mixtures were incubated at 15, 20, 25, 30 and 35oC for 180 min. The degradation of the EP
diet was evaluated by weighing the solid bodies. The nitrogen concentration of the centrifugal
supernatant in the reaction mixtures were determined according to the Kjeldahl method. The
alkaline proteases showed stable activities at 20-35oC, and the activity at 15oC significantly
decreased by 44%. The decomposition of the EP diet was enhanced by addition of both proteases at
20oC. These results indicated that the addition of proteases from microorganisms is effective toenhance the decomposition of the EP diet at low temperature. Amberjack, S. dumerli was fed with
EP diet with or without alikaline protease (control group) for 60 days. After 60 days of rearing, theaverage fish body weight was higher in the group with alikaline protease as compared with that in
the control group. These results showed that it is possible that the addition of protease enhance thegrowth rate of S. dumerili in winter season.
Key words: Seriola dumerili, protease, digestibility, growth rate, feed, low temperature,
aquaculture

Annotated Bibliography of Key Works
Satoh K. 2005. Study on improvement of composed diet for yellowtail culture. Bulletin of Oita
Institute of Marine and Fisheries Science, 6, 19-77 (in Japansese)
To date, Japanese aquaculture, mainly for yellowtail Seriola quinqueradiata, has been developed
using raw-fish as their feedstuff, such as sardine and mackerel obtained from adjacent sea, thatused to be abound and available with low cost. The reduction of sardine resources definitely
56

caused the nessesity of composed diet in yellowtail culture. Therefore, the studies examined to
improve and to improve accommodate the composed diet for yellowtails as to feedstuff, protein
digestibility, feed additives, and feeding regime.

57

P-15
Amino acid profile of thraustochytrids cells and potential of application toaquafeed
Kenya Horii*, Masahiro Hayashi, and Yousuke Taoka**
Department of Marine Biology and Environment Sciences, Faculty of Agriculture, University ofMiyazakiE-mail: * gd12020.at.student.miyazaki-u.ac.jp, ** yousuketao.at.cc.miyazaki-u.ac.jp
Fishmeal is used as protein sources for aquaculture feeds. However, the price is drastically increased
to around 170,000/ ton in 2013 during recent 10 years because of the decrease of fish resources,
anchovy. This is serious problem for sustainable aquaculture. Therefore replacement of fishmeal toanother resource is urgently needed. Thraustochytrids are marine protists and classified to one ofStramenopiles. They are widely distributed in marine environment and accumulate large number oflipids in cell bodies. Therefore, thraustochytrids have attracted strong interests for production ofvaluable lipids as single cell oils (SCOs) such as biodiesel and omega-3 fatty acids. In the processof lipid extraction from the cultured cells, some solids (extract residue) are produced as byproducts.
It is considered that this extract residue except for lipids is mainly consists of protein. From theviewpoints of industrial application of thrautochytrid cells as protein sources, we have planned touse the byproducts as resources instead of fishmeal for aquafeeds. In this study, we evaluated therecovery rate of protein in a thrausotchytrid, Aurantiochytrium limacinum strain mh0186 known asa docosahexaenoic acid (DHA) producer under the lipid extraction process. For selection ofadequate strains for protein production, thraustochytrids were isolated from marine environment.
A. limacinum strain mh0186 was cultured in a GY broth. The cultured cells were collected bycentrifugation and lyophilized for proximate analysis. The content of protein, lipid and ash in thelyophilized cells was determined according to Kjeldahl method, Folch method and heat-ashingmethod, respectively. The composition of amino acid and fatty acid were analyzed by liquid
chromatography-mas spectrophy (UF-Aminostation, Shimazu Co. Ltd., Japan) and gas
chromatography (GC-2014, Shimazu Co. Ltd., Japan), respectively. In the process of lipid extraction,
the extract residue was collected and re-lyophilized. The protein content and amino acidcomposition were analyzed by same method described as above. Seawater, sands, leaves, seaweed
were collected for the isolation of thraustochytrids from coastal area in Miyazaki, Kumamoto andOita, Kyushu, Japan. Thraustochytrids were isolated on a B12 Culture Agar “Nissui” plate mediumby pine pollen-baiting method. The isolates were cultured in a GY broth, and the cultured cells werecollected by centrifugation for analysis of the composition of amino acid and fatty acid. Isolates
were identified at genus level based on the 18S rRNA sequence analysis. The content of crude
protein lipid and ash per g of the cultured cell of mh0186 strain were 333 mg, 440 mg and 42 mg.
On the one hand, the protein content of the extract residue obtained from 1g of the cultured cells
was 226 mg (recover rate, 68 %). In both samples of the cultured cells and the extra residue, glycine,leucine, isoleucine, glutamate and arginine were mainly detected.
One hundred twenty
thraustochytrids strains were isolated. In some isolates including A. limacinum strains SR21 andmh0186, T.aureum ATCC34304, Schizochytrium aggregatum ATCC28209, glycine, leucine

58

glutamate, arginine and were detected as major amino acids. Strain Tak2 specifically accumulate
25 % cystathionine to total amino acids.
Key words: aquaculture, feeds, fishmeal, protein source, thraustochytrid, amino acid composition,
byproducts.

59

P-16
Diurnal changes in frequency of the burst swimming behavior of adult Pacificbluefin tuna (Thunnus orientalis) in a land-based tank
Akiko Tsujita* , Toshinori Takashi , Kentaro Higuchi , Tatsuru Kadota ,Koichiro Gen , Ayako Suzuki , Junpei Konishi , Keiichi Mushiake and Masakazu Oka
Research Center for Tuna Aquaculture, Seikai National Fisheries Research Institute, FRA*E-mail: tsujiaki .at. affrc.go.jp
In aquaculture of Pacific bluefin tuna, Thunnus orientalis(PBT), development of a stable system of
artificially-reared fingerlings is needed to ensure the sustainability of aquaculture through areduction on the reliance on wild captured juveniles. Therefore, we constructed two large indoor
land-based PBT broodstock tanks at Seikai National Fisheries Research Institute, FRA, Japan, andare examining the environmental cues such as water temperature and photoperiod which areessential for successful and stable spawning of PBT. However, heavy mortality of adult PBTs occur
in the tank due to collisions with the tank wall. It is considered that the collision deaths are associatedwith the burst swimming behavior. In this study, to clarify the process of the collision death, we
examined diurnal changes in frequency of the burst swimming behavior of three-year-old PBTs in
the land-based tanks. Nineteen three-year-old PBTs were reared in the land-based tanks (20 m indiameter, 6 m in depth) and their swimming behaviors were recorded using a video camera for six
days. A day was compartmentalized into six periods defined as dawn (7:00 to 9:00), daytime-I(11:00 to 13:00), daytime-II (15:00 to 16:00), dusk (16:00 to 18:00), night-I (20:00 to 22:00) andnight II (0:00 to 2:00) according to changes in illumination. We counted the frequency of the burst
swimming behavior at each period from the recorded video imagery. As a result, the frequency ofthe burst swimming behavior at dawn was significantly higher than that in other time periods.
Notably, the burst swimming behaviors were frequently observed in 30 min just after illumination(from 32 to85 lux) during dawn. Additionally, the frequency of the burst swimming behavior of
multiple PBTs in the tank like “a panic” were often observed during dawn, whereas solitary burst
swimming behaviors were observed at the other time periods. These results suggest that collision
deaths of PBT in the tank were caused by burst swimming behavior associated with sudden
increases of light intensity during dawn.
Key words: Pacific bluefin tuna, collision death, burst swimming behavior, diurnal change,
broodstock tank

60

P-17
Interval and spawning frequency of Pacific bluefin tuna Thunnus orientalis
in a land-based tank
Ayako Suzuki1*, Koichiro Gen1, Tatsuru Kadota1, Kenji Saitoh2, Takuma Sugaya3,
Masakazu Oka1 and Keiichi Mushiake1
1

Research Center for Tuna Aquaculture, Seikai National Fisheries Research Institute, FRA,
Research Center for Aquatic Genomics, National Research Institute of Fisheries Science, FRA,
3
Research Center for Marine Invertebrates, National Research Institute of Fisheries andEnvironment of Inland Sea, FRA.
*
E-mail: ayakos.at.affrc.go.jp
2

Recently, the exploitation of natural stocks of Pacific bluefin tuna Thunnus orientalis (PBT) has
increased to dangerous levels though increased fishing pressure. The scarcity of this species and its
high commercial value, together with its very high growth rates, makes it a potential candidate formarine aquaculture. Therefore, the artificial propagation and seed production techniques for PBT
represent objective for sustainable utilization of this resource. Fundamental information on the
spawning ecology of PBT is essential for the development of production techniques. However, thisinformation is still largely lacking. In this study, we investigated spawning frequency of PBT bycomparing mitochondrial DNA D-loop region haplotypes of broodstock fish, with those of fertilized
eggs and hatched larvae. Broodstock PBT, fertilized egg and hatched larval samples were obtained
from a land-based tank at Research Center for Tuna Aquaculture, Seikai National Fisheries
Research Institute in Nagasaki, Japan. Spawning activity in a land-based tank was observed over a
98 day period during May 16 to August 28 in 2014. The sampling of fertilized eggs and hatchedlarvae was conducted for a total of 15 days within the 98 days. A total of 15 samples were collected
for an initial consecutive 3 day period and then at approximately 1 week intervals from May 16 toAugust 28 in 2014. The 36 broodstock individuals and 659 eggs and hatched larvae were observed
to have 3 haplotypes. These haplotypes were named A, B and C, respectively. Among thebroodstock fish, haplotype B was detected at high frequencies, secondly haplotype C, and thirdly
haplotype A. The number of each broodstock individuals was 32, 3, and 1, respectively. Eachhaplotype of eggs and hatched larvae was detected on 14 days, 1 day and 8 days, respectively, during
the 15 sampling days. Haplotype B occurred in the 3 days of consecutive spawning. This isconsistent with genetic or histological observations that other wild Thunnus species spawn multiple
times and on consecutive days. These results demonstrate that PBT in a land-based tank may havethe potential to spawn consecutively and multiple times.
Key words: Pacific bluefin tuna (Thunnus orientalis), spawning frequency, mtDNA D-loop
region, haplotype

61

P-18
Effect of timing of restricted feeding on sexual maturation in the yellowtail,
Seriola quinqueradiata
Kentaro Higuchi1, 2*, Kazunori Yoshida1, Koichiro Gen1, Toshinori Takashi1, Kiyoshi Soyano2,
Keiichi Mushiake1
1

Seikai National Fisheries Research Institute, FRA, 1551-8 Taira-machi, Nagasaki, Nagasaki 8512213, Japan, 2Institute for East China Sea Research, Nagasaki University, 1551-7 Taira-machi,
Nagasaki, Nagasaki 851-2213, Japan*E-mail: higuken.at.affrc.go.jp
In aquaculture of large sized marine fish such as bluefin tuna and yellowtail, extensive amounts offish feed are needed for the broodstock management because of their large body size. In order tosave on the feeding cost for their broodstock management, development of a restricted feeding
technique without affecting reproductive performance is required. However, available information
about the effect of food supply on reproduction is limited in fish. In this study, we examined theeffect of restricted feeding during the gonad immature and vitellogenic phases on sexual maturationin females of the yellowtail, Seriola quinqueradiata. Two -year -old cultured yellowtail females,
which the average body weight was 5.0 kg, were divided randomly into three sea cages on
November 2012 and reared until the next spawning period (April 2013) under natural conditions at
the Goto station, Seikai National Fisheries Research Institute, FRA, Japan. The feeding regimes ineach cage were defined as follows: control group fed to satiation three times a week throughout theexperimental period, two restricted groups fed 30% of the amount of feed given to the control group(100%) during the immature (from November to January) or vitellogenic phase (from February to
April), respectively. At the end of the experimental period, the average body weights were 6.6 kg in
the control group, 5.8 kg in the restricted group during the immature phase and 5.9 kg in the
restricted group during the vitellogenic phase, which shows that the restricted feeding reduced
approximately 50% of somatic growth throughout the experimental period. Interestingly, the gonad
weights in the restricted feeding group during the vitellogenic phase were low as compared with thecontrol group and the restricted feeding group during the immature phase. Histological observations
revealed that females in all groups had oocytes that completed the accumulation of yolk globules atthe spawning period. However, the mean diameter of most advanced ovarian follicles in the
restricted feeding group during the vitellogenic phase was significantly smaller as compared withthe other groups. Furthermore, plasma estradiol-17β levels in the restricted feeding group during
the vitellogenic phase were significantly lower as compared with the other groups at the spawningperiod. These results indicate that the restricted feeding during the vitellogenic phase alters the
gonadal development in relationship with the plasma estradiol-17β levels in the yellowtail females.
Key words
Yellowtail (Seriola quinqueradiata), Sexual maturation, Restricted feeding, Broodstock
management

62

P-19
Evaluation of nitrogen excretion in young, immature and adult Pacific bluefin
tuna (Thunnus orientalis) measured in the land-based tankToshinori Takashi1*, Takeshi Eba2, Tadashi Imai3, Tetsuo Morita3, Yoshihisa Yamamoto3, Hiroyuki
Matsunari4, Junpei Konishi1, Kazuki Kumon2 , Koichiro Gen1 and Masakazu Oka1
1

Seikai National Fisheries Research Institute, FRA, 8-1551 Taira, Nagasaki, Nagasaki 851-2213,
Japan, 2Amami Laboratory, Seikai National Fisheries Research Institute, FRA, 9555
Hyousakiyamahara, Oshima Setouchi, Kagoshima 894-2414, Japan, 3Yashima Laboratory,
National Research Institute of Fisheries and Environment of Inland Sea, FRA, 234 Yashimahigashi-machi, Takamatsu, Kagawa 761-0111, Japan, 4National Research Institute of Aquaculture,
FRA, 422-1 Nakatsuhamaura, Minamiise, Mie, 516-0193, Japan*E-mail: ttakasi at fra.affrc.go.jp
Ammonia excretion in marine teleosts accounts for 70 to 90 % of their total nitrogen excretion.
Ammonia is toxic to fish and is a major factor limiting fish biomass and stocking density in intensive
culture systems and aquariums. Quantification of ammonium nitrogen is important for estimatingstocking biomass/density, water flow and size of the biological filter in the culture system. Several
studies have suggested that ammonia excretion is affected by several factors such as species, bodyweight, water temperature and ration size. Although several tuna species such as Pacific bluefin
tuna (PBT) and yellowfin tuna have been reared in aquariums and research facilities including
Seikai National Fisheries Research Institute, there is a lack of studies about ammonia excretion intunas. In this study, we investigated the effects of body weight and ration level on nitrogen excretion
for PBT in captivity. Young (0.67 ± 0.14 kg, n = 50), immature (14.4 ± 1.88 kg, n = 2) and adult(42.9 ± 6.5 kg, n = 2) PBTs were introduced to experimental land-based tanks from net cage orrearing tank, and thereafter they were acclimated to running sea water conditions. Experimental
tank size varied according to fish size (20 kl for young fish; 65 kl for immature fish and 150 kl for
adult fish). The PBTs were fed raw fish or artificial feed until the experiments. Before the fastingand postprandial experiments, the fish were deprived of food for 48 hours. Rearing water was
sampled every 2 h for the first 12 h, and at 4 h intervals from 12 to 24 h. In the postprandial
experiment, the experimental fish were fed bait fish (chub mackerel, Scomber japonicas, or
sandlance, Ammodytes personatus) or artificial feed. Fish feces were collected from the tank bottom
after 24 h. Determination of fecal nitrogen and concentration of ammonium, nitrite and nitrate
nitrogen was carried out on each fecal and water samples. Weight-specific ammonia excretion
rates of fasted fishes showed an inverse relationship with body weight (W). The relationship for
total ammonia nitrogen (TAN) was: TAN (mg N W−1 d−1) = 297.4·W−0.36 (r2=0.99). The ammonia
excretion rate at 10 kg in PBT was twice as much as it is for red seabream (unpublished data).
Although postprandial ammonium excretion rate was in relation to the ration size (R, mg feed-N W1 −1d ), linear regression analysis indicated that TAN excretion rates increased with ration size:
TAN (mg N W−1 d−1) = 332.4·R−179.6 (r2=0.87). There was no significant difference in the rates
between PBT fed the bait fish and the artificial feed. Postprandial fecal nitrogen was positively
correlated with ration size. Fecal nitrogen was excreted at a level of 0.9-1.9 % for the baitfish and
0.3-1.3 % for the artificial feed. Comparing the results of PBT with other fish, these values were63

lower than that of red seabream (12.8%) and pufferfish (16.1%). This study highlights the needfor effective evaluation of nitrogen loading and water quality management in PBT rearing facilities
and aquaculture grounds.
Key words
Pacific bluefin tuna (Thunnus orientalis), nitrogen, ammonia excretion rate, ration size

64

P-20
A high density genetic linkage map for yellowtail(Seriola quinqueradiata) containing 6,275 EST-based SNPs
Akiyuki Ozaki1*, Jun-ya Aoki1, Kazuo Araki1, Hironori Usuki1, Kazunori Yoshida2, Tsutomu
Noda2, Takuro Hotta 2, Hirotaka Mizuochi2, Taro Chujo2, Yasuhiro Shima1,2,
1National Research Institute of Aquaculture, FRA, 422-1, Nakatsuhamaura, Minamiise-cho,
Watarai-gun, Mie, 516-0193, Japan, 2 Seikai National Fisheries Research Institute, FRA, 122-7,
Nunoura, Tamanoura-machi, Goto-shi, Nagasaki, 853-0508, Japan*E-mail: aozaki at affrc.go.jp
The marine products industry has developed as majority of the fishery, which are captured anddirectly using of aquatic resources. Only recently the breeding are considered as important researchbecause available of aquatic resources are restricted gradually. The expectation of aquaculture
research is getting higher in response to the prediction of aquatic resources depletion. Also thegenetic improvement of economic traits are needed, it hopes apply to superior fish breeding, because
artificial juvenile have a possibility to improve the phenotype for suited to aquaculture condition in
every generation. We are researching practical application about selection of economic important
traits from natural genetic resources using yellow tail (Seriola quinqueradiata) as target species.
High density SNP arrays have become the tool of choice for QTL mapping, Genome-wide
association studies, marker-assisted selection (MAS) and genomic selection (GS). More recently,
high-density linkage maps generated by SNP array data have proven to be crucial for the accurate
assembly of scaffolds and contigs in whole-genome sequencing efforts. Earlier mapping studies
have identified QTL for important commercial traits including parasite disease resistance, andcombining the resources of a high density genetic map with genome sequence data will facilitate
the fine mapping of these loci and the identification of candidate genes. In this study, Affymetrix
SNP array was used to genotype 460 samples collected across five families from wild population incoastal waters of Goto Fukue-island. To establish EST (expressed sequence tag) -based SNP array,a cDNA library was generated from pooled RNA samples extracted from 11 tissues from a single
individual. Sequencing on Roche/454 GS FLX platform generated 1,353,405reads. The sequencingof SNP identification produced 570,846 raw reads derived from the full-length library and 456,482
raw reads derived from the 3’-anchored library derived from 5 hundred juveniles. Quality –based
variant calling using CLC Genomics Workbench detected 9,356 biallelic putative SNPs in 6,025
contigs, with a minor allele frequency (MAF) >25%. A Linkage analysis was performed using
application package of LINKMFEX version 2.3. This application can separate originated alleles
from male or female. In order to avoid the error of genotyping, the accuracy of genotypes in theirprogenies was checked from parental male and female alleles. Genotype data were converted to abackcross format as though the grandparent genotype was unknown. Pairwise analysis was
performed, and markers were sorted in linkage group at a minimum LOD threshold of 5.0. Linkage
phases were determined retrospectively by examining the assortment of alleles among linked
markers. A total of 6,275 EST-based SNPs were mapped to 24 linkage groups. The total distancecovered by the male and female maps were 1,230cM and 1,031cM. This map is currently being
used to map QTL for a number of commercially important traits, and will be used to improve the65assembly of the yellowtail genome. It is possible to rapidly develop domesticated strains having
commercially important traits in yellowtail aquaculture.
Keywords: Yellowtail（Seriola quinqueradiata;）; EST-based SNPs ; Genetic linkage map;
quantitative trait loci (QTL); Affymetrix.

66