Preface
The UJNR (The U.S.-Japan Cooperative Program in Natural Resources) Aquaculture Panel wasestablished in 1968, and the business meeting and symposium have been held every year since1971. Through the long history of UJNR, Aquaculture Panel has contributed to the development ofaquaculture researches of both countries by means of various cooperative activities, such as theexchange of scientists and literatures, and the promotion of joint research projects.

The

Aquaculture Panel is highly appraised as one of the most active UJNR panels in both countries.
The 43rd Joint Meeting of the UJNR Aquaculture Panel was conducted in Nagasaki, Saga andOita Prefectures, the northern Kyushu Island, Japan from November 9-14, 2015; while the ScientificMini-Symposium was held in Nagasaki University from November 10-11. The symposium themewas "Evaluation of the impact of breeding organisms on the ecosystem and aquaculture industry",
which was under the 9th Three-Year Plan, “Genetics and Breeding Studies in Aquaculture
Industry", commenced in 2014. Sixteen oral presentations were made on topics such as the impactof breeding organisms on the ecosystem and aquaculture industry, selective breeding techniques,
and bioinformatics, and twenty poster presentations were also made on broader topics related toaquaculture during the two-day symposium.
The proceedings of the 43rd UJNR Aquaculture Panel Scientific Mini Symposium “Evaluation of
the impact of breeding organisms on the ecosystem and aquaculture industry” is published as the
special issue of the Bulletin of Japan Fisheries Research and Education Agency.

With great

pleasure, this UJNR proceedings containing high quality papers authored by selected Americanand Japanese aquaculture scientists will hopefully help in the improvement of genetic/breeding
programs, which is expected to contribute to the development of the aquaculture industry in boththe United States and Japan.
Finally, I would like to express my sincere gratitude to the staff at Nagasaki University andcolleagues involved in the UJNR Aquaculture Panel for their efforts in the preparation andorganization of the symposium. I would also like to deeply thank the editorial board members for
publishing the proceedings.

Fuminari Ito
Chair of UJNR Aquaculture Panel
Executive Director
Japan Fisheries Research and Education Agency

Participants in 43rd UJNR Aquaculture Panel Symposium, held in Nagasaki University, Nagasaki, Japan,
November 10 – 11, 2015

CONTENTS

Preface ……………………………………………………………………………………………………………

1

Group Photo ………………………………………………………………………………………………………

2

Contents ……………………………………………………………………………………………………………

3

Program for The 43 Scientific Symposium of UJNR Aquaculture Panel ………………………

5rdContributed Papers and Abstracts
Preliminary Study on Triploid of Yellowtail (Yukinori Shimada, Hiroyuki Nagoya, Hiroyuki Okamoto,Toshiya Yamaguchi, Nariaki Inoue, Takashi Ishikawa, Kazuhisa Hamada, Kazuharu Nomura,
Kazunori Yoshida, and Hironori Usuki) …………………………………………………………………

9Application of Germplasm Preservation in Breeding Programs for Molluscan Shellfish Aquaculture andRestoration (Huiping Yang) ……………………………………………………………………………… 15
Improving Aquaculture Production in Haliotis Species through the Development of a Genomic Toolkit(Catherine Purcell1, Andrew Severin, Paula Sylvia, Nick Wegner, and John Hyde)
Culture Protocols and Production of Triploid Purple-Hinge Rock Scallops (Paul Olin)…………… 21………………… 29

Big Data in Agriculture and the USDA/ARS Initiative (Jeffrey Silverstein) ……………………………… 33
Genomic Selection in Aquaculture (Sho Hosoya, Kiyoshi Kikuchi, Hiroshi Nagashima, Junichi Onodera,
Kouishi Sugimoto, Kou Satoh, Keisuke Matsuzaki, Masaki Yasugi, Atsushi J. Nagano, Akira
Kumagayi, Kenichi Ueda, and Tadahide Kurokawa) ………………………………………………… 35
Genetic Selection on Animals Using Pedigree, Phenotypic, and Genomic Information (Shogo Tsuruta)………………………………………………………………………………………………………………… 41
Exploring Transcriptomic Patterns in Slow- and Fast-growing Seriola dorsalis Larvae (Catherine Purcell1,
Andrew Severin, Vince Buonaccorsi, Mark Drawbridge, Kevin Stuart, and John Hyde) ……… 47
Modeling the Variable Effects of Using Wild and Cultured Broodstock on the Fitness Risk Due toEscaped Farmed Fish (Kristen M. Gruenthal, Jason D. Volk, Gregory R. Blair, Gavin Key, andMichael B. Rust) …………………………………………………………………………………………… 57
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, Tadahide
Kurokawa, Hideki Nikaido, Daisuke Muraoka, and Yoshiro Okuda) ………………………………… 63
Evaluation of the Tsunami Impact on the Genetic Diversity of the Marbled Flounder Pseudopleuronectes
yokohamae in Sendai Bay, Miyagi, Japan (Yuki Minegishi, Minoru kedaI, Yutaka Kurita, Hiroyuki
Togashi, Yukinori Nakane, and Akihiro Kijima) ……………………………………………………… 69
Experimental Hybridization and Competition between Atlantic Salmon (Salmo salar) and Native Salmonid
Species in Japan (Kazuo Araki, Tetsuji Masaoka, Hiroyuki Okamoto, Hiroyuki Nagoya, Hidefumi
Maramura, Kouji Muto, Hiromi Oku, Jun-ya Aoki, and Toshiya Suzuki)………………………… 75

Hybrid Striped Bass National Breeding Program: Research Towards Genetic Improvement of a NonModel Species (S. Adam Fuller, Benjamin H. Beck, Steven D. Rawles, Bartholomew W. Green, Chao
Li, Eric Peatman, Catherine J. Childress, T. Gibson Gaylord, Frederick T. Barrows, and Matthew E.
McEntire) …………………………………………………………………………………………………… 89

Production of Benedenia-resistant Yellowtail (Seriola quinqueradiata) Families –A Preliminary Approachto the Candidates– (Tsutomu Noda, Kazunori Yoshida, Takurou Hotta, Hirotaka Mizuochi, Taro
Chujo, Hideaki Aono, Akiyuki Ozaki, Kazuo Araki, Mitsuru Yoshimoto, Hiroki Ogawa, Yasuhiro
Shima, and Yuichiro Fujinami)………………………………………………………………………… 101

Coho Salmon Broodstock Development: A Case Study of the Domsea Coho Salmon (1977 to 2015) (Robert
N. Iwamoto, Richard Towner, James Myers, Greg Hudson, and Patricia Munsell) ……………… 107
Development of Improved Catfish Germplasm at the Warmwater Aquaculture Research Unit, DSDAARS (Brian Bosworth and Geoffrey Waldbieser) ……………………………………………………… 113
Abstracts of Poster Presentations
List of Presentations ……………………………………………………………………………………………… 123
Abstracts of Poster Presentations ……………………………………………………………………………… 125

Program
The 43rd Scientific Symposium of UJNR Aquaculture Panel
Evaluation of the impact of breeding organisms on theecosystem and aquaculture 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, andthe importance of genetics and breeding studies has increased to meet various needs.
Although genetic improvement is common in agriculture and the resulting benefits
unquestioned, there are few se1ective1y bred commercially available strains in theaquaculture industry except in inland aquaculture.
Moreover, we should pay attention to the ecological impacts of breeding technology. At
the same time selection may increase risks when selected animals escape and breed withwild stocks unless proper safeguards are put into place. Integration of scientific knowledgeof 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 the development of the aquaculture industry in both countries ofJapan and United States.
This Symposium consists of oral session and poster session. In oral session, we will deal
with the subjects relating to ‘genetics and breeding’ such as: the impact of breedingorganisms on the ecosystem and aquaculture industry; selective breeding techniques;bioinformatics, etc. In poster session, we will present and discuss broader topics relating toAquaculture.

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)…………………………………………………………９：30- ９：35

Aim of the Symposium
Fuminari Ito (Japan Chair, Fisheries Research Agency) …………………………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. D
 id 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
Yuki Minegishi (Tohoku Ecosystem-Associated Marine Sciences, Tohoku University)………………………………………………………………………………………14：45-15：10
12. C
 ompetition between Atlantic salmon (Salmo salar) and Japan’s native salmonids.
Kazuo Araki (National Research Institute of Aquaculture, FRA)…………15：10-15：35

Break ……………………………………………………………………………………15：35-15：50
Session IV. Genetic improvement 2(Moderators: M. Ototake & H. Yang)
13. H ybrid Striped Bass National Breeding Program: Research Towards Genetic
Improvement of a Non-Model Species
Adam Fuller (USDA Agricultural Research Service) …………………………15：50-16：10
14. P roduction of Benedenia-resistant Yellowtail (Seriola quinqueradiata) Families –A
Preliminary Approach to the Candidates–
Tsutomu Noda (Seikai National Fisheries Research Institute, FRA) ………16：10-16：35
15. C
 oho Salmon Broodstock Development: A Case Study of the Domsea Coho (1977 to 2015)
Bob Iwamoto (Spring Salmon LP) ………………………………………………16：35-16：55
16. D
 evelopment of Improved Catfish Germplasm at the Warmwater Aquaculture Research
Unit, USDA-ARS
Brian Bosworth (USDA Agricultural Research Service)……………………16：55-17：15

Discussion……………………………………………………………………………17：20-17：50(Moderators: F. Ito & M. Rust)

Closing remarks……………………………………………………………………17：50-18：00(Michael B. Rust, U.S.A. Panel Chair)水 研 機 構 研 報， 第 45 号，9 － 14， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，9－14，2017

9Preliminary Study on Triploid of Yellowtail
Yukinori SHIMADA＊1, Hiroyuki NAGOYA＊2, Hiroyuki OKAMOTO＊2, Toshiya YAMAGUCHI＊1,
Nariaki INOUE＊1, Takashi ISHIKAWA＊2, Kazuhisa HAMADA＊3, Kazuharu NOMURA＊2,
Kazunori YOSHIDA＊4, and Hironori USUKI＊5

Abstract: The possibility of a large-scaled cold shock induction of triploidy, viability of triploid fishand growth performance were investigated in yellowtail Seriola quinqueradiata. Induction of
triploidy by a large-scaled cold shock (approximately 0.1 million fertilized eggs) was successful in our
preliminary test, and approximately 40 thousand hatched larvae were obtained. Some of the hatchedlarvae were tested for polyploidy, the results of which indicated that fish treated with two different
cold shocks (two trials) were 100% triploid. Then, the growth performance of the triploid and diploidfish was investigated in both mixed and separate rearing. At the age of 100 and 122 days, the bodyweights of the triploid and untreated control fish were not significantly different in both mixed andseparate rearing in two trials. However, as they grew larger, the growth rate of the untreatedcontrol was significantly higher than that of the triploid fish in both rearing conditions. Thus, thetriploid fish at early development (at least half a year) showed a delay in growth. Also, the triploidfish showed longer term viability. In future studies, we will investigate growth performance atlarger size and infertility (gonadal developmental stage) at the age of maturation.
Key words: Seriora quinqueradiata, triploid, cold shock, growth performance

Introduction

et al., 1997; Piferrer et al., 2000). Recently, the basis
for triploid induction by a cold shock treatment was

Yellowtail (Seriola quinqueradiata) is one of theestablished for yellowtail at the small-scale (Shimada

most important species for aquaculture in Japan.

unpublished data; Nagoya et al., unpublished data). It

Under culture conditions, however, mature yellowtail

showed that cold shocks for 5 – 20 min duration at 0

are remarkably reduced in body weight after the– 5 °C within 5 min after fertilization resulted inspawning season (mainly summer). This is a serious

approximately > 80% triploid rate.

concern for the market. Thus, there is an interest in

Our goal is to achieve industrial use of triploidy inthe production of sterile yellowtail. This can be

yellowtail, but there remain several issues to resolve,

achieved by chromosome set manipulation techniquessuch as developing a large-scaled cold shock(Thorgaard, 1983; Felip et al., 2001), including thetechnique and determining viability, growth

production of triploids (e.g. Purdom, 1972; Garrido-

performance and infertility of triploid fish, and also

Ramos et al., 1996; Holmefjord and Refstie, 1997; Felipmaking a basic law system for triploid fish culture in

2017年２月28日受理（Received on February 28, 2017）	Kamiura Laboratory, National Research Institute of Aquaculture, Fisheries Research and Education Agency, Tsuiura, Kamiura, Saiki, Oita 8792602, Japan＊2	
National Research Institute of Aquaculture, Fisheries Research and Education Agency, Nakatsuhamaura 422-1, Minami-ise, Watarai, Mie 5160193, Japan＊3	
Komame Laboratory, National Research Institute of Aquaculture, Fisheries Research and Education Agency, Komame 330, Otsuki, Kochi 7880315, Japan＊4	
Goto Laboratory, Seikai National Fisheries Research Institute, Fisheries Research and Education Agency, Huura 122-7, Tamanoura, Goto,
Nagasaki 853-0508, Japan＊5	
Shibushi Laboratory, National Research Institute of Aquaculture, Fisheries Research and Education Agency, Natsui 205, Shibushi, Kagoshima
899-7101, Japan	E-mail: yukinoris@affrc.go.jp＊1

Yukinori SHIMADA, Hiroyuki NAGOYA, Hiroyuki OKAMOTO, Toshiya YAMAGUCHI, Nariaki INOUE,
10 Takashi
ISHIKAWA, Kazuhisa HAMADA, Kazuharu NOMURA, Kazunori YOSHIDA, and Hironori USUKI
a sea cage. To determine viability of triploid fish in a

Rearing and growth of fish

long-term culture, a large-scaled cold shock method is

Treated and untreated fertilized eggs wererequired to produce a lot of fertilized triploid eggs.

separately maintained in 1000 L tanks until theyThe objectives of the present study are (1) to examine

hatched. Approximately 5,000 hatched larvae werethe efficacy of large-scaled cold shock induction ofreared in a 500 L tank with flow-through sea water.

triploidy, (2) to determine viability of triploid fish in

Tanks were maintained in a 4000 L water-bath tolong-term rearing, and (3) to compare a growthminimize variance of water temperature among them

performance of diploid and triploid fish.

(water temperature > 20 °C). Fish were fed theL-type rotifer Brachionus plicatilis from day 2 to 25,

Materials and methods

Artemia spp. from day 21 to 35 with DHA and EPA
enrichment (Hyper-gross, Mrinetech Co. Ltd., Aichi),

Artificial fertilizations and cold shock treatments

and dry pellets after day 30.

Fish were size

Artificial fertilization of yellowtail was performed

segregated and transferred to 1500 L tanks during

at two facilities, Kamiura Laboratory and Komame

days 35 to 40. On day 100 or 122 in each trial, bodyLaboratory of National Research Institute ofweights of all fish were measured after anesthesia

Aquaculture, in February (trial 1) and April (trial 2)with 2-phenoxyethanol (Wako Co. Ltd., Osaka). Then,

2015, respectively. Fertilized eggs were treated by

the body weights in the mixed rearing werecold shock 3 min after fertilization in 15 L containers

measured on day 151, 185, 212, 245 and 275, whereas

equipped with nets at the bottom. No temperature

those in the separate rearing were measured only on

difference was observed between the container andday 150 and 186 because all triploids died in ansurrounding water bath. The conditions of the cold

accident on day 186. In addition, all fish in the mixed

shock treatment were 5 min duration at 0 °C atrearing were injected with a syringe on day 122 with

Kamiura Laboratory, and 20 min duration at 5 °C at

a PIT tag placed into the ventral part of the body

Komame Laboratory, and the sea water temperaturecavity.

was regulated by sea water ice and / or a coolingequipment (AZ-280X, Iwaki Co. Ltd., Tokyo).

Polyploidy of fish
To determine the polyploidy of treated andFig. 1. Examples of polyploidy determination for untreated control (upper) and cold shock treated yellowtail(lower). 2 N and 3 N indicate diploid and triploid, respectively. The mean DNA contents of 3N are approximately
1.5 times larger than those for 2N.

11

triploid of yellowtail
untreated larvae, a total of 49 individuals were

measurement. In trial 1, 106 out of 121 treated fishsampled on day 0 and fixed and preserved in 100%

were triploid (87.6%), but all untreated control fishethanol at 4 °C. These individuals were subjected towere diploid (100%). In trial 2, treated and untreatedflow cytometry (Partec GmbH, Germany) for relative

control fish were triploid (11 / 11 fish) and diploid (15DNA content of the whole body cells in order to/ 15 fish), respectively.

determine their ploidy status as shown in Fig. 1.
Growth performance of triploid and diploid fish
Statistics

Growth performances of diploid and triploid fish

The data were expressed as the mean ± standard

are shown in Fig. 2. In the separate rearing, triploiderror of the means (SEM), and analyzed by an

and diploid fish were not significantly different in

independent t-test.

body weight on day 100 (df = 240, t = - 1.636 and p =0.1032), but at older ages diploid fish showed
Results

significantly larger body size than triploid fish
(day150; df = 204, t = 4.454 and p < 0.0001, day 186; df

Cold shock treatments and ploidy status= 94, t = 5.659 and p < 0.0001, Fig. 2A).

This

Large-scaled cold shocks were performed on

phenomenon was also observed in the mixed rearing

approximately 0.1 million fertilized eggs in trial 1(df = 24, day 122; t = 0.646 and p = 0.5246, day 151; t(separate rearing) and 2 (mixed rearing). Hatching= 2.057 and p = 0.0507, day 185; t = 2.829 and p =rates in cold shock treatments for 5 min at 0 °C and

0.0093, day 212; t = 2.547 and p = 0.0177, day 245; t =20 min at 5 °C, were 18.5% and 36.9%, respectively,

2.210 and p = 0.0369, and day 275; t = 2.285 and p =whereas those in untreated controls were 78.2 and0.0315).

22.9%. The triploid rates in cold shock treatments
were 100% (N = 8 and 12), whereas those in untreated

Discussion

controls were 0 and 11.8% (N = 15 and 17) on day 0.
In addition, we confirmed ploidy status of all

The most salient finding of this study was that a

individuals using their fin clips at the final

large-scaled triploid induction (0.1 million fertilized

Fig. 2. Growth performance of triploid (open circle) and diploid (black circle) under separate rearing (A) and mixed
rearing (B) conditions. All statistics were done by independent t-test. Symbols of n.s., *, ** and *** mean not
significant, P < 0.05, P < 0.01 and P < 0.001, respectively.

Yukinori SHIMADA, Hiroyuki NAGOYA, Hiroyuki OKAMOTO, Toshiya YAMAGUCHI, Nariaki INOUE,
12 Takashi
ISHIKAWA, Kazuhisa HAMADA, Kazuharu NOMURA, Kazunori YOSHIDA, and Hironori USUKI
eggs) was possible and triploid fish were viable inoocytes (Yamaguchi et al., unpublished data) as in thelong-term rearing in yellowtail. However, a growthcase of tilapia (Razak et al., 1999). Also, in the aspect

performance of triploid fish was lower than that ofof rearing technique Oppedal et al. (2003) reported

diploid fish in both separate and mixed rearing. In

that triploid salmon tended to display enhanced

what follows, we will discuss these results as well as

weight gain compared to diploid ones when given

some methodological issues in detail.

continuous lighting conditions. Thus, by improving
rearing techniques and/or controlling fish maturation,

A large-scaled cold shock treatment
In a large-scaled cold shock for fertilized eggs,
temperatures in the equipped container andthe growth of triploid yellowtail might be enhanced
comparable to the existing production system in
diploid yellowtail.

surrounding water bath were the same, suggesting
Conclusionthat the equipment used may be applied to larger
volumes of fertilized eggs.

To summarize, the results of this study
Triploid viability

demonstrated the feasibility of large-scale production

In this study, hatching rates of cold shock treatedof triploid yellowtail and their use in aquaculture.

eggs were 18.5 and 36.9% in trial 1 and 2, respectively,

However, in a comparison between separate andwhereas those of untreated control were 78.2 and 22.9

mixed rearing, we found that growth performance of%. Usually, survival of triploids is about 70 to 80% of

triploid fish was lower than that of diploid fish. Thus,

that of the controls (Felip et al., 2001). However, thewe need to develop appropriate rearing methods forpresent study showed varying results ranging from

triploid yellowtail.

24% to 161% of the hatching rates of untreatedAcknowledgment

controls. Most probably, we still need to improve themaintenance method of fertilized eggs in order toresolve the surprising result.

We thank Mr. Hiroki Ogawa and his colleagues in

Triploid fish were observed in untreated controls

Aquafarm Inc. for providing parental yellowtail. We(trial 2). So far, it is reported in a case study that overthank also Mr. Yasuhiro Shima at National Researchripening of eggs after ovulation increases triploid

Institute of Aquaculture, and Yuichiro Fujinami Ph.

rates in Anguilla japonica (Nomura et al., 2013).

D., Mr. Takuro Hotta, Mr. Tsutomu Noda and their

Therefore, over ripening might be one of the reasonscolleagues at Seikai National Fisheries Researchfor occurrence of triploids in untreated controls of

Institute for providing high quality fertilized eggs foryellowtail.

our preliminary study.
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13

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Purdom C. E., 1972: Induced polyploidy in plaice(3) Benfey T. J., 1999: The Physiology and Behavior of
Triploid Fishes. Rev. Fish. Sci. 7(1), 39-67.

(Pleuronectes platessa) and its hybrid with the

Induced triploidy is widely accepted as the mostflounder (Platlchthys flesus). Heredity 29, 11-24.

effective method for producing sterile fish for

Razak S. A., Hwang G-L., Rahman M. A., and Maclean

aquaculture and fisheries management. ArtificiallyN., 1999: Growth performance and gonadal

produced triploids generally differ from conspecificdevelopment of growth enhanced transgenic

diploids in three fundamental ways: they are moretilapia Oreochromis niloticus (L.) following heat-

heterozygous, they have larger but fewer cells inshock-induced triploidy. Mar. Biotechnol. 1, 533-

most tissues and organs, and their gonadal

544.

development is disrupted to some extent. Despite

Thorgaard G. H., 1983: Chromosome set manipulationthese basic biological differences, triploids are similarand sex control in fish. In: Fish Physiology (ed.

in most respects to diploids when examined at the

By Hoar W. S., Randall D. J., and Donaldson E.M.),

whole animal level. The only clear differences relatecol. IXB. Academic Press, New York, pp. 405-434.

to the effects of impaired gametogenesis on theYukinori SHIMADA, Hiroyuki NAGOYA, Hiroyuki OKAMOTO, Toshiya YAMAGUCHI, Nariaki INOUE,
14 Takashi
ISHIKAWA, Kazuhisa HAMADA, Kazuharu NOMURA, Kazunori YOSHIDA, and Hironori USUKI
reproductive physiology and behavior of triploids,

when reared under suboptimal conditions.

especially in females. Other apparent differences

causes of these latter two problems are poorlyThe

include reduced aggressiveness, occasional specificunderstood but must be addressed if triploids are tomorphological abnormalities, and inferior performance

be used more extensively.

水 研 機 構 研 報， 第 45 号，15 － 20， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，15－20，2017

15

Application of Germplasm Preservation in Breeding Programs for
Molluscan Shellfish Aquaculture and Restoration
Huiping YANG＊

Abstract: 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 andfor livestock as a tool for breeding programs worldwide. For fish and shellfish, cryopreservation has beenstudied in more than 200 species for preservation of natural resources and conservation of endangered
species. For molluscan shellfish aquaculture, this technology can have the following potential applications:
1) Preservation of specific lines or strains. Ongoing breeding programs have yielded specific strains andlines, such as disease resistant oysters. Cryopreservation can be used to preserve these valuable strains
and provide gametes for assistance of 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 themost valuable resources for breeding programs but difficult to produce and require years of repeatedcrossing 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 andcryopreservation, self-fertilized lines can be created by using cryopreserved sperm and oocytes from thesame individual after sex reversal; 4) Preservation of sperm from tetraploid 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 and provide cost savings and security for maintaining tetraploids.
In addition, cryopreservation of tetraploids produced each year can offer benefit for maintenance of thetetraploid 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, establishmentof mutant lines by TILLING and sperm cryopreservation will benefit aquaculture and research on oyster
functional genetics, and 6) Assistance of aquaculture hatchery practice for regular and hybrid seedproduction. The cryopreserved germplasm materials can function as a reservoir to meet the need forregular and hybrid seed production. For example, the hard clam hybrid offspring of Mercenaria mercenaria
with Mercenaria campechiensis showed fast growth and higher survival.
Key words: Germplasm, cryopreservation, shellfish aquaculture

2017年２月28日受理（Received on February 28, 2017）
	School of Forest Resources and Conservation, Institute of Food and Agricultural Sciences, University of Florida. 7922 NW 71st Street,
Gainesville, FL 32653, U.S.A.
E-mail: huipingyang@ufl.edu＊

16

Huiping YANG
Introduction

usually activated immediately and ready for
fertilization within a few hours.

Germplasm is the living genetic resources which

Germplasm collection methods usually can be: 1)

are inheritable and can generate new generations,

For gametes: natural spawning, suspension ofsuch as seeds for plants, or germ cells, gametes,

dissected gonads, or biopsy of gonads. The last twoembryos, or larvae for animals. Preservation of

methods are only useful for certain species, such as

germplasm can serve as repository (seed banks, genesome oysters and clams, in which gametes frombanks or sperm banks) for animal and plant breeding.

dissected gonad can remain fertile. 2) For embryos

Usually, germplasm preservation can be achieved

and swimming larvae: collection is usually performed

through cold storage (for plant seeds) or

by filtering through 25 - µm screen and re-suspending

cryopreservation (for animal germplasm materials).

in fresh seawater or buffer.

The technology of cryopreservation has been appliedfor human artificial reproduction as a clinicalViability analysis

treatment for infertility (Bernard and Fuller, 1996; Di

For almost all germplasm cryopreservation

Santo et al., 2012) and for livestock as a tool for

programs, fertilization and survival have been

breeding programs worldwide (Curry, 2000). For fishconsidered the main criteria for evaluation ofand shellfish, cryopreservation has been studied in

cryopreservation success. Additionally, considering

more than 200 different species with different

the time and resources required for in vitro

applications (Tiersch et al., 2007), such as preservation

fertilization, other effective and fast viability assaysof natural resources to maintain biodiversity andespecially for sperm also have been developed andconservation of endangered species.

used widely. Viability of fresh or post-thaw sperm

For molluscan shellfish, the application of

can be evaluated by observing motility with a bright-

germplasm preservation can also serve as germplasm

field light microscope and plasm membrane integrity

repository for selective breeding (Adams et al., 2008),

and mitochondria membrane integrity by flow

hybrid breeding, commercial seed production,

cytometry analysis. For oocytes, viability evaluation

preservation of natural resources, and conservation

can be assisted by observation of the size andof endangered species. In addition, due to themorphology besides fertility test. For embryos andcharacteristic of molluscan shellfish, application oflarvae, survival and growth can be used for viability

germplasm cryopreservation can be expanded toevaluation.

certain specific areas such as creation of selffertilization inbred oyster lines (Yang et al., 2015),

Cryopreservation process of germplasm

aquaculture of triploid-tetraploids (Dong et al., 2005)

Cooling rate is one of the most critical factors forand possible creation of mutant lines as that forsuccess of germplasm cryopreservation, usuallyzebrafish (Lawson, 2016).

varies with germplasm materials and the working
species, and is highly related to the volume, materials,

Materials and Methods

and shape of the packaging container. Accuratecooling rate can be achieved by using liquid nitrogen

Sample collection
In general, collection of germplasm samples foror dry ice with the following methods: 1) Computercontrolled programmable freezer.

The coolingcryopreservation is based on the reproductive

process is controlled by spraying of liquid nitrogen

biology of the working species. Currently, mostwhich is pre-set by a programmable computer toaquaculture molluscan species are bivalves (oysters,

achieve accurate cooling rates.

clams, scallops, and mussels) and a few gastropods,

cryokits composed of Styrofoam boxes with liquidsuch as abalones. Those species are primarily free

nitrogen.

spawning with external fertilization, spiral holoblastic

determined by measuring the temperature with aembryo development, and a swimming larval period

digital thermocouple, and cooling rates can be

until metamorphosis. Upon release, gametes areadjusted with the depth of liquid nitrogen, size of the2) Home-madeThe cooling rates need to be pre-

17

Germplasm preservation for shellfish aquaculture
Styrofoam box and floating boat (for holding the(Table 1). Those studies were majorly on aquaculture

samples to be frozen). 3) Others, such as nitrogenspecies, including bivalves (oysters, mussels, pearl

vapor in the storage or shipping dewar or aoysters, scallops, and clams) and gastropods

Styrofoam box filled with dry ice. Upon reaching -80(abalones), and most of those were on oyster species°
C, frozen samples can be plunged into liquid nitrogenfrom the Family Ostreidae (43 publications). A recent

for long-term storage in storage dewar.

publication reviewed the research on germplasm

For sample packaging containers for germplasm
cryopreservation, many different types have been

cryopreservation of oysters with detailed summaries(Hassan et al., 2015).

used for molluscan germplasm cryopreservation in

Overall, the germplasm materials cryopreserved

previous publications, such as French straws (0.25 ml,

include sperm, oocytes, embryos, and larvae, covering

0.5 ml, and 5 ml), cryovials, and cryo-plastic bags.

almost all of the life forms of germplasm (Table 1).

The choice of packaging containers need to be

However, no research has been found in molluscan

facilitated with the accuracy of cooling rates, sample

shellfish on cryopreservation of primordial germ cells

biosecurity, high-throughput processing, freezing andor gonad tissues, which has been developed in fish tothawing process, and efficiency of storage space.

generate gonads through transplantation of germ
cells in surrogate or sterile mothers (Okutsu et al.,Results and discussion

2007; Majhi et al., 2009). This is probably because of
the advantages of mollusks over finfish for egg,

Since the first report of sperm cryopreservation inembryo or even larval cryopreservation. For fish,

the Pacific oyster Crassostrea gigas in 1971 (Lannan,

egg or embryo cryopreservation still remains as a1971), a total of 79 publications have been publishedhuge challenge due to the size, yolk contents, andon germplasm cryopreservation in molluscan shellfish

meroblastic embryo cleavage (Yang and Tiersch,

Table 1. Summary about publications on molluscan shellfish germplasm cryopreservation
FamilyOstreidae

Mytilidae

Pteriidae
Pectinidae
Veneridae
Mactridae

Haliotidae

Species
Crassostrea gigas
Crassostrea virginica
Saccostrea cucullata
Crassostrea tulipa
Crassostrea iredalei
Crassostrea rhizophorae
Saccostrea glomerata
Ostrea edulis
Choromytilus chorus
Mytilus edulis
Perna canaliculus
Mytilus galloprovincialis
Mytilus trossulus
Pinctada fucata martensii
Pinctada margaritifera
Argopecten purpuratus
Pecten maximus
Tapes philippinarum
Meretrix lusoria
Spisula sachalinensis
Haliotis gigantean
Haliotis discus
Haliotis diversicolor
Haliotis iris
Haliotis rufescens
Haliotis midae
Haliotis laevigata

Number of
publication
30
9Materials for
Cryopreservation
Sperm, oocytes, embryos, larvae
Sperm and larvae

1971-2014
1973-2014

1

Sperm

1991

1
1
2
1
1
5
3
1
6
4
1
1
1
1
1
1
1
6
1
1
1
3

Sperm and embryos
LarvaeSperm and trochophores
Embryos
Embryos
Sperm, oocytes, trochophores
Sperm, trochophores
Trochophores
Sperm and larvae
Sperm
Sperm
Sperm
LarvaeEmbryos and larvae
LarvaeSperm
Sperm
Sperm, oocytes, and embryos
Sperm
Sperm
Embryos
Sperm

2005
2008
2011, 2012
1988
1989
2009-20142009-20132009
2003-20122005-2013
2010
2014
1992
1997
2008
1983
1983
1992-2013
2000
2005
2008
2014

Year

18

Huiping YANG
References

2009). For molluscan shellfish, a recent study with
the Pacific oyster showed the success of larval
cryopreservation, and post-thaw trochophore larvae

Adams S. L., Smith J. F., Roberts R. D., Janke A. R.,

grew into mature adults and spawned naturally

King N. G., Tervit H. R., and Webb S. C., 2008:(Suquet et al., 2014).

Application of sperm cryopreservation in
selective breeding of the Pacific oyster,

Outlook for future research

Crassostrea gigas (Thunberg). Aquac. Res. 39,
1434-42.

Although many publications have reported

Bernard A. and Fuller B. J., 1996: Cryopreservation ofresearch on molluscan shellfish germplasm

human oocytes: A review of current problems

cryopreservation, most research still has limitations

and perspectives. Hum. Reprod. Update 2, 193-

that need to be addressed through future research.

207.

The limitations could be generalized as: 1) only very

Curry M. R., 2000: Cryopreservation of semen fromsmall laboratory-scale trials were reported in almost

domestic livestock. Rev. Reprod. 5, 46-52.

all of the reports; 2) post-thaw fertilization data was

Di Santo M., Tarozzi N., Nadalini M., and Borini A.,

not included in most research to test the effectiveness2012: Human sperm cryopreservation: Update onof cryopreservation protocols; 3) no standardized

techniques, effect on DNA integrity, andsample packaging containers were used for possible

implications for ART. Adv. Urol. 2012, 12.

high-throughput application, and 4) most factors in

Dong Q., Huang C., Eudeline B., and Tiersch T. R.,

the freezing process were descriptive without

2005: Systematic factor optimization for

quantification for protocol standardization andcryopreservation of shipped sperm samples ofrepetition, such as sperm concentration, sample

diploid Pacific Oysters, Crassostrea gigas.

volume, and cooling rate. These aspects will be thefuture research directions in this field.
Since the initiation of sperm cryopreservation in

Cryobiology 51, 176-97.
FAO. 2012. Year book, Fishery and Aquaculture
Statistics. URL http://www.fao.org/fishery/

the Pacific oyster in 1971, it has been about 40 years.

statistics/programme/publications/all/en.

However, cryopreservation of molluscan shellfish

Hassan M. M., Qin J. G., and Li X. X., 2015: Spermspecies still remains essentially a research activity

cryopreservation in oysters: A review of its

with little commercial application. For livestock

current status and potentials for futurespecies, sperm cryopreservation has grown into aapplication in aquaculture. Aquaculture 438, 24-

billion-dollar global industry in about 50 years since32.

its initiation. Compared to livestock and fishes, most

Lannan J. E., 1971: Experimental self-fertilization of

aquaculture molluscan shellfish have the potential

Pacific oyster, Crassostrea gigas, utilizing

advantage for oocyte, embryo and larva
cryopreservation.

cryopreserved sperm. Genetics 68, 599-601.

Therefore, germplasm

Lawson N. D., 2016: Reverse genetics in zebrafish:

cryopreservation will have great application in

Mutants, morphants, and moving forward.

research projects, breeding programs, commercial

Trends Cell Biol. 26, 77-9.

industry, and natural resource conservation.

Majhi S. K., Hattori R. S., Yokota M., Watanabe S., andMolluscan shellfish have traditionally been a major

Struessmann C. A., 2009: Germ cellcomponent of world aquaculture, its production intransplantation using sexually competent fish:

2012 (15.2 million tonnes) accounted for about 22.8% of

An approach for rapid propagation of endangered

the total (inland and marine) aquaculture production

and valuable germlines. Plos One, 4.

and 60.3% of the world marine aquaculture production

Okutsu T., Shikina S., Kanno M., Takeuchi Y., and(FAO, 2012). It is predictable that germplasm

Yoshizaki G., 2007: Production of trout offspring

preservation will be a useful technology to enhanceand boost the molluscan shellfish industry.

from triploid salmon parents. Science 317, 1517-.
Suquet M., Labbe C., Puyo S., Mingant C., Quittet B.,
Boulais M., Queau I., Ratiskol D., Diss B., andGermplasm preservation for shellfish aquaculture

19

Haffray P., 2014: Survival, growth andtreatment for infertility, and bull sperm

reproduction of cryopreserved larvae from acryopreservation which is now a huge industry for its

marine invertebrate, the Pacific oyster

breeding programs worldwide. For fish, the first

Crassostrea gigas. Plos One 9.

report was published after this report in 1953 inTiersch T. R., Yang H., Jenkins J. A., and Dong Q.,

herring (Blaxter J. H. S., 1953: Sperm storage and2007: Sperm cryopreservation in fish andcross-fertilization of spring and autumn spawningshellfish. In: Spermatology (Soc Reprod Fertil

herring.

Supplement 65) (eds. by E.R.S. R and Gomendio

preliminary communication showed that it wasM), Nottingham University Press, Nottingham,possible to cross-fertilize the two spawning ‘types’ of

pp. 493-508.

herring Clupea harengus found in the north-east

Yang H. and Tiersch T. R., 2009: Current status of

Nature, 172, 1189 – 1190), and thisAtlantic by using the cryopreserved sperm.

sperm cryopreservation in biomedical researchfish models: zebrafish, medaka, and Xiphophorus.

(3) Tiersch T. R. and Green C. C., 2011:

Comp. Biochem. Physiol. C, Pharmacol. Toxicol.

Cryopreservation in Aquatic Species, World

Endocrinol. 149, 224-32.

Aquaculture Society, Baton Rouge, LA, pp.1034.

Yang H., Wang Y., Guo X., and Tiersch T. R., 2015:

This is the 2nd version of the book published

Production of inbred larvae through self-

electronically by the World Aquaculture Society

fertilization using oocytes and cryopreserved(www.was.org). The first version is the volume 7 in

sperm from the same individuals after sex

the series of publication “Advances in World

reversal in eastern oyster Crassostrea virginica.

Aquaculture” published in 2000. The revised version

Aquac. Res., 46, 2153-65.

is designed as a comprehensive single compendiumof information on cryopreservation in aquatic species,

Annotated bibliography

and provides a broad overview of the principles,
procedure and perspectives which are necessary for(1) Fuller B., Lane N., and Benson E., 2004: Life in thedevelopment and application of cryopreservation

Frozen State, CRC Press, New York, 672pp.

technology. It includes 101 chapters (compared to

Cryobiology is a research field encompassing

the 55 in the first edition) organized into 11 sections.

disciplines including mathematics, biophysics, cellThis book can assist with teaching, research andbiology, molecular biology, and metabolism

program development, and is available for readers tophysiology. This book brings together the knowledge

purchase through the World Aquaculture Society

about cryobiology from these aspects into one

with a very affordable price for students andplatform for readers to understand this research, andresearchers.

is probably the first major textbook on cryobiology.
It includes four themes with a total of 23 chapters,(4) Yang H., Hu E., Cuevas-Uribe R., Supan J., Guo X.,

and the four themes are Fundamental Aspects, Lifeand Tiersch T. R., 2012: High-throughput spermand Death at Low Temperatures, Freezing andcryopreservation of eastern oyster Crassostrea

Banking of Living Resources, and Medicalvirginica. Aquaculture 344-349, 223–230.

Applications.

This study provided a reliable protocol for sperm
cryopreservation in the eastern oyster Crassostrea(2) Polge C., Smith A. U., and Parkes A. S., 1949:

virginica with potential for high-throughput

Revival of spermatozoa after vitrification andprocessing. In this study, DMSO yielded the highest

dehydration at low temperatures. Nature 164, 666.

post-thaw motility at a cooling rate of 20 °C/minThis is the first paper to report the successfulwhen thawed at 30 or 40 °C among the three tested

cryopreservation and cold-drying of sperm from fowl

cryoprotectants. Further evaluation of cooling rates

by using glycerol as cryoprotectant. The finding inof 10, 15, 20, 25 and 30 °C /min showed that 20 or 25

this one-page short report opened the door for human°
C /min yielded the highest post-thaw motility (34 ±sperm cryopreservation which is now a major clinical5%) and fertility (77 ± 12%) for French straws and20

Huiping YANG

CBS straws (28 ± 3% and 69 ± 14%). Equilibration

use of non-lethal sperm collection andtimes of 10 to 60 min did not cause significant

cryopreservation. In this study, small (~1 year old)

differences in post-thaw motility when freezing withand large (~2–3 years old) oysters were biopsied for

10% DMSO at a cooling rate of 25 degrees C/min.

sperm collection. Survival of the biopsied oysters

Also, sperm concentrations ranging from 1×108 to

after 1 year was 50% for small oysters and 17% for

1×10 cells/ml at freezing did not cause significant

large oysters, and sex reversal was observed

differences in post-thaw motility.

9

Finally, after

bidirectional (from female to male and also from malethawing, sperm cryopreserved from 16 males withto female). Oocytes were collected from sex-reversed

this protocol showed 58 ± 24% fertility (from 18 tofemales, and self-fertilized with cryopreserved sperm.

86%) for French straws, and 54 ± 21% fertility for

Of the 24 cryopreserved samples, 14 individuals had

CBS straws (from 18 to 95%). Overall, this research≤ 1% fertility when crossed with oocytes fromprovided an outline template for developing a basic

unrelated females, indicating that the cryopreserved

protocol for sperm cryopreservation for any othersperm had reduced fertility. The other 10 individuals

molluscan shellfish species.

had a fertility of 39 ± 25% when crossed with oocytes
from unrelated females (non-selfing), but showed a(5) Yang H., Wang Y., Guo X., and Tiersch T. R., 2015:

significantly lower success of self-fertilization (12 ±Production of inbred larvae through self-fertilization16%) (P = 0.008), while aliquots of the same oocytes

using oocytes and cryopreserved sperm from thehad a fertilization of 83 ± 11% when crossing withsame individuals after sex reversal in eastern oyster

fresh sperm. Larvae were produced in the self-

Crassostrea virginica. Aquac. Res. 46, 2153-2165

fertilized families (12–94% of the fertilized oocytes),

The authors in this study reported the production

and survived to eyed-larvae stage at days 11–14.

of self-fertilized larvae in eastern oyster Crassostrea

Genotyping with 9 microsatellite markers confirmed

virginica for the first time, and demonstrated thethat the larvae resulted from self-fertilization in four

feasibility of creating self-fertilized inbred lines byfamilies.

水 研 機 構 研 報， 第 45 号，21 － 27， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，21－27，201721

Improving Aquaculture Production in Haliotis Species through theDevelopment of a Genomic Toolkit
Catherine PURCELL＊1, Andrew SEVERIN＊2, Paula SYLVIA＊3, Nick WEGNER＊1, and John HYDE＊1

Abstract: 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
and improved 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 species
including the domestically important red abalone, H. rufescens. Recently, researchers working with
the Southwest Fisheries Science Center (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. However, transcriptomic and RAD-mapping analyses were also
limited by 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. 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 conduct comparative genomic analyses withother 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 our understanding 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 for marker assisted selection to improve aquaculture
practices in the U.S. abalone industry and elsewhere. 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.
Key words: Haliotis, Haliotis rufescens, Haliotis sorenseni, genome assembly, RNA-Seq

Introduction

Cook, 2013). Most domestic abalone farms operate in
California and utilize three native west coast species:

In the U.S., approximately 200 metric tons ofred (Haliotis rufescens) (Fig. 1), green (H. fulgens), andfarmed abalone are produced annually (Gordon andpink abalone (H. corrugata) (Allsopp et al, 2011) (Fig.2).

2017年２月28日受理（Received on February 28, 2017）	National Marine Fisheries Service, 8901 La Jolla Shores Dr., 92037-1508 La Jolla, CA, U. S. A.
＊2Iowa State Genome Informatics Facility, Ames, IA 50011, U. S. A.
＊3Port of San Diego, 3165 Pacific Hwy, San Diego, CA 92101, U. S. A.
E-mail: catherine.purcell@noaa.gov＊1

22

Catherine PURCELL, Andrew SEVERIN, Paula SYLVIA, Nick WEGNER, and John HYDE

The dominant culture species, red abalone, grows

Conte, 1996).

quickly and reaches a large size in culture; popular in

Although abalone culture is rapidly growing, it has

the U.S. market, they are also one of the mostbeen hindered by several bottlenecks that limit

valuable species in the mollusk industry globally

production capacity and efficiency in this industry.

(Robertson, 2012; Aguilar-Espinoza et al., 2014;

Improvements to the abalone stock in disease

Brokordt et al., 2015b). Of the three species, redresistance and the other economically important

abalone is the most temperate while the green andtraits are imperative to reducing production costs

pink abalone have more southern distributions andand to accelerating growth of the abalone industry,

are of greater interest for aquaculture production inparticularly in the United States (Arai and Okamura,Southern California and Mexico, where they can be2013). The ability to overcome these bottlenecks willgrown at higher water temperatures (McBride andbe significantly improved with better genomic
resources for abalone. When correctly applied, thesetechniques may rapidly improve broodstock selection,
characterize variation (both beneficial anddetrimental), and provide methods to directly
improve the value, efficiency, and production in thetarget species.
Considerable genetic research has been conducted
for these species.

Genetic markers have been

generated to characterize variation in wild andfarmed populations, characterize variation in traits ofinterest, and identify QTLs for important traits (e.g.,Kang et al., 2010; Rhode et al., 2012; Aguilar-Espinoza
et al., 2014; De Wit and Palumbi, 2013; Gruenthal et al.,Fig. 1. Red abalone (Haliotis rufescens)2014).

SWFSC partners have been developing

Fig. 2. Green abalone (H. fulgens) (a), white abalone (H. sorenseni) (b), pink abalone (H. corrugata)(c), and black abalone (H. cracherodii) (d)Development of a genomic toolkit for Haliotis species

23

suitable parentage and kinship microsatellite panels,

an important step toward understanding of the

genotyping broodstock animals, and applying thesegenetic basis of traits that currently limit the growthmarkers to monitor survival in outplanted greenof domestic abalone culture (i.e., infectious disease,abalone.

Genetic linkage maps have also been

thermal tolerance, slow growth, and the difficulty inconstructed for several abalone species using various

determining sex). This research will greatly advancegenetic methods (e.g., amplified fragment length

the state of knowledge of abalone culture andpolymorphisms (AFLPs), microsatellites, and single

improve productivity in the U.S. abalone industry.

nucleotide polymorphisms (SNPs)) (Liu et al., 2006;

The work will also provide insight for recovery

Sekino and Hara, 2007; Vervalle et al., 2013). Whileefforts for the decimated endangered white and black

linkage-mapping and other genetic studies haveabalone populations.

started to improve culture practices for abalone, theMethods

usefulness of these studies is restricted by the low
marker density that limits an understanding of
mechanisms controlling traits of interest (Jones et al.,2013).

Mature and sexed live red abalone specimens will
be sampled for both the genomic and transcriptomic

Next generation sequencing has opened the door forsequencing. Sampling for the transcriptomic work

extensive and explorative genetic research on abalone,

will involve collection of several tissue types (e.g. footand several studies were recently published examining

muscle, gonad, digestive gland, epipodium). Sexed

transcriptomic data for species including the(male and female) pink, green, white and black

domestically important red abalone (Franchini et al.,abalone specimens were collected from the abalone

2011; Bester-Van der Merwe et al., 2013; De Wit andculture tanks at the SWFSC and from the California

Palumbi, 2013). Recently, researchers working with the

Department of Fish and Wildlife.

SWFSC used restriction site associated DNA

Genomic DNA samples were stored in vials

sequencing (RAD-Seq) methods to identify genome-

containing 95% ethanol; RNA samples submerged inwide SNP markers in green abalone and examine

RNAlater (Qiagen) and stored at -20 °C until use.

population structure in wild populations (Gruenthal et

Genomic DNA and RNA were extracted usingal., 2014). However, transcriptomic and RAD-mapping

DNeasy, Genomic Tip, and RNeasy kits (Qiagen),analyses were also limited by the paucity of genomic

respectively.

information available for abalone; without knowledge of

Following preparation of the abalone specimens

the genomic structure, it is very difficult to ascertain

and quality assessment, the DNA and RNA samples

coverage depth in these studies (Franchini et al., 2011;were sent to the DNA facility at ISU (Ames, IA) for

Arai and Okamura, 2013). Additionally, abalone species

Illumina and PacBio library preparation andare poorly represented in public genomic resource

sequencing (Fig. 3). Sequencing will be conducted

databases compared with other aquaculture species

with the Illumina Hiseq 2500 on two lanes of 150 bp(Franchini et al., 2011), further limiting usefulness of thepaired-end read data and four lanes of Illumina mate-available data.

paired end libraries, with large insert sizes of

To develop the genomic resources that haveapproximately 12 kb, for at least 100X genomic

benefited other breeding programs, we are working

coverage. Twenty smart cells of PacBio data will beto generate a de novo genome assembly for thecollected for approximately 5x coverage of thecommercially valuable red abalone, generate tissue

genome with expected average read lengths of 6 kb.

specific transcriptomes in red abalone, and conduct

The resequencing of the other abalone species willcomparative genomic analyses through resequencing

involve four lanes of 100bp paired-end Illumina

the pink, green, black (H. cracherodii) and white (H.

sequencing. For all sequencing data, half of thesorenseni) abalone. Genomic resource development inlanes/smart cells will be male and half will be female.

red abalone will help identify and characterize

Raw sequencing reads will be assembled witheconomically and/or biologically relevant variation

AllpathsLG (Gnerre et al., 2011) and MaSuRCA (Nealeamong the abalone species. This information will be

et al., 2014). The PacBio reads will either be error

24Catherine PURCELL, Andrew SEVERIN, Paula SYLVIA, Nick WEGNER, and John HYDE

Fig. 3. The sequencing strategy using Illumina and PacBio technologies to generate thered abalone (H. rufescens) genome
corrected and included in the genome assembly as
long “Illumina” reads, or used in super scaffolding

Results and discussion

after assembly. Transcripts will be assembled denovo from the available transcriptome data using

Abalone samples are currently being prepared for

Trinity (Haas et al., 2013). These transcripts will besequencing and therefore no sequencing results arealigned to the draft genome produced in the firstavailable at this time. However, it is expected thatstep. Transcripts that map uniquely to the ends ofthe de novo assemblies of the resequenced species

two scaffolds will be used as evidence to join the two

will be explored for gene loss or gain of candidate

scaffolds. If the transcript alignment spans multiple

genes that are known or suspected to be involved insmall scaffolds (one or two exons per scaffold),sex, disease resistance, thermal tolerance, and growthmultiple small scaffolds could be joined. We will use

rates (Brokordt et al., 2015a; Choi et al., 2015; Liang et

L_RNA_scaffolder (Xue et al. 2013) to perform thisal., 2014; Zippay and Hofmann, 2010; Klingbunga et al.,step. Gene models will be generated using MAKER

2009; Liu et al., 2006). Comparison of synonymous and(Holt and Yandell, 2011) genome annotation pipeline.

nonsynonymous nucleotide substitution rates in

The final set of genes will be predicted combining thehomologous genes will be performed to measure theresults from all the predictors, EST evidence, protein

evolutionary distance among the five abalone speciesalignment and BLAST similarity to other closely

and detect genomic regions under selective pressure.

related genomes.

Genes undergoing selective pressure will be

Raw reads from the other four species will beinteresting from an aquaculture perspective as theyaligned to the red abalone genome using GSNAP (Wumay relate to local adaptation and relevant genetic

and Nacu, 2010). Single Nucleotide Polymorphisms

variation among the species (e.g. thermal tolerance,(SNPs) and Insertions/Deletions (InDels) will be called

disease resistance).

using GATK (McKenna et al., 2010). A pseudogenome

Identification of sex determining regions that areof each of the four species will be generated using thespecific to one sex will also be explored using thealigned read file and the SNPs file. The pseudo

coverage depth of aligned reads to the red abalone

genome assemblies will be directly comparable togenome assembly. Since one male and one female

each other and have annotations that correspond towill be sequenced for each species, regions of theannotations in red abalone, which will provide

genome that show reduced coverage (typically one-straightforward cross-referencing among red, green,

half of the coverage for each sex, or one-fourth of thepink, white, and black abalone gene models.

coverage of the combined male/female raw reads)

Development of a genomic toolkit for Haliotis species

25

compared to the rest of the genome are prime

and Watts E., 2011: Abalone culture: In Recent

candidates for a sex-determining region (Vicoso et al.,advances and new species in aquaculture (eds.

2013). Markers in these regions will be designed andFotedar R. K. and Phillips B. F.), Wiley-Blackwell,tested by SWFSC to determine if a sex specific

Oxford,. 416pp.

marker can be identified. This will be done for all

Arai K. and Okumura S. I., 2013: Aquaculture-

five species where male and female individuals canoriented genetic researches in abalone: Current

be identified for sequencing.

status and future perspective. African J. Biotech.

To achieve the greatest impact on sustainable

12, 4044-4052.

abalone aquaculture, we will create a genomic

Bester-Van Der Merwe A., Blaauw S., Du Plessis J.,

toolbox for abalone species that facilitates theand Roodt-Wilding R., 2013: Transcriptome-wide

integration of very large sequencing data sets,

single nucleotide polymorphisms (SNPs) for

molecular markers, QTL data and genetic maps into

Abalone (Haliotis midae): validation andan easy-to-use web interface.

The website will

application using GoldenGate medium-include a Genome Browser utilizing JBrowse (Skinner

throughput genotyping assays. Int. J. Mol. Sci.

et al., 2009) for visualization of the red abalone

14, 19341-19360.

genome assembly and tracks that display the raw

Brokordt K., González R., Farías W., and Winkler F.,

read and de novo assembled alignments of the other2015a: Potential response to selection of HSP70 as

species. The genome assembly for red abalone anda component of innate immunity in the abalone

the pseudo genome assemblies for the other abalone

Haliotis rufescens. PloS One 10, e0141959.

species will be available for download for each species

Brokordt K. B., Winkler F. M., Farìas W. J., González

and deposited in the appropriate databases (e.g.

R. C., Castaño F., Fullsack P., and Herbinger C.

NCBI-GenBank).

M., 2015b: Changes of heritability and genetic

These tools will enable abalone aquaculture tocorrelations in production traits over time in red

move past the primary bottlenecks in production by

abalone (Haliotis rufescens) under culture.

moving toward a marker assisted selection model

Aquac. Res. 46, 2248-2259.

with breeding and/or hybridization programs that

Choi M., Kim D., Kim J. and Lim H., 2015: Differentially-

exploit natural diversity in abalone species toexpressed genes associated with faster growth of

improve production capacity and efficiency. By

the Pacific abalone, Haliotis discus hannai. Int. J.

selecting individual abalone or developing hybrids

Mol. Sci. 16, 27520-27534.

with desirable traits (e.g., disease resistance, thermal

De Wit P. and Palumbi S., 2013: Transcriptome-wide

tolerance, and growth rate) the value of thepolymorphisms of red abalone Haliotis rufescens)aquaculture product will be greatly improved. In

reveal patterns of gene flow and local adaptation.

support of conservation and restoration efforts, theMol. Ecol. 22, 2884-2897.

developed genetic toolkit can be used to monitor theFranchini P., van der Merwe M., and Roodt-Wilding

health and viability of wild populations and guideR., 2011: Transcriptome characterization of thebreeding programs for stock replenishment.

South African abalone Haliotis midae using
sequencing-by-synthesis. BMC Res. Notes 4, 59.

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Xue W., Li J.-T., Zhu Y.-P., Hou G.-Y., Kong X.-F.,

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assembly that resulted in 27,761 contigs with anscaffolder: scaffolding genomes with transcripts.

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Annotated bibliography

sequences were assigned to functional categories
using GO and KOG databases. The authors were also

(1) De Wit P. and Palumbi S., 2013: Transcriptome-

able to use this data to identify thousands of SNPs,

wide polymorphisms of red abalone (Haliotis

and out of those, they developed 420 primer sets.

rufescens) reveal patterns of gene flow and local
adaptation. Mol. Ecol. 22, 2884-2897.

(3) Gruenthal K., Witting D., Ford T., Neuman M.,

The authors of this study tested whether signals of

Williams J., Pondell, D., Bird A., Caruso N., Hyde J.,

environmental selection could be detected in samples

Seeb L., and Larson W., 2014: Development andof red abalone (Haliotis rufescens) collected fromapplication of genomic tools to the restoration of

three locations in California: Monterey Bay, Sonoma,

green abalone in southern California. Conservat.

and north of Cape Mendocino. These particular areas

Genet. 15, 109-121.

are especially distinct in terms of their temperature,

The authors describe the use of next generation

aragonite saturation, exposure to hypoxia stress, andsequencing technology to develop an extensive set of

disease pressure; as such, the authors hypothesizedmarkers to test population structure and effectivethat genes related to shell biomineralization,

population size in green abalone in Southern

resistance to hypoxia, temperature tolerance, andCalifornia. In this study, RADSeq restriction site

resistance to pathogens would show the strongest

associated DNA sequencing) was used to generatesignals of local adaptation. The authors tested this by

millions of short sequences, from which, many

conducting RNA-Seq analyses on mantle tissue of 39

thousands of SNPs may be identified that span a

red abalone individuals from the above locations. A

greater proportion of the genome compared tototal of 21,579 SNPs were genotyped for eachprevious types of marker development. A total ofindividual, and out of these 691 showed significant

1209 polymorphic SNPs were developed from thisdifferentiation. From this set of 691, 163 loci could besequencing. While the extensive set of markers did

identified through BLAST annotation; many of thesenot detect population structure in green abalone ingenes had functions related to biomineralization,

the range that was sampled, they were able toenergy metabolism, heat-, and disease- or hypoxia-

estimate an effective population size Ne) of 1,100-3,600

tolerance.

individuals.

These genes are now candidates forfurther studies to look for signals of local adaption.

Importantly, this work generated

valuable genomic resources that can be used tofurther build the set of tools available to study(2) Franchini P., van der Merwe M., and Roodt-

Haliotis species.

水 研 機 構 研 報， 第 45 号，29 － 31， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，29－31，2017

29

Culture Protocols and Production of Triploid Purple-Hinge RockScallops
Paul G. OLIN＊

Abstract: 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 thenative 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 are also
genetic concerns associated with rearing native species for aquaculture using hatchery-reared seedthat 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 Davis in
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 early as a limiting factor. Subsequent efforts relied on amixed diet of C-Isochrysis sp., Nannochloropsis sp., Pavlova sp., Chaetoceros sp., and Thallasiosira 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. Theoptimal dosage of 6-DMAP for production of 3N scallops by inhibition of second polar body extrusion
has been determined to be 425 uM.
Key words: Purple-hinged rock scallop, Crassadoma gigantea, triploid, 6-DMAP

Annotated bibliography

Canadian Technical Report of Fisheries and Aquatic
Sciences, 1694pp.

(1) Bourne N., Hodgson C. A., and White J. N. C., 1989:
A manual for scallop culture in British Columbia.

A comprehensive manual for hatchery productionof scallops.

2017年２月28日受理（Received on February 28, 2017）	California Sea Grant, UCSD / Scripps Institution of Oceanography, 133 Aviation Blvd., Suite 109, Santa Rosa CA 95403, U. S. A	E-mail: polin@ucsd.edu

＊1

30

Paul G. OLIN
of sea scallop spat (Placopecten magellanicus) in(2) Helm M.M. and Bourne,N., 2004: The hatcheryculture of bivalves: a practical manual. FAO Fisheries
Technical Paper, 471pp.
http://www.fao.org/docrep/007/y5720e/y5720e09.
htm.
A comprehensive manual for hatchery productionof bivalve shellfish.

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. Four annual productionseasonal 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(3) University of Bergen, 2004: Mortalities in a2 – 25% of the planned target of 20 million spat per

hatchery of the great scallop, Pecten maximus.

year. This presentation will report our experiences

University of Bergen, Department of Biology, Bergen,in 1998 regarding algal culture, broodstock

Norway.

conditioning, larval growth, spat settlement andA bacteriological study was carried out in a scallop

growth, and bacteriological monitoring of water

hatchery in western Norway. The hatchery had

quality. Algae (flagellates and diatoms) were grown

suffered severe mortalities during the larval stages,

in a continuous culture system purchased fromand prophylactic use of antibacterial agents was

Seasalter Shellfisheries in England. The intent was tonecessary to produce larvae. A number of bacterial

provide scallop larvae and spat with a diet of mixed

strains were isolated from the hatchery. A challenge

species of varied lipid and carbohydrate content. The

test was performed with the isolates. Six of thefollowing flagellates and diatoms were cultured instrains caused mortalities not statistically different

500-L polyethylene bags under continuous light at 22

from Vibrio pectenicida, a known scallop pathogen.

degree C with the addition of CO sub(2): Isochrysis

From 16s rDNA analysis on these strains, thegalbana, T.Iso, Pavlova lutheri, Tetraselmis suecica,phylogenetic tree indicated two groups of apparent

Chaetoceros calcitrans, C. ceratosporum, C. muelleri,pathogens; one strain that resembles theThalassiosira pseudonana, T. weissflogii, andAlteromonas/Pseudoalteromonas group and a cluster

Chroomonas salina.

of strains that resemble the species Vibrio splendidus.

approximately 0.30 and 0.35 divisions/day for the

Since the antibacterial agent used in the hatcheryflagellates and diatoms respectively.

The growth rate waswas chloramphenicol, which is now banned in
Norway and Europe, application of alternative(5) Meng Q., Bao Z., Wang Z., Wang S., and Hu J., 2012:

antibacterial agents were investigated. In this study

Growth and reproductive performance of triploidthe minimum inhibitory concentration (MIC) values

Yesso scallops (Patinopecten yessoensis) induced byof chloramphenicol, florfenicol, fluemequine, thehypotonic shock. J. Shell. Res. 31, 1113-1122.

combination trimethoprim/sulfadiazine,

The successful induction of triploid embryos or

oxytetracycline, oxolinic acid and Pyceze on bacterial

larvae has been performed in Patinopecten yessoensis

strains isolated from scallop larvae were investigated.

during the past two decades. However, no research has

Based on these MIC values, procedures for the

been reported about the performance of triploid P.

treatment of scallop larvae with antibacterial agents

yessoensis cultured in the field. This study induced

were evaluated. Since the therapeutic procedures

triploidy in P. yessoensis by hypotonic shock andwill be used in a marine environment, thecompared the growth and reproductive performance ofantagonizing effect of seawater on some of thetriploids and diploids reared under commercial

antibacterial agents was measured. For fluemequineconditions for up to 24 months. The main results of thisand trimethoprim/sulfadiazine the average MIC

study are as follows: Triploid scallops were smaller invalues increased significantly when using seawater

size and weight compared with diploids and had awith salinity of 25 ppt compared to 2% NaCl.

retarded absolute growth rate (AGR). After 24 monthsof cultivation, the mean shell height, shell length, shell(4) Caines J. and Crocker K. 1999: Hatchery production

width, and body weight of triploids were 9%, 10%, 9%,Triploid rock scallop aquaculture31

and 25% less than diploids, respectively (P < 0.01).

concentrations of CB were evaluated for ability to

Although normal in sex ratio, the reproductive potential

produce 3N larvae. The efficacy of current andof triploids was significantly reduced. Only 87% of the

proposed 3N induction techniques (i.e., 4N x 2N

triploids exhibited sex-discernible gonads during thecrosses) and the commercial potential of 3N bay

breeding season. None of the male triploids spawned,

scallops are discussed.

and the percentage of female spawners among thetriploid population was only 27% of that for the diploid(7) Ruiz-Verdugo C. A., Allen S. K., and Ibarra A. M.,

population. The relative fecundity of triploid females

2001: Family differences in success of triploidwas only 4% of diploid females. Triploid eggs produced

induction and effects of triploidy on fecundity ofmostly aneuploid larvae and had an extremely small

catarina scallop (Argopecten ventricosus). Aquaculture

chance of generating viable offspring when fertilized by

201, 19-33.

sperm from diploid males. Most aneuploid larvae died

Mass induction of triploidy in the catarina scallop

before the D-shaped stage, and no survival exceeded(Argopecten ventricosus) results in low success in theseven days. The potential to yield viable offspring frompercentage of triploids produced. To understandthe triploid population was estimated to be only 4% of

whether this is a treatment effect affecting all eggs

that of the diploid population. Despite the growthequally, families were individually induced to triploidy

disadvantage of triploids, this study may support, inwith cytochalasin-B (CB), comparing their survival frompart, the energy reallocation hypothesis because triploid

egg to D-larvae and spat and the percent of triploidy

AGR was similar to diploid AGR (2% variance) during

within families. Differences in percent triploidy successthe sexual maturation season. Our results also indicate

were evident between families, obtaining some with nothat there would be no growth advantage, but instead a

triploids and some with high triploidy. Among thedisadvantage, for triploid P. yessoensis growing at thepossible causes for these differences are quality of eggs,

experiment site. In addition, this research provides thedifferent developmental rates, and differences infirst evidence that viable triploid molluscs can besusceptibility to the treatment (CB or DMSO) itself.

induced by hypotonic shock, of which the practical andRegardless of those differences, overall triploidy

evolutionary implications are also discussed.

production was increased by inducing individually eggs
of each scallop rather than in mixed egg batches. In the(6) Cogswell A. T., Roach S. E., and MacDonald B. W.,

first experiment, it was improved by 17%, and in the

2006: Triploid bay scallops (Argopecten irradians):

second experiment by 42%, as indicated by the

induction methodology, early gonadic development

weighted mean of triploids among the families andand growth. Canadian Technical Report of Fisheries

when compared with previous results with this sameand Aquatic Sciences, 2635pp.

species, where triploidy success was 58%. In a second

Triploid (3N) Pacific oysters (Crassostrea gigas)

experiment with three different families, the growthaccount for more than 50% of total oyster production

and fecundity of triploid and diploid catarina scallopin the US. The success of this species has created an

were evaluated. The growth superiority of triploids

interest in producing triploids of other commercial

was confirmed. The results indicated that triploidshellfish species, including scallops, clams andcatarina scallop had a significantly reduced fecundity

mussels. Here, we report on 3N induction trials withwhen compared with diploid scallop. The reduced

the bay scallop, Argopecten irradians. The most

fecundity appears to be mostly of a random nature,commonly employed 3N induction technique involves

possibly associated with a reduced capability to produce

exposing early embryos to chemicals (e.g.,balanced gametes. Whereas the successful production

Cytochalasin B (CB)). CB inhibits the release of theof tetraploid catarina scallop from fertile triploid scallop

second polar body immediately following fertilization,is in principle possible, the low number of eggs shed bycausing retention of both pairs of female chromosomes

triploid catarina scallop could diminish that success rate,

in addition to the male chromosome set. It does thiseven more if single triploid females are required toby disrupting actin polymerization. Four

optimize tetraploid induction.

水 研 機 構 研 報， 第 45 号，33 － 33， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，33－33，2017

33

Big Data in Agriculture and the USDA/ARS Initiative
Jeffrey SILVERSTEIN＊

Abstract: In recent years, technological achievements have permitted the relatively inexpensive andrapid production of vast amounts of data. The large and often complex datasets produced in thescientific sphere demand new approaches to gain value and to turn data into information.
Management of the growth 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, ourcomputational infrastructure has primarily been based on meeting administrative needs andsecurity requirements, whereas computational, analytical, and sharing of scientific data wereregarded as secondary priorities. The Big Data problem has required a reassessment of scientificcomputing 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 havedeveloped 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 constructeda high-performance computing (HPC) system with high memory, high processing capacity and thepotential to burst computational workloads to commercial resources when necessary. SCInet andthe HPC have been largely constructed and connected in the first 18 months of the initiative.
Finally, we are developing a virtual research support core of individuals who will provide scientificcomputational and informatics support. These individuals will work with agency scientists tofacilitate 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, and4) controlling costs due to restrictive budgets are challenges that will be shared and discussed.
Key words: Big Data, Internet2, informatics

2017年２月28日受理（Received on February 28, 2017）	Southeast Area, US Department of Agriculture, Agricultural Research Service, P.O. Box 225, Stoneville MS 38776, U.S.A.
	
E-mail: jeff.silverstein@ars.usda.gov＊1

水 研 機 構 研 報， 第 45 号，35 － 39， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，35－39，2017

35

Genomic Selection in Aquaculture
Sho HOSOYA＊1, Kiyoshi KIKUCHI＊1, Hiroshi NAGASHIMA＊2, Junichi ONODERA＊2,
Kouishi SUGIMOTO＊2, Kou SATOH＊2, Keisuke MATSUZAKI＊2, Masaki YASUGI＊3,
Atsushi J. NAGANO＊3,4, Akira KUMAGAYI＊2, Kenichi UEDA＊2, and Tadahide KUROKAWA＊5

Abstract: Recent advances in genotyping and sequencing technology facilitate handling thousandsof SNPs from hundreds of samples. This enables us genomic prediction of breeding values for
economically important traits not only for livestock and plants but also for aquaculture species. In
this article, we review six empirical studies on genomic prediction in aquaculture. Implementationof genomic selection for aquaculture is steadily progressing with no doubt, and we will see thetangible and practical outcomes of genomic selection within several years.
Key words: Genomic selection, NGS, selective breeding, SNP

Introduction

relatives of individuals of the training group. There
are two major methods for marker effect estimation:

Genomic selection (GS) introduced by Meuwissen et

GBLUP and BayesB. To use these methods, a prioral. (2001) is a form of marker-assisted selection where

distribution of marker effects is required, and one of

the genomic estimated breeding value (GEBV) isthe major differences between the two methods ispredicted as the sum of additive genetic effects of

the assumption of the distribution and variances ofgenome-wide dense genetic markers.

All loci

marker effects. Normal distribution with constantaffecting the trait (e.g. quantitative trait loci; QTL)

variance is assumed for GBLUP, and therefore theare supposed to be in linkage disequilibrium with atmodel is equivalent to a conventional BLUP (PBLUP)

least one marker. Therefore, to predict marker

animal model; a numerator relationship matrix

effects for highly polygenic traits, such as humanestimated from pedigree information is substituted

height and disease (Yang et al., 2015; Abraham et al.,by a realized relationship matrix estimated from2015), hundreds of thousands of genetic markers are

genome-wide SNP information in GBLUP. On therequired. When the idea was proposed for the firstother hand, non-normal distribution is assumed fortime, it was highly challenging to genotype thousands

BayesB, where only a subset of markers has effects

of markers from hundreds of specimens. However,and these effects follow a reflected exponential

recent advances in genotyping technology put it into

distribution.

practice.

prediction accuracy of these two methods superior to

Simulation data revealed that the

Succinctly, GS consists of two steps. The first stepthat of traditional BLUP (Meuwissen et al., 2001).

is estimation of marker effects in a test (training)This is mainly because genomic prediction takes thegroup, and the second step is prediction of GEBV of

Mendelian sampling term into account (Daetwyler etselection candidates (validation group), sibs and/oral., 2007). Because of its high prediction accuracy,

2017年２月28日受理（Received on February 28, 2017）
	Fisheries Laboratory, Graduate school of agricultural and life sciences, University of Tokyo. Hamamatsu, Shizuoka 431-0214, Japan.
	Miyagi Prefecture Fisheries Technology Institute,Freshwater Fisheries Experimental Station. Taiwa, Miyagi 981-3625, Japan.
＊3	Centerfor Ecological Research, Kyoto University, Otsu, Shiga 520-2113, Japan＊4
	Faculty of Agriculture, Ryukoku University, Otsu, Shiga 520-2194, Japan＊5
	Kushiro Laboratory, Hokkaido National Fisheries Research Institute, Japan Fisheries Research and Education Agency. Kushiro, Hokkaido 0850802, Japan.
E-mail: ahosoya@mail.ecc.u-tokyo.ac.jp＊1＊2

HOSOYA, Kiyoshi KIKUCHI, Hiroshi NAGASHIMA, Junichi ONODERA, Kouishi SUGIMOTO, Kou SATOH,
36 KeisukeSho
MATSUZAKI, Masaki YASUGI, Atsushi J. NAGANO, Akira KUMAGAYI, Kenichi UEDA, and Tadahide KUROKAWA
Table 1. Summary of species, traits, genotyping platform and prediction method researches about genomic
selection for aquaculture
Species

Trait

Genotyping platform

Prediction method

Citation

Atlantic salmon

parasite resistance

SNP array

GBLUP

Ødegård et al. (2014)(Salmo salar)fille color

Rainbow trout

body size

SNP array

GBLUP

Tsai et al. (2015)

parasite resistance

SNP array

GBLUP

Tsai et al. (2016)

disease resistance

SNP array, RAD

ssGBLUP, wssGBLUP, BayesB,

Vallejo et al. (2016)(Onchorhynchus mykiss)

BayesC

Yellow croaker

body size

Genotyping-by-Sequencing(Larimichthys crocea)

fatty acid composition(GBS)

Yesso scallop

body size

2d RAD-seq(Patinopecten yessoensis)
Coho salmon

GBLUP, emBayesB

Dong et al. (2106)

GBLUP, LASSO, Bayesian LASSO,

Dou et al. (2016)

BayesA, BayesB, rrBLUP
body size

ddRAD-seq

GBLUP

Hosoya et al. (submitted)(Onchorhynchus kisutch)* adjusted prediction accuracy: the correlation between the GEBV of the test population and the actual phenotypes divided by the square toot of theheritability

genomic selection is now widely used in livestock and2016). Additionally, dense SNP chips (Axiom® Salmon

plants, but there has been limited realization in

Genotyping Array, Affimetrix) are commerciallyaquaculture.

available. All those three studies used SNP chips forWith the aids of new genotyping technologies, suchgenotyping. Analyzed traits for this species are

as SNP array and restriction enzyme associated DNA

salmon lice (Lepeophtheirus salmonis) resistance (LR:sequencing (RAD-seq), genomic selection becomes

estimated heritabilities = 0.14) and fillet color

applicable not only for the major livestock species(estimated heritabilities = 0.43) (Ødegård et al., 2014);

and plants but also for aquatic species. So far, at least

weight (estimated heritabilities = 0.5) and length

six studies have reported the possibility of genomic(estimated heritabilities = 0.6) (Tsai et al., 2015); LR

selection in aquatic species (Table 1). In this report,(estimated heritabilities = 0.2 - 0.3) (Tsai et al., 2016).

we review these studies. Five are about finfish, and

GEBV for these traits were predicted by GBLUP.

the other is about scallop. Three of the studies wereRelatively high prediction accuracy was generated

done by researchers from North Europe, one wasfor traits with higher heritability. Interestingly, whilefrom North America, and the other from Mainlandover 50k SNPs are required to gain better prediction

China. All these studies have been published within

accuracy for livestock species, 5k SNPs are sufficient

the last couple of years, and more attempts of GS forfor Atlantic salmon within-family prediction.

a wide range of aquatic species will be published

However, for prediction across populations or year

from all over the world.

groups, where genetic relationships are more distant
between training and validation groups, accuracy

Atlantic salmon

was substantially low even when 30k SNPs wereAs mentioned above, there are three articles onused for the prediction (Tsai et. al., 2016). In such

Atlantic salmon, and one of them (Ødegård et al.,cases, using larger population sample sizes and2014) was the first report attempting to incorporate

higher-density SNP genotypes will improve

GS in aquaculture. Atlantic salmon is one of the most

prediction accuracy. Thus, phenotyping training

important aquaculture species as its estimated global

populations consisting of animals closely-related toeconomic values was $ 7.8 billion in 2010 (FAO, 2010).

the selection candidates and genotyping relatively

It is also known as a genomic model species forlower-density of SNPs (5k) will gain better costaquaculture as its chromosomal-level genome

performance for commercial salmon breeding

assembly (GCA_000233375.4) is available (Lien et al.,schemes. This strategy is supported by simulation

37

Genomic selection in aquaculture
studies (e.g. Sonesson and Meuwissen, 2009) andlarvae and the maintenance of separate families.

generally works for populations from mostSince the number of selection candidates is large andaquaculture breeding programs since relatively

the value of the selection candidate is low for

limited numbers of broodstock are used in most of

aquaculture species, the cost of pedigree recording

the aquaculture breeding programs.

may not be negligible, and this will be a major
obstacle to implement ssGBLUP into selective

Rainbow trout

breeding programs in aquaculture.

Vallejo et al., (2016) reported an attempt to
implement genomic selection for bacterial cold water

Large yellow croakerdisease (BCWD) resistance to the National Center for

Genomic selection is also attempted on non-

Cool and Cold Water Aquaculture (NCCCWA) BCWD

salmonid fish. Dong et al. (2016) reported theresistance breeding line. Several major resistancefeasibility of genomic selection in the traits of growth

QTL (including a QTL of PVE = 58%) have beenrate and meat quality (i.e. the percentage of n-3

detected for the trait using the same breeding line

highly unsaturated fatty acids (n-3HUFA) in muscle)(Palti et al., 2015). However, because of complex

of large yellow croaker (Larimichthys crocea). They

genetic architecture and high genetic variation, thefirst estimated heritability and then comparedauthors postulated that GS gives better performance

predictive ability between GBLUP and emBayesB.

for genetic improvement in BCWD resistance thanemBayesB is an alteration of BayesB. The Markov

the marker-assisted selection based on QTL analyses.

Chain Monte Carlo (MCMC) technique (i.e. Gibbs

Genomic predictions for survival days and survival

sampling) used in BayesB for the model fitting

status were done using single step GBLUP (ssGBLUP),

requires large computational time with dense SNP

weighted ssGBLUP (wssGBLUP), BayesB anddata. On the other hand, MCMC is replaced by the

BayesC. ssGBLUP is an integrated version of GBLUP

Expectation-Maximization (EM) algorithm in theand PBLUP. While GBLUP and PBLUP use either

emBayesB approach.

genomic or pedigree information to construct a

accurate GEBV prediction (Shepherd et al., 2010).

This enables us fast but

relationship matrix (i.e. G matrix and A matrix),

Estimated heritability for body weight, body length

ssGBLUP uses both types of information (H matrix)

and n-3HUFA were 0.604, 0.586 and 0.438, respectively.

(Aguilar et al., 2010). wssGBLUP is an extended

GBLUP was superior to emBayesB in the predictive

version of ssGBLUP, where QTL effects are weighted.

abilities for body weight and body length (GBLUP:

Overall predictive abilities were similar among the

BW = 0.41 and BL= 0.4; emBayesB: BW = 0.37 andGS models and PBLUP. ssGBLUP showed slightly

BL= 0.37), but not for the n-3HUFA trait with

better performance compared to the Bayesian

relatively lower heritability (GBLUP: 0.30; emBayesB:

methods. This will be partly because a large number

0.32).

of individuals was used for the training population in

number of QTLs affecting the traits; the number of

ssGBLUP. Construction of H matrix for ssGBLUP

QTLs is expected to be smaller for the n-3HUFA trait

seems somewhat complicated since it includes

than that for body weight and body length. The“tuning” steps. However, this step can increase theresults suggested the importance of testing

performance of genomic prediction.

Moreover,algorithms on specific traits to gain the best

because individuals with phenotype, but without

prediction performance. The authors also estimatedgenotype, data can be included in the trainingthat at least 1000 individuals in the training

population under ssGBLUP model, it is possible topopulation are required to get prediction accuracy of

increase the sample size of training population

0.8 by fitting the curve of prediction accuracy. The

without increasing genotyping cost.

number will be affordable when GBS is used for

Therefore,

ssGBLUP will be one of the most powerful solutions

The differences are probably due to the

genotyping.

for genomic prediction of GEBV. However, pedigree
records are not typically maintained in aquaculture
because of the difficulty in the tagging of individual

ScallopA species other than finfish tested for theHOSOYA, Kiyoshi KIKUCHI, Hiroshi NAGASHIMA, Junichi ONODERA, Kouishi SUGIMOTO, Kou SATOH,
38 KeisukeSho
MATSUZAKI, Masaki YASUGI, Atsushi J. NAGANO, Akira KUMAGAYI, Kenichi UEDA, and Tadahide KUROKAWA
possibility of implementation of GS is Yesso scallop

the livestock populations. Although GBLUP and(Patinopecten yessoensis) (Dou et al. 2016). In theBayesian methods showed good performance instudy, the performance of 2b-RAD sequencing

prediction accuracy, it will be better to compare themethods (a type of RAD-seq, or genotyped-by-

performance among between prediction models

sequencing GBS), where the uniform fragments

before deciding the model to use because theproduced by type IIB restriction endonucleases are

performance is largely depending on the number ofsequenced: Wang et al., 2012) was evaluated for shellsamples, population structure and heritability of thelength, shell width and shell height. The prediction

traits.

accuracies calculated under models of GBLUP,
References

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It seems a relatively small number of SNPs (~ 5k)

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practical levels of prediction accuracy. Our result on

D. R., Zimin A., Grammes F., Grove H., Gjuvslandcoho salmon (Hosoya et al., submitted) is alsoA., Walenz B., Hermansen R.A., von Schalburg K.,

supportive of this idea. The reason is partly because

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the generation is still young and LD size is large inJ., Vigeland M.D., Caler L., Grimholt U., Jentoft S.,

many aquaculture breeding populations compared toVåge D. I., de Jong P., Moen T., Baranski M., Palti

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Tooming-Klunderud A., Jakobsen K. S., Jiang X.,

Dong L., Xiao S., Wang Q., and Wang Z., 2016:

Fan D., Hu Y., Liberles D. A., Vidal R., Iturra P.,

Comparative analysis of the GBLUP, emBayesB,

Jones S. J., Jonassen I., Maass A., Omholt S. W.,

and GWAS algorithms to predict genetic values

and Davidson W. S. 2016: The Atlantic salmon

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D., and Palti Y. 2016: Evaluation of genome-

Fernando R. L., Davis J. M., Jokela E. J., Martin T.

enabled selection for bacterial cold water disease

A., Peter G. F., and Kirst M., 2012: Accuracy ofresistance using progeny performance data ingenomic selection methods in a standard data set

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DNA sequencing. PLoS One 10, e0138435.

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水 研 機 構 研 報， 第 45 号，41 － 46， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，41－46，2017

41

Genetic Selection on Animals using Pedigree, Phenotypic, and Genomic
Information
Shogo TSURUTA＊

Abstract: Genetic selection of quantitative traits in animals has been performed for a century in the
United States. Especially, in the last two decades, field data analysis of dairy cattle, beef cattle,
swine, and chicken has contributed to livestock industries. We have analyzed genetic components
in animal production, reproduction, and disease traits using our own computer programs (so-called
BLUPF90 family). 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 predict genomic breeding values by combining pedigree, phenotypes, andgenotypes (SNP markers).
With ssGBLUP, we found that the accuracy of genomic breeding values can be increased in dairy
and beef 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/~.
In this paper, we explain general BLUP and ssGBLUP algorithms applicable to livestock and fish
breeding. Also, we show evidence of genetic progress by genetic selection and results from genomic
analyses in US Holsteins.
Genomic selection has a greater advantage when more genotyped animals are available because
predictions are more accurate and more complex models can be applied. When animals are
genotyped in the earlier stages of life, genetic progress can be accelerated and genetic gains can bemagnified. The ssGBLUP methodologies are applicable to other organisms, such as farmed fish,shellfish, anadromous fish (salmon), honey bees, and even plants.
Key words: Genetic selection, BLUP, genomic information

Introduction

analysis, least square analysis, and best linear
unbiased prediction (BLUP) with an inverse of the

Genetic selection of quantitative traits in US

relationship matrix) to estimate genetic parameters

Holsteins using genetic evaluation has been

and to predict breeding values for economically

performed for 80 years in the United States (USDA-

important traits in animals. As a result, animal

ARS Animal Improvement Program 2013). In the

productions have dramatically increased in the last

beginning, artificial selection was conducted using

half-century.

only phenotypes. Later, pedigree information was

Genetic progress can be equated to a function ofadded from parent-offspring relationships, siblings,

accuracy of the selection, genetic variation (standard

and families to all available information among

deviation), selection intensity, and generation interval.

related animals. Around the same time, advanced

To accelerate genetic progress for quantitative traits,

statistical methods were developed (e.g., regression

we need to increase the first three parameters and2017年２月28日受理（Received on February 28, 2017）
	Department of Animal and Dairy Science, The University of Georgia, Athens, GA, 30602, U. S. A.
E-mail: shogo@uga.edu

＊1

42

Shogo TSURUTA

dec r eas e the last on e, ig n orin g all p o s s ible

(empirical BLUP), assuming that those varianceinteractions. Accuracy can be increased by adding

components are known. Using matrix notation, agenomic information into traditional breeding values

general mixed model equation (MME) in animal

estimated with phenotypes and pedigrees.

breeding and genetics can be written asWe

conducted several studies with genomic information
using a single step genomic BLUP (ssGBLUP) to
increase accuracy in genomic breeding values(Aguilar et al., 2010; Tsuruta et al., 2011; Lourenco etal., 2015; Fragomeni et al., 2015). Genetic variation canbe increased by outbreeding and mutation. Selection

where b and a are unknown vectors and X and Z

intensity can be increased by increasing theare design matrices for fixed and random effects,

population size. Generation interval can be reduced

respectively. The additive genetic variance for aby evaluating an animal’s performance at the early(breeding values) and the residual variance for e arestages of its life, using genomic information.

described as

Unfortunately, in the US and in Japan, genetic
selection in fish and shellfish has not garnered much
popularity compared to other livestock. The main
reasons may include 1) recording phenotypes andidentifying individuals were expensive, 2) farming

where A is a pedigree or numerator relationshipwas much more expensive than fishing, and 3) as amatrix; I is an identity matrix; σu2 and σe2 are varianceresult, genetic analyses were not well conducted.

components for additive genetic and residual effects,

However, these conditions have been changing overrespectively. Therefore, heritability can be calculated

the years. As seafood gains popularity, overfishingas σa2 / (σe2 + σa2). To obtain solutions for b (BLUE)

becomes a problem for popular species, and theand a (BLUP) from MME, the preconditioning

balanced ocean ecosystem could collapse.

conjugate gradient (PCG) algorithm is widely used

Fortunately, a complete breeding cycle technology inowing to its stable convergence property (Strandénaquaculture for some species has been successfullyand Lidauer, 1999; Tsuruta et al., 2001).

developed. In addition, low-cost single nucleotide
polymorphism (SNP) marker information is now

For ssGBLUP (Legarra et al., 2009; Aguilar et al.,2010), simply replace A-1 by H-1 as described below:

available for genomic selection.
The objective of this paper was to show how
genetic selection has been efficiently conducted inlivestock production in the US and to explain its
potential for improving marine species as well.
Materials and Methods
For 34,506 US Holstein bulls, 42,503 SNP markers
were available to predict genomic breeding values

where G is a genomic relationship matrix defined
as follows (VanRaden, 2008):

and SNP marker effects for 305-day (d) milk yield andcow mortality, using ssGBLUP. Details are describedin Tsuruta et al. (2015).
In general, there are two options in genetic
evaluation processes: 1) estimating genetic

where pi = allele frequency at locus i; D = weightparameters (heritability and genetic correlation) andfor each locus (I if assuming the same variance); W =breeding values simultaneously or 2) estimating

a design matrix as follows:

genetic parameters first and then breeding values

43

Efficient genomic selection in animals

For a genome-wide association study (GWAS), SNP
marker effects and SNP variances can be estimatedby solving a=Wu where u is a vector of SNP marker
effects (Strandén and Garrick, 2009). The BLUPF90
family programs (Misztal et al., 2002) for predicting

Fig. 1. Phenotypic and environmental trend for 305-d
milk yield in US Holsteins

genomic breeding values and estimating variancecomponents are available at http://nce.ads.uga.
edu/~.
Results and discussion

which started in 1957 assuming no genetic trend at
first, can be distinguished as genetic progress or gain.

Selection index
A selection index is widely used when selecting

Genomic selection

animals on multiple traits. Table 1 shows various

Since the USDA-ARS started genomic evaluation

selection indexes used in US Holsteins (VanRaden

using SNP markers as genotypes in 2008, genomic

and Cole, 2014). A selection index can be weighted

evaluation is gaining popularity in other breeds anddifferently for each trait, depending on the selection

species.

goal. Each dairy farm has a unique strategy to

As previously mentioned in H -1, MME

contains three different relationship matrices: A, G,

improve their animals in that environment (e.g.,

and A22. Due to different definitions and bases on A

focusing on milk production or cheese production).

(or A22) and G, distributions of the matrix elements
look inconsistent (Fig. 2) when 42,503 SNP markers

Genetic trend

for 34,506 US Holstein bulls were analyzed (Tsuruta

Phenotypic and environmental trends of 305-d milk

et al., 2015). The inverse matrices (A22 and G-1) can

yield in US Holsteins (Fig. 1) were calculated based on

be consistent, but the base adjustment could still be

cow’s EBV published in 2015 from Council on Dairy
Cattle Breeding (CDCB 2015).

required for a large population with many

The difference

between phenotypic and environmental trends,

generations.
In Fig. 3, according to the genetic progress formula

Table 1. Relative weights (%) on four different selection indexes for US Holsteins
Trait
Protein

Relative weight (%)
Net Merit $Cheese Merit $Fluid Merit $Grazing Merit $

20

24

0

18

Fat

22

19

23

20

Milk

−1

−9

23

−1

Productive life

19

16

20

10

Somatic cell score

−7

−7

−3

−6

Udder

8

6

8

8

Feet/legs

3

2

3

3

Body size

−5

−4

−5

−4
19

Daughter pregnancy rate

7

6

7

Heifer conception rate

2

1

2

3

Cow conception rate

1

1

2

5

Caving ability ($)

5

4

5

5

44

Shogo TSURUTA

Fig. 4a. Genetic variance (%) explained by SNP
markers for 305-d milk yield in first lactation

Fig. 2. Distribution of diagonals of genomic
relationship (G) and pedigree relationship (A 22 )matrices (mean values > 1.0 in parentheses indicate
inbreeding) in US HolsteinsFig. 4b. Genetic variance (%) explained by SNP
markers for cow mortality in first lactation

Fig. 4c. Genetic variance (%) explained by SNP
markers for cow mortality in second lactation
Fig. 3. Future prediction of genetic trend for finalscore in US Holsteins

described before, future genetic trends for final scorewith heritability (0.31) in US Holsteins were calculatedwith several conditions that assume that reliability in
GEBV is 0.2 higher than that in EBV and reliability in

Fig. 4d. Genetic variance (%) explained by SNP
markers for cow mortality in third lactation.

EBV is 0.4 higher than that in parent average (PA).
The solid thick line is the highest genetic trend when

14 was significantly large (2.5%) for milk yield and themaking the generation interval a half, indicating thatvariance for cow mortality decreased as the number

reducing generation interval will increase geneticof lactations increased, suggesting that younger cows

gains significantly.

died for a reason more genetically related to milkThis becomes possible bygenotyping and evaluating animals in the early

production than older cows.

stages of their life before sexual maturity.
Genetic selection in aquaculture
GWAS

Norway has been successfully conducting genetic

Using the ssGBLUP algorithm, SNP marker effects

selection in Atlantic salmon for decades. Table 2

and variances were estimated for 305-d milk yieldshows four different breeding programs that selectand cow mortality in US Holsteins (Tsuruta et al.,animals for four to 11 traits.

2015). Figs. 4(a-d) show genetic variance (%) explained

The FAO published results for “genetic

by SNP markers on each chromosome for 305-d milk

improvement” in fish (FAO Fisheries andyield in the first lactation and cow mortality in the

Aquaculture Department 2015), showing somefirst three lactations for US Holsteins. The Manhattan

genetic progress in fish and shellfish. Selective

plots indicated that the SNP variance on chromosome

breeding improved growth rate 50% after 10

45Efficient genomic selection in animals
Table 2. Traits presently selected in the four
breeding programs for Atlantic salmon in Norway(Thomassen et al. 2007)

because predicted breeding values are more accurate

Traitgenomic selection can be applied to any quantitative

Breeding program
A

B

C

D

Growth in freshwater

√

√

√

√

Body weight at slaughter

√

√

√

√

Early sexual maturity√√

Furunculosis√√

Infectious salmon anaemia√√

Infectious pancreatic necrosis√√

Dressing percentage√√

Fillet yield√√

Fillet color√√

Fillet fat√√

Deformity√√

Body shape√than the traditional breeding values and thegeneration interval can be reduced. Genetic andtrait for marine animals, such as farmed fish, shellfish,
and anadromous fish, if accurate pedigree information,
sufficient phenotypes, and/or genotypes are available.
Aquaculture has great potential in food supply allover the world, and fish production can besignificantly increased by sustainable genetic√√

selection.
√

Acknowledgements
This paper was made possible by numerous
studies in our animal breeding and genetics group at
the University of Georgia, including current faculties

generations in coho salmon, 20% per generation ingilthead sea bream, 10 – 13% live weight in oyster,and 60% in tilapia. Maturity and time of spawning,
physiological tolerance (stress), and disease andpollutant resistances were also improved.
If genetic selection can successfully be applied, fish
farming may become the most profitable option in
the fish industry. If sufficient facilities and resources(ocean, river, or lake) are available, strong selection
pressure can be put on breeding lines because fishand shellfish produce a large number of eggs in asingle spawning (that could be many full sibs and halfsibs). A superior individual within full sibs can be
identified by using genotypes. If genetic selection issystematically conducted in a complete breeding
cycle by recording pedigrees (at least selection lines),
phenotypes, and genotypes, the genetic progress infish farming will be maximized. Genetic selection on
economically important traits for farmed fish,shellfish, and anadromous fish is environmentallyfriendly and sustainable because no gene or gamete
manipulation is involved (i.e., only gene frequencies
are changed). It also has the capacity to multiply
seafood production while maintaining productquality.

(Ignacy Misztal and Daniela Lourenco), postdocs(Yutaka Masuda and Breno Fragomeni), a former
postdoc (Andres Legarra), and a former graduate
student (Ignacio Aguilar). We thank the livestockindustry and USDA for their continued support.
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Strandén I. and Garrick D. J., 2009: Technical note:(4) Lourenco D. A. L., Tsuruta S., Fragomeni B. O.,

Derivation of equivalent computing algorithms

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水 研 機 構 研 報， 第 45 号，47 － 55， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，47－55，201747

Exploring Transcriptomic Patterns in Slow- and Fast-growing Seriola
dorsalis Larvae
Catherine PURCELL＊1, Andrew SEVERIN＊2, Vince BUONACCORSI＊3, Mark DRAWBRIDGE＊4,
Kevin STUART＊4, and John HYDE＊1

Abstract: Seriola species have traditionally been a major component of global commercial andrecreational 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 ofoffshore 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 insurvival and 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 were 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.
Key words: Aquaculture, Seriola dorsalis, RNA-Seq, growth-heterogeneity, larvae

Introduction

Southern California.

However, the feasibility ofcommercial-scale culture for this and other Seriola
In the United States, the native California yellowtail

species hinges on reliable juvenile production from

(S. dorsalis) is considered a prime candidate forbroodstock populations. In Japan, Seriola culture has

aquaculture development in Southern California andtraditionally relied on harvesting and growout of wildis the target for an aquaculture industry poised forjuveniles, but more recently, focus has shifted to

rapid growth in North America, with offshore net

closed life-cycle production to help alleviate pressure

pens in place off northern Baja California, andon natural populations and to generate a more

proposals to develop similar pens off the coast ofpredictable supply of juveniles (Ohara et al., 2005;2017年２月28日受理（Received on February 28, 2017）	Southwest Fisheries Science Center, NOAA Fisheries, 8901 La Jolla Shores Dr., La Jolla, CA 92037, U. S. A.
＊2	Genome Informatics Facility, 206 Science I, Iowa State University, Ames, Iowa 50011, U. S. A.
＊3
	Juniata College, 1700 Moore St. Huntingdon PA 16652, U. S. A.
＊4
	Hubbs Sea World Research Institute, 2595 Ingraham Street, San Diego, CA 92109, U. S. A.
E-mail: catherine.purcell@noaa.gov＊1Catherine PURCELL, Andrew SEVERIN, Vince BUONACCORSI,Mark DRAWBRIDGE, Kevin STUART, and John HYDE

48

Ozaki et al., 2013; Aoki et al., 2014).

Similar tothe selection for economically important traits (Aokiaquaculture production of other marine fish, hatcheryet al., 2014) and improve the understanding of the

production of Seriola species in the U.S. andbiological, biochemical, and molecular networks

elsewhere has been hindered by high larval mortality

involved in larval development, which could then be(Woolley et al., 2014) and a propensity for growth

used to improve culturing techniques (Benzekri et al.,heterogeneity and deformities developed during

2014; Mazurais et al., 2011).

larval and early juvenile stages that limit the
production capacity and efficiency.

In this study, we describe the generation of whole

Growthlarval transcriptomes for the California yellowtail (S.

heterogeneity may be especially problematic for

dorsalis) at three developmental stages: 2, 7, and 17

Seriola, as it has been estimated in S. lalandi, fordays post hatching (dph) using RNA-Seq on theexample, that small fish constitute up to 42% of an

Illumina HiSeq 2500 sequencing platform.

offspring population, with many of those individuals

Transcriptional profiles and differential gene

exhibiting developmental issues leading to theirexpression were also investigated for slow- and fast-demise (Moran, 2007).

growing larvae over these same stages. We will

Although other aspects of research (e.g., disease,present the findings of the transcriptomic

nutrition, environmental factors, tank conditions)investigations. Through this study, we hope to apply

have supported the development of the yellowtail

the gene expression results to generate a better

aquaculture industry, genetic improvement programs

understanding of the mechanisms and timing offor Seriola culture are in the beginning stages (Ozaki

larval development in S. dorsalis, and of the genes

et al., 2013). To date, genetic research aimed atand processes involved in the observed growth

improving Seriola aquaculture has included:

heterogeneity that has limited Seriola aquaculture

construction of linkage maps at varying levels of

production.

coverage (including a map utilizing a hybrid crossMethods

between S. quinqueradiata and S. lalandi, Ohara etal., 2005), development of genetic markers(microsatellites and single nucleotide polymorphisms

Larval yellowtail were collected from spawning(SNPs)), identification of markers associated with

events using the wild-caught broodstock population

candidate genes of interest, mapping of genes,

held at Hubbs-SeaWorld Research Institute (HSWRI)

estimation of heritability for commercially-relevant(San Diego, CA). Yellowtail larvae were sampledtraits, and identification of quantitative trait lociopportunistically at 2, 7, and 17 dph with three(QTLs) associated with disease resistance (Ozaki etreplicates for each time point, and the replicatesal., 2013; Whatmore et al., 2013; Aoki et al., 2014; Fuji

within each time point were sampled from separate

et al., 2014).

spawning events.

However, up until this point,

Depending on developmental

bioinformatics data resulting from next generation

stage, one to several larvae were placed on asequencing has not been applied toward improving

microscope slide and euthanized with a lethal dose of

aquaculture for Seriola. The development of large-

MS-222. The larvae were quickly photographed,scale genomic resources has become increasingly

measured, and sorted by size into vials containing

accessible and affordable for non-model organisms.

RNAlater® (Ambion). The vials of RNAlater® wereThese types of genetic resources have been

kept on ice until being placed in -20 °C storage at thedeveloped and used extensively in agriculture and

NMFS laboratory.

livestock breeding for decades to improve productAs a proxy for physical larval fitness (i.e., overall

quality and quantity. Only more recently have theselarval growth and robustness), the smallest andgenomic approaches been applied to selectlargest larvae, hereafter referred to as slow-growing

aquaculture species (e.g. rainbow trout, Atlantic(SG) and fast-growing (FG) larvae, were sampledsalmon, tilapia, catfish, flounder, Atlantic cod) (Terova

within each spawning event at the three time points

et al., 2013; Dunham et al., 2014). Applying theseused for this experiment, with three replicates atgenomic approaches to Seriola would greatly aid ineach time point. For each replicate, 10 whole larvae

Transcriptomics of larval yellowtail growth heterogeneity49

were pooled for the tissue homogenization. Poolingwith significant interaction terms (FDR = 0.1) were

was used to: 1) minimize differences between

selected for further analysis. Modules were sorted

individual larvae and focus on general differences inby magnitude of difference between groups in mean

larval age and fitness groups and 2) provide adequate

expression change over time. Modules with large

amounts of RNA for the analyses, which was

expression increases over time in FG fish and large

problematic in single specimens at the earlier timedecrease over time in SG (and vice versa) were ofpoints (i.e., 2 dph). Whole larvae were utilized at theparticular interest in order to identify developmental

three time points to best characterize organism-wide

gene differences or regulatory differences between

developmental changes, particularly as many organ

these two groups. Module genes were pooled bysystems were not developed at 2 dph.

pattern as described above. GO enrichment analysis

RNA-Seq was conducted on the Illumina Hiseq(FDR = 0.05) was then performed within Cytoscape (v2500, and a total of 600 million reads were generated

3.2.1) (Smoot et al., 2001; Shannon et al., 2003) using thefor the 18 samples (30 million reads/replicate). Raw

BiNGO (Maere et al. 2005) plugin, and zebrafish generead data were assembled using Trinity (Haas et al.,ontologies (http://zeogs.molgen.mpg.de/).

2013), and DESeq (Anders & Huber, 2010) was used

regulatory transcripts with ZFIN (Zebrafish

to calculate the differential expression (DE) of theInformation Network) (Sprague et al., 2005) gene IDs

genes for every combination of timepoint and groupwere used as the reference gene set for enrichment(SG and FG fish).

analysis.

For the exploration of geneNon-ontology (GO) enrichment, only transcripts with a
Results and discussion

fragments per kilobase of exon per million fragments
mapped (FPKM) value of greater than four in at least
one of the 18 samples was retained, resulting in a
total of 54,858 high confidence transcripts.

For slow-growing (SG) fish between 2 and 7 dph

GOand between 7 and 17 dph, 343 and 765 significantly

enrichment was performed on each subset of DE

enriched GO categories were identified, respectively.

genes identified by DEseq for each comparison with

In fast-growing (FG) fish, 604 and 904 GO categories

a false discovery rate (FDR) corrected q-value cutoff

were identified as significantly enriched between 2of 0.05.

GO enrichment was also performed on

and 7 dph and between 7 and 17 dph. For SG and FG

additional subsets of transcripts that had opposite

fish at both time period comparisons (2-7 dph and 7-17transcriptional trends spanning the three time pointsdph), the GO categories of metabolism and catalytic

between the SG and FG samples. The transcripts

activity contained the largest proportion of enrichedwere required to have at least 15 normalized readterms (Fig. 1).

counts across all 18 samples, and there had to be at

A large proportion of enriched terms fell under the

least a twofold change between the averaged counts

transport category for all but the 2-7 dph SG fish,of biological replicates for days two and 17, which

where it was significantly lower than otherrepresented the start and end points of the sampling.

comparisons.

GO terms were further analyzed for broad GO term

enriched terms in the cellular metabolism category

categories using CateGOrizer and MGI_GO_slim2

was significantly higher in SG fish (both time periods)

categories (Hu et al., 2008), and pathway analysis wasthan in FG fish. Within the cellular metabolism

conducted using PathVisio 3 (Kutmon et al., 2015).

category, nucleic acid metabolism was higher for 2-7

Interestingly, the proportion ofLemon Tree analysis (Bonnet et al., 2015; Joshi et al.,dph and 7-17 dph SG fish (5.1% and 4.6%, respectively)2009) was performed to identify gene co-expression

than for FG fish (1.8% and 1.9%). However, enriched

modules and assign regulators to those modules.

GO terms under primary metabolism were higher

Counts (FPKM) were normalized for library size,

between 7-17 dph in the FG fish but not in SG fishusing RSEM (Li and Dewey, 2011). All 18 samples

and not different between SG and FG fish in the 2-7

were used for clustering. Transcripts were filtereddph comparisons (Fig.1).

for having at least one sample with FPKM greater

The normalized count data for the three biologicalthan four, resulting in 47,609 transcripts. Modules

replicates at each time point were averaged. These

50

Catherine PURCELL, Andrew SEVERIN, Vince BUONACCORSI,Mark DRAWBRIDGE, Kevin STUART, and John HYDE

Fig. 1. Proportions of enriched DE GO terms assigned to GO categories between two time periods, 2
to 7 days post hatch (dph) and 7 to 17 dph, for SG and FG fish. Significant differences in GO categories
between growth category or time period are represented by * (p < 0.05) and ** (p < 0.01), and ns (notsignificant) is used to clarify significance relationships when necessary.

SG fish at 17 dph are transcriptionally more similar to
7 dph and 2 dph FG and SG fish than to FG fish at 17
dph (Fig. 2).
In the module analysis, initial clustering produced
878 modules, and 63 of these had significant
interaction terms (FDR = 0.1). Genes from thesemodules were further grouped into expression
categories: 1) up-regulation in FG and downregulation in SG fish (31 modules) and 2) up-regulationin SG and down-regulation in FG fish (12 modules).
For modules showing preferential increases in FG
fish, top GO terms (and associated top hierarchicalFig. 2. A hierarchical clustering dendogram for all
gene transcripts to visualize the overall similarity
between time points of slow-growing (SG) and fastgrowing (FG) fish.

enriched GO terms within the GO network) for
module genes were regulation of metabolic process,regulation of biological process, biosynthetic process,ribosome biogenesis, nucleobase-nucleosidenucleotide and nucleic acid metabolism, response tohypoxia, mesenchyme cell migration, cellular

averaged values were then used to generate acomponent assembly, small molecule metabolism andhierarchical clustering dendrogram using hclust

generation of precursor metabolites and energy, and(method = ”average”) (Murtagh and Legendre, 2014;transport. The percentage terms under the GO

Murtagh, 1985) to visualize the overall similarity

categories of translation, generation of precursor

between time points of SG and FG fish. Similar to themetabolism and energy, cellular nitrogen-compound

phenotypic characterizations we used to classify themetabolism, generation precursor metabolites andSG and FG fish, we note the transcriptional profiles ofenergy, biosynthetic process, and small molecule

2 dph and 7 dph cluster together, whereas by 17 dph,

metabolism were significantly higher in FG fish thanthe FG fish show a significantly different

in SG fish (Fig. 3).

transcriptional profile than the SG fish. In fact, theFor the 12 modules exhibiting differential increases

Transcriptomics of larval yellowtail growth heterogeneity

51

Fig. 3. From the significant modules, the proportions of terms identified in GO categories for genes in biologicalprocesses (A) and molecular function (B). Bar color reflects inclusion in the broader parent GO categories listed
in the key provided for each graph. GO categories significantly higher for either the FG or SG larvae are denotedby *(P < 0.05) and ** (P < 0.01).

in SG fish, enriched GO terms associated with thefish at 2 dph and a complete reversal by 17 dph, with

non-regulatory genes included response to nutrient

primarily higher gene expression in FG fish. Pathway

levels (starvation), cellular response to stimulus,

analysis within growth groups indicate that SG fish

autophagy and Schwann cell development. Terms

show decreasing gene expression in these pathways

under GO categories associated with cell adhesion,as development continues, while FG show increasing

locomotion, membrane organization, autophagy, cellgene expression over the same 15 day period.

motility, cell morphogenesis, vesicle-mediated

It is not immediately obvious why 2 dph SG larvaetransport, cell-protein modification, signal

exhibit up-regulation in oxidative phosphorylationtransduction, and cell-cell signaling were significantly

and electron-transport chain pathways compared tohigher in SG fish module genes (Fig. 3).

FG larvae at the same time point. These larvae

Pathway analysis of SG and FG larvae at the time

appear to be generating or attempting to generate

points revealed two pathways with obvious trends inhigher levels of energy that do not translate intodevelopmental progression: oxidative phosphorylation

growth. Quickly the SG larvae lag behind the FG(Fig.4) and the electron transport chain. The overall

larvae, in size and in developmental stages; instead

trend for these pathways included higher genethey up-regulate processes related to starvation

expression for most genes in these pathways in SG

responses and autophagy. One hypothesis is that one

52

Catherine PURCELL, Andrew SEVERIN, Vince BUONACCORSI,Mark DRAWBRIDGE, Kevin STUART, and John HYDE

Fig. 4. Gene expression comparison (insert expression info) in the electron transport chain
pathway between 2 dph (A) and 17 dph (B) FG and SG larvae. Gene boxes in pink/red
indicate higher gene expression in SG larvae, and gene boxes in blue indicate higher gene
expression in FG larvae.

or more of the ATP-generating pathways contain a
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Annotated bibliography

processes underlying the differential growth rates.
Of the genes identified by GenBank, ribosomal(1) Kocmarek A., Ferguson M., and Danzmann R.,

proteins were the most abundant, comprising 50% of

2014: Differential gene expression in small and large

the total genes with 17 different ribosomal protein

rainbow trout derived from two seasonal spawning

genes. The genes included nine components of thegroups. BMC Genomics 15, 57.

large ribosomal subunit, and eight components of the

In this study, the authors wanted to identify genes

small ribosomal subunit. Some of these genes werethat showed similar expression patterns between

up-regulated in the fast-growing larvae (n = 6), whilelarge and small rainbow trout from different

others were up-regulated in the slow-growing larvae

spawning seasons in two types of tissue: liver and(n = 11). Since ribosome biogenesis is a significant

white muscle. The goal in identifying these genes

metabolic cost in cell proliferation, any changes in

was to be able to develop growth-related markers forthis pathway would likely have a large effect on theTranscriptomics of larval yellowtail growth heterogeneity

55

overall energy metabolism. The authors hypothesizedays. The authors believe that these small fishthat in the slow-growing larvae there may be a highappear to be on a degenerative developmental

metabolic cost to synthesizing and degrading excess

strategy without any influence of the larger

ribosomal protein copies resulting from the higher

aggressive fish.

expression of those genes.
(3) Salem M., Silverstein J., Rexroad III C., and Yao J.,(3) Moran D., 2007: Size heterogeneity, growth

2007: Effect of starvation on global gene expression

potential and aggression in juvenile yellowtail

and proteolysis in rainbow trout (Oncorhynchus

kingfish (Seriola lalandi Valenciennes). Aquac. Res.

mykiss). BMC Genomics 8, 328.

38, 1254-1264.

The authors of this study used microarrays to

In this study, the authors are describing theidentify genes and pathways involved in theoccurrence of size heterogeneity and aggressive

starvation response and protein turnover in rainbow

behaviors in cultures of Seriola lalandi; this was done

trout, and to identify metabolic adaptations thatto examine the effectiveness of size-grading inoccur in the liver during these starvation periods.

reducing aggression and mortality, and increasingThis study was of interest because, as the authors

growth rates. To do this, graded and ungradedpoint out, examining changes in metabolism during

juveniles were compared for various measures of

starvation is an effective way to identify relationships

aggression and growth, and a RNA/DNA ratio was

between metabolic pathways and body processes.

used as a measure of growth rate. The authors foundThe experiments showed down-regulated expressionthat size heterogeneity became more pronounced atof genes involved in protein biosynthesis in the

12 days post hatch (dph) when Artemia are offered as

starved fish, but no significant changes in protein

a food source.

catabolic pathways, and a slight increase in 20S

While the large and aggressive

juveniles only accounted for 8% of the population, theproteasome activity.

small grade juveniles that received the aggression

starvation included an overall decline in geneaccounted for 42% of the population. In the ungraded

expression associated with decreasing tissue

treatment, this aggression was associated withmetabolism, a reduction in protein synthetic capacity,

mortality for most small fish. However, even without

an impairment of ATP-biosynthesis, and lower

aggression, the small-grade juveniles did not gain

expression in hepatic lipid and fatty acid transport.

weight or increase their RNA/DNA ratio after 12

Responses in the liver to水 研 機 構 研 報， 第 45 号，57 － 62， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，57－62，2017

57

Modeling the Variable Effects of Using Wild and Cultured Broodstockon the Fitness Risk Due to Escaped Farmed Fish
Kristen M. GRUENTHAL＊1, Jason D. VOLK＊2, Gregory R. BLAIR＊2, Gavin KEY＊3,
and Michael B. RUST＊1

Abstract: Various negative ecological and genetic impacts may occur when farmed fish escape andmix with wild conspecifics. Genetic impacts result due to interbreeding between wild fish andescapees and may result in reduced genetic diversity within and among populations and a loss offitness. Loss of genetic diversity and fitness compromise the adaptive potential of a mixed (wild plus
cultured) population, making it potentially less able to respond to changes in environmental
conditions (e.g. climate change).
Simplified, risk to wild population fitness due to interbreeding with escapees is a function of thenumber of escapees relative to wild census size, the genetic difference between escapees and wild
fish, and the fitness of escapees in the wild relative to wild fish. The Offshore Mariculture Escapes
Genetics Assessment (OMEGA) model simulates the magnitude of this risk. To illustrate OMEGA’s
application, we present results from evaluations of a planned pilot project for Almaco jack (Seriola
rivoliana) in Hawaii and contrast them with those obtained from evaluation of a theoretical sablefish(Anoplopoma fimbria) aquaculture program originally used to verify the model. Escape scenarios forboth 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
to wild population fitness, even a total loss of almaco jack had a negligible effect. Key differences inthe simulated fitness risk associated with each program included, but are not limited to, broodstock
source (domesticated or wild), encounter rate between wild fish and escapees, and the scale (size andlongevity) of the operation.
Key words: Almaco jack, fitness, genetic risk, offshore aquaculture, sablefish

Introduction

correlated with the level of domestication those fish
experienced in culture (Baskett et al., 2012). Genetic

The rapid development of offshore marine finfish

and fitness-related impacts to the wild populationaquaculture worldwide has raised concerns due, inthen occur when escaped individuals from thesepart, to the potential negative genetic and fitness

cultured populations interbreed with wild conspecifics

impacts escaped farmed fish may have on natural(Baskett et al., 2013). Changes in gene expression due

populations (Hindar et al., 1991; Tufto, 2010; Waples etto adaptation to the hatchery environment andal., 2012).

fitness declines in a mixed population due toFitness is measured in terms of anindividual’s relative reproductive success.

Thedomestication can occur after only one or two

fitness of escapees in the wild is often inversely

generations (Araki et al., 2008; Christie et al., 2016).

2017年２月28日受理（Received on February 28, 2017）
	Consultant with Earth Resources Technology, Inc., NOAA Fisheries Office of Aquaculture, 1315 East-West Highway, Silver Spring, MD 20910,
U. S. A.
＊2
ICF International, 710 Second Avenue Suite 550, Seattle, WA 98104, U.S.A.
＊3
Kampachi Farms, LLC, 1 Keahole Point Road, Kailua-Kona, HI 95740, U.S.A.
E-mail: kristen.gruenthal@noaa.gov＊1

58

Kristen M. GRUENTHAL, Jason D. VOLK, Gregory R. BLAIR, Gavin KEY, and Michael B. RUST

These impacts can threaten the adaptive potential ofon the mean trait value. Initially, each of the two

the wild population, which may make it less able topopulations is exposed to a separate environment

respond to and survive environmental changes(hatchery or wild) and, therefore, selective regime(Tringali and Bert, 1998).

(domestication or natural). The mean trait value of

There is little scientific data, however, that reliably

each population is equal to its environmental (andassigns the risk to fitness due to escapees, particularly

fitness) optimum, and any shift away from either

for mariculture. Thus, existing regulatory standards

environmental optimum trait value leads to aaddressing escapes are largely preventative,

reduction in fitness (Lande, 2007; Tufto, 2010).

theoretical, or qualitative rather than quantitative.

OMEGA is intended to provide insight into the

To address this knowledge gap, the NOAA Fisheries

variables affecting risk, help identify information

Office of Aquaculture (OAQ) developed a researchgaps and research priorities, explore options for

initiative designed to explore the genetic interactions

operational design or modification, and inform policy

between wild and cultured fish. As part of thisand management decisions (Volk et al., 2015). To

initiative, the OAQ solicited ICF International, Inc.,

illustrate OMEGA’s application, we present results(ICF) to develop the Offshore Mariculture Escapesfrom evaluations of a theoretical sablefish

Genetics Assessment (OMEGA) model. OMEGA is a

(Anoplopoma fimbria) program and a real world pilot

Microsoft Excel-based program designed to simulate

project for Almaco jack (Seriola rivoliana).

the risk posed to wild population fitness by escapes
Methods

from marine aquaculture programs.
Simplified, risk to wild population fitness due tointerbreeding with escapees is a function of theEscape scenarios for both species varied from low

number of escapees relative to wild census size, theto high base leak rates, cage failure probabilities, andgenetic difference between escapees and wild fish,catastrophic cage failure probabilities.

and the fitness of escapees in the wild relative to wild
fish. OMEGA is organized around three interacting

Sablefishcomponents that incorporate these factors, including

Sablefish are a highly-prized groundfish foundthe aquaculture operation itself, wild population

along the North Pacific Rim. The wild population

characteristics, and the potential for interaction

includes a single stock along the west coast of the

between escapes and wild conspecifics. Under these

U.S. Fish live up to 80 years but old fish are rare.

components, OMEGA employs a modular format to

The female spawning biomass is 79 - 82 metric tons

describe assumptions used to model the potential(mt), with maturity reached by six years. Models forinteractions and impacts of escapees in the wild.

population dynamics and harvest are well-established

There are nine modules requiring user input, ranging

in this species.

from basic settings and preferences for saving theCommercial culture of sablefish currently occurs inworkspace and running simulations to entering wild

Washington State, USA, and British Columbia,

and cultured population data. Currently, there are

Canada. The OMEGA scenarios were originally

103 possible user-defined variables (Index of Userdeveloped for model verification and explored a low

Inputs available upon request), some of which areversus a high incidence of escapes, with a highoptional and a few that represent alternatives forencounter rate between wild and farmed fish (Volk etwhich the user chooses one of two or more options.

al., 2015). A hypothetical sablefish hatchery program

Once the model has been parameterized with data

was sited in the U.S. Pacific Northwest and included

describing the three interacting components,using a domesticated broodstock to produce up to

OMEGA employs a single trait phenotypic fitness

10,000 mt of fish stocked among 50 offshore cagemodel developed by Ford (2002) to simulate risk.

sites. Culture methods were recently developed,

This underlying model is a quantitative two

with fish reaching harvest size in one year.

population analysis of differential selection regimes

OMEGA was run multiple times. For the low

and the effect of gene flow between the populations

escapes scenario, the base leak rate and cage failure

59

Predictive modeling of the fitness effects of escaped farmed fishprobability was set at a static 0.1%, regardless of fish

catches reported for all carangids in the Westernsize. The annual probability of a catastrophic event

Pacific Region reef fish trend report (2010). The

was set at 5% through the first 20 years of operation,encounter rate between wild and farmed almaco jackdecreasing to 1% by year 26 and thereafter, with ais likely to be low due to behavior characteristics (i.e.

static assumed program loss of 20% per event acrossschooling near objects).

all years. For the high escapes scenario, the basereproductive capabilities are known to be poor fromleak rate, cage failure probability, and probability andprevious pilot projects.

Escapee survival andmagnitude of losses due to catastrophic events were
Resultsincreased. Base leak rate was dependent on fish size,with 3% of the smallest fish and 0.5% of the larger fish
escaping.

Cage failure probability was 0.5% forOver the default 100-year timespan of thesmaller fish and 1% for larger fish. Meanwhile, thesimulations, the low escapes scenario for sablefish

probability of catastrophic events was set at 10%,

resulted in escapee percentage peaking at ≤ 5% ofwith a 60% loss of fish per event, in year 1, which

the total population and the spawning biomass soon

decreased to 5%, with a 40% loss, by year 16.

after each escape event, with larger and/or

Thereafter, probability was 5%, with a 20% program

successive events resulting in higher percentages,

loss.

but gradually shrinking to < 1% of the population.
High chronic escapes coupled with several acute

Almaco jackescape events, in contrast, had a longer lasting

Almaco jack (also known as kahala or kampachi inimpact on population make-up. The high escape

Hawaii) have a circumglobal distribution in tropicalscenario resulted in escapees comprising aand temperate waters. Fish tend to school near reef

significantly higher proportion of the population,slopes, offshore banks, and other objects, and they are

averaging 5 -7%. Correspondingly, fitness in the wildoften found in mixed schools with the greater

was reduced (shifted toward the hatchery optimum;

amberjack (S. dumerili). The wild population in

Fig. 1) more under the high escape scenario, whereas

Hawaii is poorly characterized, and data used forthe low escape scenario remained near the wild

populating OMEGA was supplemented with

optimum.

information from congenerics (e.g. greater amberjack

Whereas the high escapes scenario for sablefish

size at maturity of 2.5 kg and fecundity at 130

resulted in a significant impact to wild population

thousand eggs per kg body weight).

fitness, even a total loss of almaco jack had a

In terms of U.S. commercial culture, Kampachi
Farms has recently engaged in iterative open-ocean
pilot projects directed at expanding offshore farmingof almaco jack to a commercial scale near KailuaKona, HI, USA, and elsewhere.

The OMEGA

scenarios for Kampachi are based on well-developed
operational plans.

The project will use wild

broodstock as required for permitting in Hawaii.
Culture methods are established, and fish reachharvest size of 1.8 kg in one year. The F1 juveniles
will be stocked in a tethered cage locatedapproximately 11 km offshore of Kona, withproduction of about 14 mt per year for each of two
years.
OMEGA was run multiple times. The assumed
number of escapes was set at 50-100% per year. The
natural spawning and total biomass was set equal to

Fig. 1. Relative fitness effects for a sablefish (broken
red line) and an almaco jack (solid red line) escapescenario, both with a high number and frequency ofescapes. Almaco jack scenario results similar to noaquaculture. Dotted black line represents ahypothetical regulatory maximum 10% decrease in
mixed population fitness.

60

Kristen M. GRUENTHAL, Jason D. VOLK, Gregory R. BLAIR, Gavin KEY, and Michael B. RUST

negligible effect on the mean trait value (Fig. 1). The

model scenarios, evaluate model parameters, andproportion of the wild almaco jack population andvalidate the model with data from current andspawning biomass made up of escapees peaked atplanned aquaculture operations worldwide;

0.45% and 0.87%, respectively, of the over 100-year

developing targeted scenarios, such as exploring the

span of the simulation. No significant decrease inmagnitude of risk associated with 1) using wild or

fitness (< 0.02%) was predicted even when all 30,000

selected broodstock; 2) widening the fitness gap

fish escaped and survived. Fitness impacts were

between farmed and wild fish or keeping fish “wild-

realized, but still not significant, in attempts to force

like;” focused research like induced sterility (e.g.

the model to show a fitness effect (i.e. to “break the

triploidy; Hindar et al., 1991), and setting andmodel”), with a small estimated natural biomass,

enforcing spatiotemporal escape limits. Downstream

significantly more than two years of production, 100%

steps include performing a sensitivity analysis of caseannual escape and encounter rates, and 100% survival

studies, determining acceptable default parameters,

and spawning of escapees.

and developing an economic analysis module. Workwill also be directed at making OMEGA fully

Discussion

operational and friendly to a variety of user groups(e.g. managers, industry professionals, researchers),

Results were in line with expectations according topaving the way for the model to play a key role in

Ford (2002), as well as results from related research

genetic risk assessments for offshore culture in the(e.g. Baskett et al., 2013). Key differences in the

U.S. and elsewhere.

simulated fitness risk associated with each program

Aquaculture must simultaneously support

included, but were not limited to, broodstock sourcecommercial interests; increase the availability of safe

(domesticated or wild), encounter rate, and the scaleand nutritious fish, shellfish, and other products for(size and longevity) of the operation. The sablefish

consumers; and protect wild populations (IWG-A

culture program was hypothetical and used originally2014). Toward that end, more specific questions thatto verify that the OMEGA was operating as expected.

OMEGA may help user groups answer include how

Were an offshore program to be developed for thesignificant the impacts of escapes surviving to

species, results from these hypothetical scenarios

reproduce are and what the effects of our decisions

could be used as a starting point in terms of designingabout aquaculture operations, such a broodstocka low risk program. Topics that may be important tomanagement (e.g. wild or selected), may be. Moreconsider when designing a program based in part on

broadly, the intent is to use OMEGA to help define an

results from OMEGA include, but are not limited to,acceptable risk to marine resources (e.g. 5% or 10%

whether any scenarios are economically viable orfitness decrease), as well as use it as a decision-

ecologically sustainable and whether engineering or

making tool to assist in the assessment andbiological technology to minimize risk is available andmanagement of marine aquaculture operations sucheconomically feasible. In contrast, the almaco jackthat they may remain both commercially viable andpilot program produced negligible effects. These

environmentally responsible.

results were included in an environment assessment
submitted as part of the permitting process for the

Acknowledgements

proposed pilot project for Kampachi Farms. It is
recommended that reassessment of risk be

The authors would like to thank Walton Dickhoff at

performed, however, if Kampachi Farms wishes to

the NOAA Fisheries Northwest Fisheries Sciencemodify protocols or scale production up to commercial

Center; Alan Everson, retired, from the NOAA

levels.

Fisheries Pacific Islands Regional Office; Conrad

OMEGA is still a work in progress, however, andMahnken; Lars Mobrand with DJ Warren andrequires further testing. The next steps toward

Associates; and Neil Sims at Kampachi Farms, LLC.

finalizing the OMEGA model package for broader use

OMEGA v1.0 and its user guide are freely available

include fostering external collaborations to develop

for download on the OAQ website at www.nmfs.noaa.

Predictive modeling of the fitness effects of escaped farmed fish

61

gov/aquaculture/science/omega_model_homepage.

Technical Memorandum NMFS-NWFSC-119. US

html.

Department of Commerce NOAA NMFS,
Washington, D.C.
References
Annotated bibliography

Araki H, Berejikian B. A., Ford M. J., and Blouin M. S.,
2008: Fitness of hatchery-reared salmonids in the(1) Baskett M. L., Burgess S. C., and Waples R. S., 2013:

wild. Evol. Appl. 1, 342-355.

Assessing strategies to minimize unintended fitness

Baskett M. L., Burgess S. C., and Waples R. S., 2013:
Assessing strategies to minimaize unintended
fitness consequences of aquaculture on wild
populations. Evol. Appl. 6, 1090-1108.

consequences of aquaculture on wild populations.
Evol. Appl. 6, 1090-1108.
Baskett et al. (2013) address various factors
associated with the management of cultured

Christie M. R., Marine M. L., Fox S. E., French R. A.,

populations and model their potential fitness impacts

and Blouin M. S., 2016: A single generation ofshould farmed fish escape into the wild. Factors

domestication heritably alters the expression ofassessed include the origin and level of domestication

hundreds of genes. Nat. Commun. DOI: 10.1038/

in the cultured stock (i.e. degree of maladaptation),

ncomms10676.

induced sterility in the cultured stock, and theFord M. J., 2002: Selection in captivity during

magnitude and frequency of escapes (e.g. continuous

supportive breeding may reduce fitness in the

low level leakage versus rare catastrophic events).

wild. Conserv. Biol. 16, 815-825.

Results indicate that, up to a point, the magnitude ofHindar K., Ryman N., and Utter F., 1991: Genetic

the fitness impact rises as the maladaptation ofeffects of cultured fish on natural fish population.

escaped cultured fish increases; an extremely

Can. J. Fish. Aquat. Sci. 48, 945-957.

maladapted, non-local origin cultured population may

Interagency Working Group on Aquaculture (IWG-A).

actually have effects similar to a weakly diverged

2014. National Strategic Plan for Federal

stock.

Aquaculture Research (2014-2019). National

fitness consequences rapidly. Finally, it is more

Science and Technology Council, Committee on

effective to reduce low-level leakage than guard

Science, www.whitehouse.gov/sites/default/

against sporadic large-scale escape events.

Second, sterilization reduces unintended

files/microsites/ostp/NSTC/aquaculture_
strategic_plan_final.pdf.
Lande R., 2007: Expected relative fitness and theadaptive topography of fluctuating selection.
Evolution 61, 1835-1846.

(2) Ford M. J., 2002: Selection in captivity during
supportive breeding may reduce fitness in the wild.
Conserv. Biol. 16, 815-825.
Escaped cultured fish may cause a potential loss of

Tringali M. D. and Bert T. M., 1998: Risk to genetic

fitness in the wild, if breeding occurs between

effective population size should be an important

escapees and wild conspecifics. This drop in fitness

consideration in fish stockenhancement

is associated with a difference in the optimum trait

programs. Bull. Mar. Sci. 62, 641-659.

values for hatchery and natural environments. The

Tufto J., 2010: Gene flow from domesticated species tosingle-trait phenotypic fitness model in Ford (2002)

wild relatives: migration load in a model ofdescribes how mean phenotype values of the mixed

multivariate selection. Evolution 64, 180-192.

population (captive plus wild fish) may shift relative

Volk J. D., Rust M. B., Blair G. R., Mobrand L. E.,

to the optimum values for each environment, based

Mahnken C. V. W., and Dickhoff W. W., 2015:

on the presence/absence and amount of gene flow

Modeling intraspecific genetic effects for(interbreeding) between the cultured escaped andmanagement of aquaculture programs. Bull.

wild fish. The overall fitness effect depends on theFish. Res. Agen. 40, 89-96.

magnitude of the difference in optimum trait value,

Waples R.S., Hindar K., and Hard J. J., 2012: Genetic

trait heritability, and selection pressure against

risks associated with marine aquaculture. NOAA

domestication in the wild, as well as habitat capacity,

62

Kristen M. GRUENTHAL, Jason D. VOLK, Gregory R. BLAIR, Gavin KEY, and Michael B. RUST

magnitude and frequency of escape events, wild andpopulation.

captive population demographics, and the potential
for interaction between wild fish and escapees. Ford

(4) Waples R. S., Hindar K., and Hard, J. J., 2012:(2202) was used most notably in Pacific Northwest

Genetic risks associated with marine aquaculture.

salmonids, where the All-H Analyzer, or AHA, model

NOAA Technical Memorandum NMFS-NWFSC-119.

helped the Hatchery Scientific Review Group explore

US Department of Commerce NOAA NMFS,

the potential fitness consequences of supplementing

Washington, D.C.

wild populations and of cultured fish straying into
wild populations.

Waples et al. (2012) is a comprehensive overview ofthe potential genetic impacts to wild populations
associated with marine aquaculture. As such, the(3) Hindar K., Ryman N., and Utter, F., 1991: Genetic

authors “provide managers with a better

effects of cultured fish on natural fish population.

understanding of the genetic effects of marine

Can. J. Fish. Aquat. Sci. 48, 945-957.

aquaculture on natural populations,” with the intent

Hindar et al. (1991) represents one of the originalof informing policy and management decision-making.

manuscripts reviewing the genetic impact escaped

The document synthesizes relevant information andfarmed fish may have on wild populations and is one

provides key references, identifies research priorities,of the most often cited. The authors recommend

provides a risk assessment framework, and gives

several strategies for protecting the genetic integrity

recommendations for monitoring and evaluationof wild populations, many of which remain the focus

toward sustainable marine aquaculture developmentof aquaculture programs today. These strategies

in the US. Waples et al. (2012) focuses on commercial

include improved containment technology andaquaculture of marine finfish but presents it in lightrecovery, sterilization of the culture stock, and betterof decades of research on salmon hatcheries andbreeding practices, coupled with monitoring themarine stock enhancement.

genetic contribution of escaped fish to the mixed水 研 機 構 研 報， 第 45 号，63 － 67， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，63－67，201763

Did Farmed Coho Salmon Oncorhynchus kisutch that Escaped during
the Earthquake and Tsunami Disaster in 2011 Interbreed with Native
Masu Salmon Oncorhynchus masou?
Kei SASAKI＊1, Tadahide KUROKAWA＊2, Hideki NIKAIDO＊1, Daisuke MURAOKA＊1,
and Yoshiro OKADA＊1

Abstract: During the Great East Japan Earthquake on March 11, 2011, several million individuals of
farmed coho salmon (Oncorhynchus kisutch) escaped from aquaculture facilities in the Northeast
Pacific Ocean, Tohoku, Japan. In fall of the same year, sexually mature coho salmon migrated up
rivers in this area. Farmed coho salmon that migrate up rivers to breed may affect the genetic
material of native salmon species and result in weakened populations. Especially, there is a strong
concern that coho salmon may cross with the native masu salmon (Oncorhynchus masou); it is known
that hybrids of these species have survivability. In this study, we surveyed masu salmon landed at
a local fish market, using genetic and morphological methods in order to determine whether there
are hybrids. As a result, hybrids were not found in this survey. Therefore, at this moment the
impact on the genetic resources of masu salmon is considered to be low.
Key words: Farmed coho salmon (Oncorhynchus kisutch), escaped, masu salmon (Oncorhynchus
masou), hybrids, DNA fragment analysis

Introduction

northeastern Japan. In fall of the same year, sexuallymature coho salmon migrated up rivers in this area.

Coho salmon (Oncorhynchus kisutch) has been

These coho salmon may have affected the genetictransplanted from North America to Hokkaido sincematerial of native salmon species and resulted in

the 1970s (Ishida et al., 1975, 1976; Nara et al., 1979;

weakened populations. There is a strong concern

Umeda et al., 1981), but has not become naturalized inthat coho salmon may cross with the native masu

Japan. They migrate to waters off Hokkaido forsalmon (Oncorhynchus masou) because it is known

feeding, and sometimes stray and go up rivers inthat hybrids of these species have survivability (Ito et

Hokkaido (Kikuchi et al., 1998); however, they do notal., 2006). On the other hand, the ability to survive isregularly spawn in Japan.

Sea farming of coho

low for crosses with chum salmon (Oncorhynchus

salmon has been conducted around the Tohokuketa) (Foerster, 1935). The upriver season is different

Pacific coast in Japan since 1975. In recent years,

between masu salmon and coho salmon; however, theproduction has remained at over 10,000 tons per year.

spawning season overlaps for these species. The

Due to the effects of the Great East Japan

spawning season of masu salmon (Honshu pacific

Earthquake and tsunami on March 11, 2011, allregion) is from September to November (Kiso, 1995),

farmed coho salmon (more than 5 million fishes)

while that of coho salmon is from November toescaped into the Northeast Pacific Ocean off Tohoku,

January (Koseki, 2013). It is unclear how many years

2017年２月28日受理（Received on February 28, 2017）	Research Center for Coastal Fisheries and Aquaculture, Tohoku National Fisheries Research Institute, Japan Fisheries Research and
Education Agency, Miyako, Iwate, 027-0097, Japan.
＊2	Kushiro Laboratory, Hokkaido National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Kushiro, Hokkaido, 0850802, Japan.
E-mail: keis@affrc.go.jp＊1

64

Kei SASAKI, Tadahide KUROKAWA, Hideki NIKAIDO, Daisuke MURAOKA, and Yoshiro OKADA

hybrids will need to mature. However, based on thematuration age of both species being 3 years old, thepossibility that hybrids returned in spring of 2014
was considered to be high. In this study, we surveyedmasu salmon landed at a local fish market, using
genetic and morphological methods in order todetermine the presence of hybrids.
Materials and methods
We conducted visual checks of masu and coho
salmon landed at a Miyako fish market from May to
September, 2014 (approx. 2,000 individuals) (Fig. 1). In
general, it is possible to distinguish both species bychecking the gill raker and the radial silver stripes on
the caudal fin (Fig. 2). We surveyed presence ofindividuals with mixed characteristics. The reasons
we choose this market were as follows: (1) large
number of escaped coho salmon were landed at thismarket during summer to autumn season in 2011 and(2) upriver coho salmon were detected in several

Fig. 1. Map of the Tohoku Pacific Coast. Rivers
marked in gray indicate that adult coho salmon weredetected in 2011. Open circle indicates the main
farming area. “■” indicate study site.

rivers near the market. We selected 39 masu, and 5

Fig. 2. Comparison of the tail and the first gill of adult coho and masu salmon. Upper
panels shows tail of masu (a: fork length 57.0cm) and coho (b: fork length 60.6cm),
lower panels shows the first gill of masu (c) and coho (d). Radial silver stripes on thecaudal fin are obvious in coho, and the gill rakers longer and slender in coho.

Did escaped Coho salmon interbreed with native Masu salmon?

65

coho salmon (all individuals were sampled during the

intron C of the growth hormone 1 (GH-1) using asurvey) for more detailed analysis.

For theprimer set with 17 bp adapter sequence for themorphological comparison, we compared the number

fluorescent label. PCR amplified product of masu

of rays of each fin, and the number and length of thesalmon (256bp) was 34 bp less than that of cohogill rakers. We conducted sequence analysis of thesalmon (290 bp) (Fig. 3), thus making it possible toFig. 3. Alignment of the intron C of the growth hormone 1 (GH-1) between masu salmon (GenBank: AF541855)
and coho salmon (AF005925). PCR product amplified using primer set OSH3 and OSH4 of masu salmon is 34 bp
less than that of coho salmon.

Fig. 4. Box plot of the number of gill rakers (left panel), and relationship between the longest gill raker length
in the first gill and the fork length (right panel).

66

Kei SASAKI, Tadahide KUROKAWA, Hideki NIKAIDO, Daisuke MURAOKA, and Yoshiro OKADA

distinguish the two species. The amplicon was

species identification judged from the morphologicalexamined in both to see if there was a hybrid.

comparison (Fig. 5). Therefore, based on the current
survey, the impact of escaped coho salmon on the

Results and Discussion

genetic resources of masu salmon is considered to below. However, three of five of coho salmon were of

As a result of the morphological comparison, thethe 2011 brood, and their gonads were developed.

range of number of fin rays overlapped, and there

These individuals may have derived from thewas no significant difference in the two groups (Table

escaped coho salmon during the 2011 earthquake.

1). The number and length of gill rakers were larger

Therefore, it is necessary to carefully monitor theand longer in coho salmon (Fig. 4). Individuals withoccurrence of hybrids in the near future.

mixed characteristics were not found.
As a result of DNA fragment analysis, hybrids

Acknowledgment

were not confirmed. Identification of species from
DNA analysis of all the individuals agreed with theWe thank the staff of the Miyako fisherycooperative union for their cooperation in collecting

Table 1. The number of fin rays in masu salmon andcoho salmon

samples in the market. We are also grateful to T.
Kato, N. Watanabe, M. Haga, K. Ohmoto, M. Yatsuya
for their help with collecting morphological data.
References
Foerster R. E., 1935: Inter-specific cross-breeding ofPacific salmon. Trans. Royal Soc. Can. 29, 21–33.
Ishida T., Tanaka T., Kameyama S., Sasaki K., andNemoto Y., 1975: On the Coho Salmon

Fig. 5. Fragment size of the PCR amplified products of masu and coho salmon. Upper panel shows control of masu(a) and coho (b) salmon. Middle panel shows present specimens of masu (c) and coho (d). Lower panel shows
artificial cross of masu (male) and coho (female) (e), the amplicon is seen in both 256 bp and 290 bp.

Did escaped Coho salmon interbreed with native Masu salmon?

67

transplanted from U. S. A. into Yurappu River,

Oncorhynchus masou originated from rivers of

Hokkaido. Sci. Rep. Hokkaido Salmon Hatcherythe pacific coast of Northern Honshu, Japan.

29, 11-15.

Bull. Natl. Res. Inst. Fish. Sci. 7, 1-188.

Ishida T., Tsuji H., Hosokawa T., and Nara K., 1976:
On the Coho Salmon Transplanted from North
Amerika into the Shibetsu River, Hokkaido. Sci.

Koseki, Y., 2013: Profile of Salmonid-11 Coho salmon.
FRA Salmonid Res. Rep. 7, 34-37.
Nara K., Shimizu M., Okukawa G., Matsumura K., andRep. Hokkaido Salmon Hatchery 30, 47-53.

Umeda K., 1979: On the Coho Salmon

Ito D., Fujiwara A., and Abe S., 2006: Hybridtransplanted from North America into theInviability and Chromosome Abnormality inShibetsu River (Ⅱ). Sci. Rep. Hokkaido Salmon

Salmonid Fish. The Journal of Animal Genetics.

Hatchery 33, 7-16.

34(1), 65-70.

Umeda K., Matsumura K., Okukawa G., Sazawa R.,

Kikuchi M., Urawa S., Ohkuma K., and Kaeriyama M.,

Honma H., Arauchi M., Kasahara K., and Nara K.,

1998: A Record of Adult Coho Salmon

1981: On the Coho Salmon transplanted from

(Oncorhynchus kisutch) strayed into the Chitose

North America into the Ichani River. Sci. Rep.

River. Bull. Natl. Sal. Res. Cen. 1, 39-43.

Hokkaido Salmon Hatchery 35, 9-23.

Kiso, K., 1995: The life history of masu salmon

水 研 機 構 研 報， 第 45 号，69 － 73， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，69－73，201769

Evaluation of the Tsunami Impact on the Genetic Diversity of the
Marbled Flounder Pseudopleuronectes yokohamae in Sendai Bay,
Miyagi, Japan
Yuki MINEGISHI＊1, Minoru IKEDA＊1, Yutaka KURITA＊2, Hiroyuki TOGASHI＊2,
Yukinori NAKANE＊2, and Akihiro KIJIMA＊1

Abstract: Evaluating genetic diversity is a fundamental step for stock management. However, it canbe influenced by environmental and demographic changes, and thus it should be carefully
investigated, especially when drastic disturbances have occurred.

Marbled flounder

Pseudopleuronectes yokohamae is a commercially valued fish in Japan, and possibly consists ofmultiple management units due to its benthic lifestyle including low dispersal ability. In Sendai Bay,the population is thought to form a single management unit, and its spawning ground has beenprotected for stock conservation. Since this population could have been strongly affected by thehuge tsunami associated with the 2011 off the Pacific coast of Tohoku Earthquake, the tsunami
impact on the genetic diversity of the population of the marbled flounder in Sendai Bay needs to beevaluated.
We firstly developed genomic resources for this species using next generation sequencing
technology and de novo assembly, which generated a total of 525,502 contigs and 10,732,070
unassembled reads. We then designed a total of 331,368 microsatellite primer pairs from thegenomic data, and characterized sixteen primer pairs as novel microsatellite loci. Using thesemicrosatellite markers, significant genetic divergence was found between the east and west sides of
Boso Peninsula (pairwise FST = 0.02305 – 0.19784). At the east side, genetic homogeneity wasobserved over the wide area from Onagawa Bay to Choshi including Sendai Bay, except for Mutsu
Bay. The genetic variability of the Sendai Bay population did not show any considerable changes
after the tsunami from April 2012 to February 2014 (HE = 0.5061 – 0.5463) and was relatively higher
compared to those of the western populations of Boso Peninsula (HE = 0.3817 – 0.4557). These results
suggest that the tsunami impact on the Sendai Bay population of the marbled flounder is considered
to be minor but long-term monitoring may be needed.
Key words: Marbled flounder, Pseudopleuronectes yokohamae, genetic diversity, management units,
Sendai Bay

Introduction

genetic diversity such as population structure,
migration rate between populations and population

Evaluating genetic diversity of species and / orsize helps identify management units andpopulations of interest is a fundamental step fortranslocation ranges, and monitoring those alterations

appropriate stock management. The information on

provides the insights into the population vulnerability

2017年２月28日受理（Received on February 28, 2017）	Tohoku Ecosystem-Associated Marine Sciences, Graduate School of Agricultural Science, Tohoku University, Mukai 15, Konorihama, Onagawa,
Oshika, Miyagi 986-2242, Japan＊2
	Tohoku National Fisheries Research Institute, Fisheries Research Agency, Shinhama-cho 3-27-5, Shiogama, Miyagi 985-0001, Japan
E-mail: y.minegishi@m.tohoku.ac.jp＊1

70

Yuki MINEGISHI, Minoru IKEDA, Yutaka KURITA, Hiroyuki TOGASHI,
Yukinori NAKANE, and Akihiro KIJIMA

and viability (Waples et al., 2008). On the other hand,studies to develop the genomic resources and novel

genetic diversity is influenced by environmental andmicrosatellite markers for the marbled flounder, thendemographic changes like climate change, natural

demonstrate the results of population genetic

disaster and human activities. Direct effects involve

analyses of this species using newly developed

population bottleneck and varied migration between

markers, and finally discussed the impact of that

populations, whereas indirect ones cause changes intsunami on the genetic diversity of this fish.

habitat structure and community composition (Bankset al., 2013). Genetic diversity should be, therefore,

Materials and Methods

carefully investigated, especially in the situation
where drastic disturbances have occurred.
The 2011 off the Pacific coast of Tohoku

Development of the genomic resource and novel
microsatellite markers

Earthquake occurred on March 11, 2011, with a

A 200-bp library was prepared using the genomic

magnitude 9.0 generated a large tsunami that struck

DNA extracted from a single adult specimen of thethe Pacific coastline of eastern Japan (Mori et al.,marbled flounder collected in Sendai Bay in 2012.

2012). In addition to social, economic and physical

IonTorrent PGM sequencing was performed and a

impacts, ecological ones have been recently studied

single round of de novo assembly was conducted. To

and reported in this area. Urabe et al. (2015) examined

test if the assembled contigs can function as reference

the macrobenthic animal communities at ninein genomic analyses, IonTorrent RAD-seq wasintertidal flat in Sendai Bay and the Sanriku Coast

performed using six individuals of this fish and SNPs

and found that 30 – 80% of taxa disappeared after thewere discovered by mapping RAD-tag reads to thosetsunami. Hata et al. (2016) investigated the ecologicalcontigs. The detailed procedure was provided intraits of ayu Plecoglossus altivelis with a one-year

Genomic Resources Development Consortium et al.

amphidromous life history and concluded that the(2015). Using the software QDD version 3.0 (Meglécz

tsunami drastically changed the ecological traits of

et al., 2010), microsatellite sequences were searchedthis fish on the Sanriku Coast, such as hatching datefor in the obtained genome sequence data of P.

and oceanic growth period. It can be, therefore,yokohamae and primers were designed for those

expected that this environmental event also affects

microsatellites with a default configuration. The

genetic diversity of aquatic organisms in this area.

assembled contig sequences and unassembled reads

Marbled flounder Pseudopleuronectes yokohamae isfrom de novo assembly were separately used as input

a commercially valued fish across Japan. Due to thedata in the software. Based on PCR amplification,

benthic lifestyle including low dispersal ability of thevisual confirmation of polymorphisms on an agarose

marbled flounder, this species possibly consists ofgel and accurate genotyping, the primer pairs were

multiple management units over its geographic

chosen for subsequent population genetic analyses

distribution.

(Minegishi et al., 2015).

In Sendai Bay, the population isconsidered to form a single management unit, and its
spawning ground has been protected for stock

Population genetic analyses

conservation. This population, however, could have

Temporal change in genetic diversity of the

been strongly influenced by the great tsunami thatmarbled flounder after the tsunami impact was

occurred along the Pacific coast in Japan in 2011, andinvestigated using thirteen populations collected inconsequently the genetic diversity could be changed.

Sendai Bay from April 2012 to February 2014 (N =Accordingly, the temporal genetic variation of the807). Nine populations from the Pacific Ocean coasts

Sendai Bay population needs to be investigated and(Mutsu Bay, Onagawa Bay, Sendai Bay, Ibaraki,

the geographic range of the management unit should

Choshi, Tokyo Bay [Funabashi, Hagiu, Takeoka, andbe determined based on the genetic population

Uraga], Sagami Bay, Hamana Lake, and Mikawa Bay;

structure of this species inferred together with otherN = 596) were also analyzed for comparisons (Fig. 1).

Pacific Ocean populations.

Genomic DNA was extracted from fin clips andIn this study, we firstly introduce our previous

genotyping was performed at the newly developed

Evaluation of the genetic diversity of the marbled flounder71

Consortium et al., 2015). Microsatellites were found in
781,773 sequences in the unassembled reads, and a
total of 331,368 primer sets were successfully
designed.

In contrast, no microsatellites weredetected in the contigs. Screening based on PCR
amplification, visual confirmation of polymorphisms
on an agarose gel and accurate genotyping resulted
in sixteen primer pairs that are polymorphic andapplicable for population genetic analyses of thisspecies (Minegishi et al., 2015). These results indicatethat the obtained genome sequence data can be used
as a genomic resource of this species and contributes
to finding novel DNA markers such as SNPs andother microsatellites and the genes associated withvarious useful phenotypes including faster growthand high resistance to virus infection.
Fig. 1. Distribution of the marbled flounder (boldlines) and sampling sites in the present study. The
black circle shows Sendai Bay. Boso Peninsula
locates between Choshi and Tokyo Bay.

Evaluation of the genetic diversity of the Sendai Bay
population of the marbled flounderPopulation genetic analyses using genotyping datafrom the microsatellites developed above foundsignificant genetic divergence between the east andsixteen microsatellite loci. As the presence of nullalleles was suggested at locus Psyo075, this locus was
excluded from further analyses. Population genetic
analyses were conducted using Arlequin ver. 3.5(Excoffier et al., 2005). The detailed experimental
conditions for genotyping were described inwest sides of Boso Peninsula (pairwise FST = 0.02305– 0.19784). On the east side of Boso Peninsula, theMutsu Bay population was differentiated (F ST =0.03829, P < 0.001) from other populations and geneflow was observed over the wide area from Onagawa
Bay to Choshi including Sendai Bay. The genetic

Minegishi et al. (2015).
Results and Discussion
Development of the genomic resource and novel
microsatellite markers
IonTorrent PGM sequencing and de novo assembly
generated a total of 525,502 contigs (N50 = 1994) and
10,732,070 unassembled reads. The summed lengthof the contigs was 547.8 megabases, corresponding to
81.8% of the genome of this species (670 megabases;
Ojima and Yamamoto, 1990). This suggests that thisgenome sequences data contains most information of
the genome of this species. RAD-seq reads (91.92 –95.34% of the reads) were successfully mapped tothese contig sequences, which subsequently yielded
75,472 SNPs at maximum, indicating the utility of thisgenome data as a reference sequence in the genomescale analyses (Genomic Resources Development

Fig. 2. Temporal change of the average
heterozygosity over 15 microsatellite loci of the
Sendai Bay population sampled from April 2012 tolate February 2014. Dark and light grey indicates
expected and observed values, respectively.

72

Yuki MINEGISHI, Minoru IKEDA, Yutaka KURITA, Hiroyuki TOGASHI,
Yukinori NAKANE, and Akihiro KIJIMA
Banks S. C., Cary G. J., Smith A. L, Davies I. D.,
Driscoll, Malcolm Gill A., Lindenmayer D. B., andPeakall R., 2013: How does ecological disturbanceinfluence genetic diversity? Trends Ecol. Evol.
28, 670–679.
Excoffier L., Laval G., and Schneider S., 2005: Arlequinver. 3.0: An integrated software package forpopulation genetics data analysis.

Evol.

Bioinform. Online 1, 47–50.
Genomic Resources Development Consortium,
Arthofer W., Bertini L., Caruso C., Cicconardi F.,
Delph L. F., Fields P. D., Ikeda M., Minegishi Y.,
Fig. 3. Comparison of the average heterozygosity
over 15 microsatellite loci of nine populations from
Mutsu Bay to Mikawa Bay. Dark and light grey
indicates expected and observed values, respectively.

Proietti S., Ritthammer H., Schlick-Steiner B. C.,
Steiner F. M., Wachter G. A., Wagner H. C., andWeingartner L. A., 2015: Genomic Resources
Notes accepted 1 February 2015 – 31 March2015. Mol. Ecol. Res. 15, 1014–1015.
Hata M., Kawakami T., and Otake T., 2016: Immediatevariability of the Sendai Bay population did not show
any drastic changes during the sampling period from
April 2012 to February 2014 (HE = 0.5061 – 0.5463)(Fig. 2) and no signal of a genetic bottleneck was
observed. Moreover, the genetic diversity of the
Sendai Bay population was relatively higher
compared to those of the western populations of Boso
Peninsula such as Tokyo Bay and Mikawa Bay (HE =0.3817 – 0.4557) (Fig. 3) . These results indicate that
the Sendai Bay population should be treated as asingle management unit together with otherpopulations in the area ranging from Onagawa Bay to
Choshi for stock management. In addition, unlike
macrobenthic animals and ayu mentioned earlier, thetsunami impact on the Sendai Bay population of themarbled flounder is considered to be minor, but
further monitoring may be needed since a long-term
effect of the tsunami impact on marine ecosystems
could emerge in the future.
Acknowledgments
This study was financially supported by Tohoku
Ecosystem-Associated Marine Sciences from the
Ministry of Education, Culture, Sports, Science andTechnology of Japan.
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impact of the tsunami associated with the 2011
Great East Japan Earthquake on the Plecoglossus
altivelis altivelis population from the Sanriku
coast of northern Japan. Environ. Biol. Fish 99,
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Meglécz E., Costedoat C., Dubut V., Gilles A., Malausa
T., Pech N., and Martin J.-F., 2010: QDD: a userfriendly program to select microsatellite markers
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projects. Bioinformatics 26, 403–404.
Minegishi Y., Ikeda M., and Kijima A., 2015: Novel
microsatellite marker development from theunassembled genome sequence data of themarbled flounder Pseudopleuronectes yokohamae.
Mar. Genomics 24, 357–361.
Mori N., Takahashi T., and the 2011 Tohoku
earthquake tsunami joint survey group., 2012:
Nationwide post event survey and analysis of the2011 Tohoku Earthquake Tsunami.

Coast.

Engng. J. 54, 1250001.
Ojima Y. and Yamamoto K., 1990: Cellular DNA
contents of fishes determined by flow cytometry.
La Kromosomo II 57, 1871–1888.
Urabe J., Suzuki T., Nishita T., and Makino W., 2013:
Immediate ecological impacts of the 2011 Tohoku
Earthquake Tsunami on intertidal flat
communities. PLoS ONE 8, e62779.
Waples R. S., Punt A. E., and Cope J. M. 2008:

Evaluation of the genetic diversity of the marbled flounder73

Integrating genetic data into management ofDeveloping this understanding is now critical because

marine resources: how can we do it better? Fishdisturbance regimes are changing rapidly in a

Fish. 9, 423–449.

human-modified world.

Annotated Bibliography(2) Waples R. S., Punt A. E., and Cope J. M., 2008:
Integrating genetic data into management of marine(1) Banks S. C., Cary G. J., Smith A. L, Davies I. D.,

resources: how can we do it better? Fish Fish. 9,

Driscoll, Malcolm Gill A., Lindenmayer D. B., and423–449.

Peakall R., 2013: How does ecological disturbance

Molecular genetic data have found widespreadinfluence genetic diversity? Trends Ecol. Evol. 28,

application in the identification of population and

670–679.

conservation units for aquatic species. However,Environmental disturbance underpins theintegration of genetic information into actualdynamics and diversity of many of the ecosystems ofmanagement has been slow, and explicit andthe world, yet its influence on the patterns andquantitative inclusion of genetic data into fisheries

distribution of genetic diversity is poorly appreciated.

models is rare. In part, this reflects the inherent

We argue here that disturbance history may be thedifficulty in using genetic markers to draw inferences

major driver that shapes patterns of genetic diversity

about demographic independence, which is generally

in many natural populations.

We outline how

the information of the greatest short-term interest to

disturbance influences genetic diversity throughfishery managers. However, practical managementchanges in both selective processes andconstraints, institutional structures anddemographically driven, selectively neutral processes.

communication issues have also contributed to the

Our review highlights the opportunities andlack of integration. This paper identifies some of thechallenges presented by genetic approaches, such asorganizational, conceptual and technical barriers thatlandscape genomics, for better understanding andhave hampered full use of genetics data in stock

predicting the demographic and evolutionary

assessment and hence fishery management andresponses of natural populations to disturbance.

outlines how such use could be enhanced.

水 研 機 構 研 報， 第 45 号，75 － 87， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，75－87，201775

Experimental Hybridization and Competition between Atlantic Salmon(Salmo salar) and Native Salmonid Species in Japan
Kazuo ARAKI＊1,2, Tetsuji MASAOKA＊1, Hiroyuki OKAMOTO＊1, Hiroyuki NAGOYA＊1,
Hidefumi NARAMURA＊3, Kouji MUTO＊3, Hiromi OKU＊4, Jun-ya AOKI＊1, and Toshiya SUZUKI＊3

Abstract: Atlantic salmon (Salmo salar) is a leading aquaculture species that has been extensively
bred in Norway using selective DNA marker technologies. The commercial application of a highgrowth transgenic Atlantic salmon (namely AquAdvantage® Salmon) was approved by the UnitedState Food and Drug Administration in November 2015. AquAdvantage salmon is a strain ofAtlantic salmon that has been genetically modified by the integration of a growth-hormone
regulating gene from a Chinook salmon and the antifreeze protein gene promoter from the ocean
pout. The GM strain grows at least twice as fast as regular farmed Atlantic salmon. Should it
become a candidate for commercial production in Japan, there is not enough available informationon the biology and ecology of Atlantic salmon in Japanese waters for comparison. To remedy this inpart, we evaluated the maturation of experimentally reared Atlantic salmon in Japan, and tested thepotential of the species for hybridization and competition with four native salmonid species.
Seasonal water temperature was a primary determinant of maturation of the Atlantic salmon.
Crossed with Amago salmon, Masu salmon, and Biwa trout, most of the hybrid embryos ceased todevelop between mesoderm induction and axis formation. However, a relatively low number of theAtlantic salmon and native white potted char (Salvelinus leucomaenis) hybrid, as confirmed throughrestriction fragment length polymerase analysis, survived for 2 years. In addition, we reared
different life stages of Atlantic salmon and three native salmonid species in tanks or ponds todetermine the extent of interspecific and intraspecific competition for food and space, as well as
related changes in body mass. We found that competition was dependent on the combination of
salmonid species and life stage of the different species.
Key words: Atlantic salmon (Salmo salar), competition, interspecific crosses, maturation, native
salmonids in Japan

Introduction

European and Baltic. As with other salmonids, theanadromous life cycle of this species is complex (Mills,

The distribution of Atlantic salmon (Salmo salar)

1989, 1991; Hendry and Cragg-Hine, 1997; Urke et al.,depends on water temperature and is native to the2010). Young Atlantic salmon spend one to four

North Atlantic, where it is generally considered toyears in their natal river. When they develop acomprise three populations: North American,

highly evolved chemotactic system, they migrate

2017年２月28日受理（Received on February 28, 2017）	Research Center for Aquatic Breeding, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 224-1
Hiruda, Tamaki, Watarai, Mie 516-0423, Japan＊2
	Mie University Graduate School & Faculty of Bioresources, 1577 Kurimamachiya, Tsu, Mie 514-8507, Japan＊3
	Research Center for Freshwater Fisheries, National Research Institute of Fisheries Science, Fisheries Research and Education Agency, 2482-3
Chugushi, Nikko, Tochigi 321-1661, Japan＊4
	Research Center for Aquaculture System, National Institute of Aquaculture, Japan Fisheries Research and Education Agency, 224-1 Hiruda,
Tamaki, 516-0423, Watarai, Mie 516-0423, Japan
E-mail: arakin@affrc.go.jp＊1

76

Kazuo ARAKI, Tetsuji MASAOKA, Hiroyuki OKAMOTO, Hiroyuki NAGOYA,
Hidefumi NARAMURA, Kouji MUTO, Hiromi OKU, Jun-ya AOKI, and Toshiya SUZUKI

toward the Atlantic Ocean and follow on sea surfacechange among hatchery-reared fish is a form of

currents and feed on plankton or juveniles of otherselection, with differential mortality among thefish species such as herring. After a year to up to

genotypes, with brood stock chosen based on

five years of good growth, they move to the sea

commercial production traits such as high adult bodysurface currents that transport them back to theirmass and fast growth rate (Perrier et al., 2013).

natal river. It is a major misconception that salmon

Escaped male salmon are often larger than the wild

swim thousands of kilometers in the sea (Hendry andfish, making them more attractive to females andCragg-Hine, 2000; Jonsson and Jonsson, 2007; Øysteinmore successful in spawning, even though they may

et al., 2010).

be less fit genetically (Hendry and Cragg-Hine, 2000).

Since the mid-1960s, Atlantic salmon aquaculture

Numerous researchers have described interspecifichas grown well beyond the native range of theand intraspecific competition between Atlantic

species, with Norway, Chile, Scotland and Canada

salmon and other salmonid species for all life stages

currently the largest producers (Marine Harvest,(Fausch, 1998; Bremset and Heggenes, 2001; Volpe et2015). Atlantic salmon can now be farmed throughal., 2001b; Heggenes et al., 2002; Van Zwol et al., 2012).

their entire life cycle, with brood stock selected for a

Most studies to date have suggested that the

number of traits, including rapid growth, hardinessperformance of wild steelhead, rainbow and brownand good edible yield, and the adults are often

trout is superior to that of Atlantic salmon, even with

cultured in marine net pens or sea cages.

significant behavioral differences observed among

Nearly one third of the total world production ofthese species. In experimental tank environments,

Atlantic salmon occurs in regions where this species

the behavior of Atlantic salmon has been described tois exotic. There is an evidence of successful spawning

be influenced by that of other salmonid species in a

in three streams in British Columbia, Canada, butmanner similar to that occurs in natural riverwhether escaped Atlantic salmon have establishedenvironments (Stradmeyer et al., 2008; Berg et al.,breeding populations along the North American West2014).

Coast remains uncertain (Volpe et al., 2001a).

Fletcher et al. (2004) reported genetically modified

Spawning of escaped farmed Atlantic salmon has not(GM) Atlantic salmon to grow faster than non-GM

been documented in Chile or Tasmania. Outside its

farm-raised Atlantic salmon. The GM salmon containnative range the Atlantic salmon is a poor colonizer.

an rDNA construct comprising a growth hormone

Because Atlantic salmon are attacked by othergene from Chinook salmon, under the control of asalmonid fishes, such as native brown trout (Heggenes

gene promoter derived from the ocean pout

et al., 2002), the probability of escaped Atlantic(Macrozoarces americanus). The GM salmon havesalmon establishing populations in exotic

proven to grow faster before the pre-smolt stage andenvironments seems low, although the possibility of

can reach market weight one year faster than thethis occurring cannot be ruled out. It is difficult toregular farmed Atlantic salmon (Du et al., 1992; Cook

predict if or how Atlantic salmon adapt to a new

et al., 2000; Fletcher et al., 2004; Deitch et al., 2006).

environment, partly because research on the

AquaBounty Technologies Inc. first applied to sellpotential impacts in such a habitat is limiteda GM strain of Atlantic salmon (AquAdvantage ®

(McGinnity et al., 2003).

Salmon) in North America. Based on the proposed

Released or escaped cultured salmon have thephysical and biological containment measures, thepotential to compete with the wild stocks for food,

United States Food and Drug Administration deemedspace, and breeding partners (Van Zwol et al., 2012).

that the likelihood of the GM salmon escaping from

As a result of the morphological, physiological,

land-based facilities (in Canada and Panama),

ecological, and behavioral changes that may occur

thereafter surviving in the ocean or freshwater andamong artificially reared salmon, their competitive

interbreeding with wild-type salmon, is remote.

ability is likely to differ from that of the wild fish.

Accordingly, they issued a ‘Finding of No Significant

These changes are partly phenotypic and partly

Impact’ (FONSI) on November 19, 2015, thereby

genotypic (Perrier et al., 2013). For instance, genetic

approving the company’s application to produce theArtificially reared Atlantic salmon and native salmonids in Japansalmon commercially.

77

between October and the following January to

The only report of farmed Atlantic salmon in Japandecrease the water temperature to approximately

comes from Aomori Prefecture for 1983–1988

10°
C. A separate batch of immature Atlantic salmon(Yoshida and Matsuzaka 1991). Thus, there is scantwere cultured indoors throughout the year at Nikko

information describing the biology and ecology ofstation in spring water maintained at approximately

Atlantic salmon in Japanese conditions for comparison

10°
C. Between late November and December, oncewith AquAdvantage salmon, which would help inthe salmon female had matured, we squeezed eggs

determining whether AquaBounty Technologies canfrom females and sperm from males, and thenapply for commercial production of the GM strain in

artificially fertilized the salmon to ensure the quality

Japan.

Therefore, to partly remedy the lack ofof the egg and sperm. Male Atlantic salmon werecomparative data, we evaluated the maturation of

maturated from the end of October at Tamaki

Atlantic salmon cultured at two locations in Japan.

station, and we exposed the sperm of mature Atlantic

Next, we tested the potential of the species forsalmon to the eggs of mature Amago salmon, Masu

hybridization with four native salmonid species, andsalmon, Biwa trout and white-spotted char to induce

meanwhile sought to evaluate the levels of

hybridization.

intraspecific competition and interspecificcompetition, with three native salmonid species,

Competition between juvenile Atlantic salmon, andunder contained conditions.

with Amago salmon and Biwa trout
To estimate intraspecific and interspecificMaterials and methods

competition between juvenile fish, ten individuals
each of Atlantic salmon, Amago salmon and Biwa

Experimental salmonids

trout of average weight of 30 g were reared for 6

To investigate maturation of Atlantic salmon

months in separate 2 m × 50 cm × 25 cm tanks with

(Salmo salar) reared in Japan, we obtained hatchedtransparent fronts.

fry from the Hokkaido University Nanae Freshwater

Atlantic salmon with five Amago salmons, and five

Station (Hakodate) and transferred them to the

Atlantic salmon with five Biwa trout for 3 months.

National Research Institute of Aquaculture’s Tamaki

During this period, we used time-lapse video to(Mie Prefecture) and to Nikko inland station (Tochigi

record the position of each fish at 5 - minute intervals

Prefecture) (the latter being in the coldest region on

for 1 hour each day, and we weighed the juvenile fish

Honshu Island during winter).

approximately every 2 weeks.

To investigate

In addition, we reared fivecompetition and the potential for crossbreeding withnative salmonids, Biwa trout (Oncorhynchus masou

Competition between the adult Atlantic salmon andrhodurus) were sourced from Lake Biwa Museum

three native salmonid species(Shiga Prefecture) and transferred to Tamaki station;

To quantify interspecific and intraspecific

Masu salmon (O. masou masou) and white-spottedcompetition between adult fish, 6 individuals each ofchar (S. leucomaenis) were sourced from Nikko

Atlantic salmon, Amago salmon and Biwa trout ofstation and transferred to Tamaki station; and theaverage weight of 250 g, were reared in separate 5 m

Amago salmon (O. masou ishikawae) were bred at

× 1.5 m × 1 m ponds. Additionally, we reared 3

Tamaki station.

individuals each of Atlantic, Amago and Masu salmon
and Biwa trout together for approximately 2 months.

Crossbreeding the Atlantic salmon with four native

For 1 hour per day over a 2-week period, the position

salmonid speciesof each fish was recorded using two underwater

We monitored the water temperature for thevideo cameras.

And, each day for 2 weeks, we

immature Atlantic salmon cultured in outdoor tanks

counted the number of fish consuming the food

(15 m × 2.5 m × 1 m) at Tamaki station from January

pellets offered, using the camera footage for 30

to September. The immature salmon were thenminutes each feeding. Lastly, the fish were weighed

transferred to an indoor 2 tons tank for rearing

every 2 weeks during the approximately 2 - month

78

Kazuo ARAKI, Tetsuji MASAOKA, Hiroyuki OKAMOTO, Hiroyuki NAGOYA,
Hidefumi NARAMURA, Kouji MUTO, Hiromi OKU, Jun-ya AOKI, and Toshiya SUZUKI

period.

15 °C, after which it decreased to approximately 10°
C from late October to November (Fig. 1B). In order
Results and Discussion

to mature Atlantic salmon at water temperaturesimilar to that in 2015, Atlantic salmon were reared

Maturation of Atlantic salmon reared on Honshu

outdoor from January to September at water

Island, Japan

temperatures from 12 to 15 °C, after which they were

Seasonal water temperature was an importanttransferred to an indoor 2 tons black breeding tank,determinant of the maturation of the experimentallyfrom October to December, where the temperature

reared Atlantic salmon. At the more northern Nikko

was maintained at approximately 10 °C (Fig. 1C).

inland station, the salmon could be bred year-round in

These conditions produced survival to the fry stage.

relatively cold water, maintained at approximately 10

The natural breeding grounds of Atlantic salmon°
C (Fig. 1A). However, the survival of the fry grown

are rivers in Europe and the east coast of North

from artificially fertilized eggs was < 0.1% over a five-

America (Øystein et al., 2010). The bulk of individuals

year period (2011–2015). Only at the more southernof the ocean-run form live in freshwater for the first

Tamaki station, in 2012, did survival of similarly

2 years of life, after which they smolt and migrate toderived fry reach 4%. In 2012, the water temperature

the Atlantic Ocean to feed, grow and mature for 1 or

between January and September ranged from 13 tomore years. The adults utilize the ocean surface

Fig. 1. Water temperature at Tamaki and Nikko stations, and an Atlantic salmon × white-spotted char hybrid fry.
A: Water temperatures at Nikko station. B: Water temperatures at Tamaki station. C: Regulated watertemperature in the indoor 2-t water black breeding tank at Tamaki station. D: Atlantic salmon and white-spottedchar hybrid fry.

Artificially reared Atlantic salmon and native salmonids in Japan79

currents (Hendry and Cragg-Hine, 2000) to return tointron of the aromatase gene (Masaoka et al., 2015). In

their natal river to spawn, generally from April toour experiments, the surviving fry achieved a

September (with peak spawning in October andrelatively short body length and had a slightly

November in Canadian waters). Atlantic salmon

irregular body form (Fig. 1D); however, we did notcomplete their maturation in the sea, and return todetermine to what life stage the hybrids might havetheir natal river before or during summer withsurvived or whether they could have ever reachedhigher water temperatures (Bromage and Roberts,

maturity.

1994; Jonsson and Jonsson, 2007). Rapid reductions in

Ban et al. (2013) crossed Atlantic salmon withwater temperature during October or November

several salmonid species nativ to Japan with a low

induce spawning (Taranger et al., 2010; Good, 2015).

level of success, and almost all the hybrid embryos

In 2012, the year that we successfully managed towere gynogenetic. Likewise, those authors reported

artificially fertilize the salmon and hatch out embryos

an even lower level of success with hybrid embryos

to the fry stage, the culture water temperatures wereof male Atlantic salmon crossed with female Japanesesimilar what to this species would encounter in thechar. We similarly observed poor survival of thewild in its native range. Natural populations ofAtlantic salmon and white-spotted char hybrid fry.

Atlantic salmon live at high latitudes, with mostThus far, the potential for natural hybridizationaquaculture facilities also traditionally located at highbetween these species in Japan appears to belatitudes (e.g., Norway, Chile, Canada, Unitedunlikely, indicating that escaped farmed Atlantic

Kingdom, and Alaska in the US). Both experimental

salmon have little potential to impact the genetic

stations in our study, however, are situated atfitness of the wild salmonid stocks in Japan.

somewhat lower latitudes.

The maturation of

On the other hand, interspecific hybridization

Atlantic salmon is understood to be a complex

between Atlantic salmon and brown trout wasprocess variously affected by seasonal water

possibly the first hybrid fish ever described; natural

temperature, photoperiod, growth rate while at sea,

hybridization of these species was mentioned by

and age (Taranger et al., 2010). Future research on

Willughbeii in 1686 (Makhrov, 2008), and artificial

these aspects will be required to determine the total

hybridization of these species has been known sinceappropriate conditions without water temperature

the early 19th century (Leaniz and Verspoor, 1989;

for the artificial breeding of Atlantic salmon in Japan.

Jansson and Ost, 1997). Today, the hybridization ofAtlantic salmon and brown trout provides a goodHybridization between the Atlantic salmon and fourmodel for studying the factors and consequences of

salmon species native to Japan

interspecific hybridization among salmonids (Garcia-

We attempted to crossbreed Atlantic salmon withfour native salmonid species, at both Tamaki and
Nikko station.

Vazquez et al., 2004; Quilodrán et al., 2003; Ban et al.,2013).

Most cross-fertilized embryos ofAtlantic salmon with Amago salmon (Oncorhynchus

Interspecific competition among juveniles

masou ishikawae), Masu salmon (Oncorhynchus

Prior to our experiments, no published information

masou masou), Biwa trout (Oncorhynchus masou

was available on competition between Atlantic

rhodurus), and white-spotted char (Salvelinus

salmon and salmonid species native to Japan. To

leucomaenis) died between mesoderm induction andevaluate competition for food and space, and theaxis formation because mesoderm was induced andresulting differences in body mass, we reared twocell cycle rate closed to that of somatic cells at thedifferent life stages (juvenile and before maturation)

stage. However, some embryos of female white-of Atlantic salmon together with three native

spotted char crossed with male Atlantic salmon did

salmonid species.

hatch.

These hybrid fry have been previously

Most of the Atlantic salmon juveniles (average

identified using restriction fragment length

weight 20 g) reared in a 2 m × 50 cm × 25 cm

polymorphism (RFLP) analysis of a PCR-amplified

aquarium gained weight during the same seasonalfragment of the 16S rRNA gene and the second

period but with different individual growth rates.

80

Kazuo ARAKI, Tetsuji MASAOKA, Hiroyuki OKAMOTO, Hiroyuki NAGOYA,
Hidefumi NARAMURA, Kouji MUTO, Hiromi OKU, Jun-ya AOKI, and Toshiya SUZUKI

Fig. 2. Food competition between Atlantic salmon, Amago salmon, Biwa trout and Masu salmon. A: Competition
for food between Atlantic salmon and Amago salmon. B: Competition for food between Atlantic salmon and Biwa
trout. C: Competition for food between Atlantic salmon and Masu salmon. X-axis: time (days) from commencementof experiment; Y-axis: total number of food pellets consumed by 3 individual salmon.

This also occurred among ten juvenile Amago

Kept in sympatry, most of the five individuals eachsalmons (average weight 20 g) reared in a single-of Atlantic salmon and Amago salmon gained weightspecies tank, however their individual growth rates

during the same period, but with overall differenceswere greater than that of the juvenile Atlantic

in growth likely caused by intraspecific competition

salmon. Interspecific competition for food between

among one or the other of the species.

juvenile Atlantic salmon and Amago salmon

interspecific competition between juvenile Atlantic

appeared weak; intraspecific competition for foodsalmon and Amago salmon appeared weak. Whenamong juvenile Amago salmon appeared stronger

we reared five individual Atlantic salmon together

than what was observed among Atlantic salmon.

with five Biwa trout, the weight of one Biwa trout

Among ten Biwa trout juveniles (average weight 30

individual increased substantially, whereas theg) reared in one aquarium, the weight gain of one wasweights of the other juvenile Biwa trout and theconsiderably large, and that of another much less so

Atlantic salmon increased relatively slowly. The

pronounced. Finally, intraspecific competition forlargest Biwa trout was aggressive toward thefood appeared stronger among the juvenile Biwa

Atlantic salmon to the point of damaging the fins ofsalmon than among either Amago salmon or Atlantic

the latter. Moreover, the Atlantic salmon appeared

salmon.

to avoid the Biwa trout by aggregating in a corner ofThus,

Artificially reared Atlantic salmon and native salmonids in Japan81

Fig. 3. Body weight change in the competition test between Atlantic salmon and Amago salmon. A: The bodyweight change of 6 individual Atlantic salmon reared in a single-species pool. X-axis: time (day); Y-axis: body weight(g). B: The body weight change of 6 individual Amago salmon reared in a single-species pool. C: The body weightchange of 3 Atlantic salmon and 3 Amago salmon reared and fed together in one pool.

the aquarium. Thus, we surmise that interspecificdifference in growth rate (Fig 3B). Biwa trout raisedcompetition between juvenile Biwa trout and Atlantic

in a single-species pond gained weight slowly (Figsalmon was strong in the containment conditions.

4B). For the six Masu salmon raised in a singlespecies pond, the weight increased considerably in

Intra and Interspecific competition among sub-three individuals and decreased slowly in one

adultsindividual (Fig 5B). Our results suggest stronger

W hen w e i nv est ig at ed ou t d oor in t er- a nd
intraspecific competition between Atlantic salmon

intraspecific competition in Masu salmon than
Amago salmon and Biwa trout.

and Amago salmon, or Biwa trout or Masu salmon

We investigated interspecific competition by

before maturation, Atlantic salmon raised in single-

observing three individual Atlantic salmon reared

species ponds increased weight at the same period

together with three Amago salmon, three Biwa trout,with little difference in growth rate (Fig.3A, 4A, 5A).

or three Masu salmon, before maturation, in 5 m × 1.5

Subadult intraspecific competition was deemed weak.

m × 1 m outdoor ponds. When we cultured Atlantic

We also deemed intraspecific competition between

salmon and these native species of salmon in one

subadult Amago salmon was weak because Amago

pond, the amount of food that Atlantic salmon could

individuals increased their weight with little

eat changed depending on the salmon species

82

Kazuo ARAKI, Tetsuji MASAOKA, Hiroyuki OKAMOTO, Hiroyuki NAGOYA,
Hidefumi NARAMURA, Kouji MUTO, Hiromi OKU, Jun-ya AOKI, and Toshiya SUZUKI

Fig. 4. Body weight change in the competition test between Atlantic salmon and Biwa trout. A: The body weightchange of 6 individual Atlantic salmon reared in a single-species pool. X-axis: time (day); Y-axis: body weight (g).
B: The body weight change of 6 individual Biwa trout reared in a single-species pool. C: The body weight changeof 3 Atlantic salmon and 3 Biwa trout reared and fed together in one pool.

combined with Atlantic salmon (Fig.2). We foundthe growth of Atlantic salmon exceeded that of Biwa

that the overall growth of the Amago salmon

trout (Fig. 4C). Kept together in one pond, the two

exceeded that of the Atlantic salmon regardless of

species tended to divide themselves in the habitat.

the fish being kept in separate single-species ponds

Hence, we surmised a minimal level of interspecificor in sympatry (Fig. 3). Kept together in one pond,competition between these species at this life stage,

the Amago salmon tended to distribute themselves

but recognized that Atlantic salmon would possibly

throughout the pond, whereas the Atlantic salmon

dominate Biwa trout with age and growth.

appeared more restricted in the space use. Based on

When three individual Atlantic salmon and threethese observations, we surmise that interspecific

Masu salmon were reared together, we observed thatcompetition was limited between these two species at

Masu salmon tended to occupy the center of the pond

this life stage, although we recognize that Amagoand fed easily, whereas the Atlantic salmon tended tosalmon tended to dominate the Atlantic salmon instay near the bottom and in shade. Also, kept in

sympatry.

sympatry, Masu salmon consumed more food pellets

Reared together in one pond, the three Atlantic

than Atlantic salmon (Fig. 2C) and their body mass

salmon and three Biwa trout consumed a comparable

increased advantageously (Fig.5C). We conclude that

number of food pellets each day (Fig. 2B). However,interspecific competition between Masu salmon andArtificially reared Atlantic salmon and native salmonids in Japan83

Fig. 5. Body weight change in the competition test between Atlantic salmon and Masu salmon. A: The bodyweight change of 6 individual Atlantic salmon reared in a single-species pool. X-axis: time (day); Y-axis: body weight(g). B: The body weight change of 6 individual Masu salmon reared in a single-species. C: The body weight changeof 3 Atlantic salmon and 3 Masu salmon reared and fed together in one pool.

Atlantic salmon was strong in the experimental

Atlantic salmon and consume more food thanconditions, and that the level of competition depends

Atlantic salmon. Habitat segregation is an importanton the combination of species at this life stage (sub-means for juvenile Atlantic salmon and brown trout

adult).

to avoid competition in rivers and lakes (Berg et al.,Many accounts of interspecific and intraspecific2014), with Atlantic salmon juveniles tending tocompetition describing dominance, feeding behaviors,

inhabit deeper waters than brown trout (Heggenes etand growth rates of Atlantic salmon and otheral., 2002). Small, naturally reproducing populations ofsalmonid species exist, particularly as the habitat

Atlantic salmon may exist in some rivers inniche of Atlantic salmon overlaps with that of brown

Vancouver Island on the Pacific coast of Canada,

trout and rainbow trout, especially in juvenile stage

where they are believed to segregate within their(Fausch, 1998; Bremset and Heggenes, 2001; Volpe ethabit when in sympatry with other salmon species,al., 2001b; Stradmeyer et al., 2008; Van Zwol et al.,such as steelhead trout (Maitland and Campbell, 1992;2012).

Volpe et al., 2001b).

Most reports suggest that brown trout

In this study, the Atlantic

dominates and maintain a favorable growth ratesalmon reared in a pond with either Amago salmon

regardless of which other salmonid species areor Biwa trout occupied the deeper areas, seemingly

present, and also that rainbow trout will dominate

to avoid the other species, and ate only food pellets

84

Kazuo ARAKI, Tetsuji MASAOKA, Hiroyuki OKAMOTO, Hiroyuki NAGOYA,
Hidefumi NARAMURA, Kouji MUTO, Hiromi OKU, Jun-ya AOKI, and Toshiya SUZUKI

fallen on the pond floor. Moreover, when reared with
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effect of competition among three salmonids on

In recent years, there has been a great deal of

dominance and growth during the juvenile life

interest in how growth hormone (GH) transgenesisstage. Ecol. Freshw. Fish 21, 535–540

affects fish physiology. However, the results of these

Volpe J. P., Taylor E. B., Rimmer D. W., and Glickman

studies are often difficult to interpret because theB.W., 2001a: Evidence of natural reproduction oftransgenic and non-transgenic fish had very different

aquaculture-escaped Atlantic salmon in coastal

environmental/rearinghistories. This study used a

British Columbia river. Conserv. Biol. 14(3),stable line of size-matched GH Atlantic salmon (Salmo

899–903salar) that were reared in a shared tank with controls

Volpe J. P., Anholt R. R., and Glickman B. W., 2001b:(at 10°C, for ~9・months) to perform a comprehensive

Competition among juvenile Atlantic salmon

examination of the cardiorespiratory physiology of(Salmo salar) and steelhead (Oncorhynchus

GH transgenic salmon, and serves as a novel test of

mykiss): relevance to invasion potential in British

the theory of symmorphosis. The GH transgenic

Columbia. Can. J. Fish. Aquat. Sci. 58, 197–207

salmon had a 3.6 faster growth rate, and 21 and 25%

Yoshida H. and Matsuzaka H., 1991: A summary ofhigher values for mass-specific routine and standard

the exchange commission businesses, such as

oxygen consumption (MO2), respectively. However,Japan and the Soviet Union fishery cooperation

there was no concurrent increase in their maximum

seedling.

Aomori Prefectural Fisheries

MO2, which resulted in them having an 18% lower

Experiment Station Business Overview at First

metabolic scope and a 9% reduction in criticalYear of Heisei. 13-15

swimming speed. This decreased metabolic capacity/ performance was surprising given that the

Annotated bibliographytransgenics had a 29% larger heart with an 18%
greater mass-specific maximum in situ cardiac(1) Du S.-J., Gong Z.-Y., Fletcher L., Shears M. A., King

output, a 14% greater post-stress blood haemoglobin

M.-J., Idler D. R. , and Hew C.-L., 1992: Growthconcentration, 5 – 10% higher red muscle and heartenhancement in transgenic Atlantic salmon by the

aerobic enzyme (citratesynthase or cytochromeuse of an "all fish" chimeric growth hormone geneoxidase) activities, and twofold higher resting and 1.7

construct. Biotechnology (N Y) 10(2), 176–181

higher post-stress, catecholamine levels. However,We have developed an "all fish" growth hormone

gill surface area was the only cardiorespiratory(GH) chimeric gene construct by using an antifreeze

parameter that was not enhanced, and our dataprotein gene (AFP) promoter from ocean pout linked

suggest that gill oxygen transfer may have been

to a chinook salmon GH cDNA clone.

After

limiting. Overall, this research: (1) shows that there

microinjection into fertilized, nonactivated Atlantic

are significant metabolic costs associated with GH

salmon eggs via the micropyle, transgenic Atlantictransgenesis in this line of Atlantic salmon; (2)salmon were generated.

The presence of theprovides the first direct evidence that cardiac

transgene was detected by polymerase chain reaction

function is enhanced by GH transgenesis; (3) shows(PCR) using specific oligonucleotide primers.

A

that a universal upregulation of post-smolt (adult) GH

number of these transgenic fish showed dramatictransgenic salmon cardiorespiratory physiology, as

increases in their growth rate. At 1 year old, thesuggested by symmorphosis, does not occur; and (4)Artificially reared Atlantic salmon and native salmonids in Japan87

supports the idea that whereas differences in arterial

suggests that this species is now naturally

oxygen transport (i.e.cardiac output and blood

reproducing in Vancouver Island Rivers.

oxygen carrying capacity) are important

objective was to quantify the performance of eachdeterminants of inter-specific differences in

species in intra- and inter-specific competition by

aerobicity, diffusion-limited processes must be

assessing the competitive ability of Atlantic salmon

enhanced to achieve substantial intra-specificsympatric with native niche equivalent steelhead –improvements in metabolic and swimming

rainbow trout (Oncorhynchus mykiss). Significant

performance.

behavioural differences, particularly with respect toOuragonism, were observed between species; however,(3) Volpe J. P., Anholt B. R., and Glickman B. W., 2001:

the status of an individual as resident or challenger

Competition among juvenile Atlantic salmon (Salmo

was the best predictor of performance. Resident fishsalar) and steelhead (Oncorhynchus mykiss): relevancealways outperformed challengers, regardless of

to invasion potential in British Columbia. Canadian.

species. Thus, we suggest that Atlantic salmon may

Can. J. Fish. Aquat. Sci. 58, 197–207.

be capable of colonizing and persisting in coastal

Atlantic salmon (Salmo salar) are routinely
captured in both freshwater and marine environmentsof coastal British Columbia (Canada). Recent evidence

British Columbia river systems that are underutilizedby native species, such as the steelhead.

水 研 機 構 研 報， 第 45 号，89 － 100， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，89－100，2017

89

Hybrid Striped Bass National Breeding Program: Research Towards
Genetic Improvement of a Non-Model Species
S. Adam FULLER＊1, Benjamin H. BECK＊1,5, Steven D. RAWLES＊1, Bartholomew W. GREEN*1,
Chao LI＊2, Eric PEATMAN＊2, Catherine J. CHILDRESS＊1, T. Gibson GAYLORD＊3,
Frederick T. BARROWS＊4, and Matthew E. McENTIRE＊1

Abstract: 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 theparent species of the HSB (white bass: WB, Morone chrysops; striped bass: SB, M. saxatilis) have been
fairly limited in scope. At the USDA-ARS HKD Stuttgart National Aquaculture Research Center(HKDSNARC), 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 HKDSNARC are: 1) the development of genomic resources for WB and
SB; 2) the molecular and physiological consequences of alternative production and broodstock dietson HSB and parental species; and 3) the molecular and physiological consequences of exposure todifferent production environments. An overview of these findings will be discussed.
Key words: Morone, selective improvement, transcriptome, RNA-seq, performance testing

Introduction and backgroundreplaced by the more easily spawned reciprocal
cross, or sunshine bass (white bass female × striped

The white bass (Morone chrysops) and the stripedbass male) (Garber and Sullivan, 2006).

bass (M. saxatilis) are temperate basses with highIn general, the HSB commercial production cycle isecological, recreational, and commercial value incomposed of four distinct phases (Hodson, 1995;

North America. The hybrid striped bass (HSB) was

Harrell and Webster, 1997). Following the hatcheryfound to exhibit improved performance in captivity

phase (Phase 0), 3 - 5 day-old larvae (fry) are stockedwith regard to growth, survival, hardiness andinto fertilized, outdoor ponds where they feed on

disease resistance, presumably through hybrid vigor

natural zooplankton. Approximately 30 - 45 days after(heterosis) resulting from the crossing of the twostocking, the fry are recovered as Phase I fingerlings,

parent species, white bass and striped bass (Kerby

graded, trained to feed on prepared diets andand Harrell, 1990; Harrell, 1997). Commercial

restocked at approximately 50 mm length and1g

production of HSB began in the early 1980s with the

body weight. Phase I is the interval from stocking oforiginal HSB cross, or palmetto bass (striped bass

larvae to harvest of the juvenile fingerlings, whichfemale × white bass male), which has been mostly

typically extends from the spawning season in April-2017年２月28日受理（Received on February 28, 2017）
	United States Department of Agriculture (USDA), Agricultural Research Service (ARS) - Harry K. Dupree Stuttgart National Aquaculture
Research Center (HKDSNARC), 2955 Highway 130 East, Stuttgart, Arkansas, 72160-1050, U.S.A.
＊2
	School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, 36849, U.S.A.
＊3
	U.S. Department of the Interior, Fish and Wildlife Service, Bozeman Fish Technology Center, 4050 Bridger Canyon Road, Bozeman, MT 59715,
U. S. A.
＊4
	USDA/ARS - Small Grains and Potato Germplasm Research Unit, Hagerman Fish Culture Experiment Station, 3059F National Fish Hatchery
Road, Hagerman, ID 83332, U. S. A.
＊5
	Present address; United States Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, Auburn, AL,
36832, U. S. A.
E-mail: adam.fuller@ars.usda.gov＊1

FULLER, Benjamin H. BECK, Steven D. RAWLES, Bartholomew W. GREEN, Chao LI, Eric PEATMAN,
90 S. Adam
Catherine J. CHILDRESS, T. Gibson GAYLORD, Frederick T. BARROWS, and Matthew E. McENTIREMay until harvest of the Phase I fingerlings.

diets, many of these alternative “replacement”

Production Phase II begins when the Phase I

formulations have resulted in poorer fish performance

fingerlings are restocked into ponds for growout until

due to an array of reasons, including limited amounts

late winter or early spring, typically February orof essential amino acids (EAA) needed for optimum

March, when the fish are harvested as Phase II

growth (Small and Soares, 1998; Francis et al., 2001;

fingerlings, usually 90 - 225 g in body weight. Phase

Cheng et al., 2003; Glencross et al., 2007). Muscle isII fingerlings are harvested, graded and restocked

the largest tissue compartment in fish, comprising ~into ponds for growout until they weigh 0.68 kg or

60% of the total fish mass and is the desired consumer

more, when they are ready for harvest. At harvest

end product. Hence, it is reasonable to postulate thatsize, the fish are considered to be Phase III HSB.

the muscle amino acid profile provides an ideal model

Typically, the entire growout time from hatching tofor practical diet formulation and for optimizing

harvest is from 18 - 24 months for most HSB

muscle growth. In fishmeal replacement diets, three

producers. In intensive tank culture systems, as

EAAs —lysine, methionine, and threonine — areopposed to pond systems, seasonal influences are

typically identified as first limiting (Keembiyehetty

largely irrelevant, and the HSB production phases

and Gatlin, 1992; Webster, 2002; Encarnação et al.,are defined by mesh size as opposed to the length of

2004; Gatlin et al., 2007).

time in or season of production. In general, at the end

Two growth differentiation factors that areof each production phase, the HSB must be

secreted as part of the regulatory process ofenumerated, measured, graded and restocked or soldmyogenesis are myostatin and myogenin. In cattle,

to alter total densities, make feed adjustments andmyostatin has been shown to act primarily on the

reduce variability in fish size.

muscles by inhibiting myoblast proliferation andWhile HSB is among the most importantdifferentiation; however, when myostatin is mutatedaquacultured species in the United States, mostand non-functional, a double muscling effect results

producers are still reliant on wild-origin broodstock,(Bass et al., 1999; Thomas et al., 2000; Langley et al.,meaning they are not able to take advantage of2002). On the other hand, myogenin is required forgenetic gains available from a selectively improved

the differentiation of myoblasts and fusion ofbroodstock (Garber and Sullivan, 2006). Given the

myogenic precursor cells to existing fibers, or toimportance of HSB to American aquaculture, it iscreate new fibers during myogenesis (Johansen andcritical to continue to develop research results andOverturf, 2005).

genetic resources that can be incorporated into a

Previously, we conducted a growth trial to test the

selectively improved broodstock available tohypothesis that ideal protein theory accurately

American farmers. Following is a brief synopsis ofpredicts first-limiting amino acids and optimum lysine

several studies conducted by my lab.

level for a fishmeal-free, commercial-grade dietformulated for hybrid striped bass (Rawles et al.,Effect of amino acid supplementation on gene2013). In that work we concluded that even though

expression in hybrid striped bass lean muscle

lysine was not first-limiting in our replacement test

Fishmeal represents one of the largest influences

diets, lysine concentration in the muscle of hybrid

on the price of feed, leading to an increase in

striped bass accurately predicted the appropriate

operating expenses for producers that affects the

dietary levels (between 2.7 and 4.1 g Lys / 100g)

long-term sustainability of the industry. This isneeded for improved fish performance with respect

because ocean supplies of fishmeal are finite and at

to a number of response variables, including protein

the maximum sustainable yield, which limits market

accretion and muscle ratio (Rawles et al., 2013). The

availability and results in intense competition among

mechanisms by which dietary amino acidall animal feed industries for this commodity (Naylor

supplementation affect nutrient utilization and

et al., 2000; Tacon and Metian, 2008; Naylor et al.,muscling in fish is unclear, but post-prandial

2009). As research continues to explore minimizing

elevations in plasma amino acids have been shown toor eliminating fishmeal in commercial aquaculture

stimulate protein synthesis in muscle fibers (Frank etHybrid striped bass breeding research program overview

91al., 2006). Moreover, cell culture studies suggest that

stored in RNAlater solution (Ambion, Inc, Foster

there are multiple possible levels of crosstalk in theCity, CA, USA) at -80 ˚C until RNA extraction. Realsalient signaling pathways mediated by mammalianTime RT-PCR methods are described in Childress ettarget of rapamycin (mTOR) – (Proud, 2004;

al. (2015).

Sarbassov et al., 2005; Wullschleger et al., 2006). While

Our goal in the current study was to examine how

a significant body of research has focused on

dietary lysine supplementation of a fishmeal-free dietestablishing the role of amino acids in regulating

influenced the interplay of two muscle genes,

protein synthesis in livestock (Suryawan and Davis,

myostatin and myogenin, with critical roles in

2003, 2005; Suryawan et al., 2001, 2003, 2004, 2006),

myogenesis in HSB and to explore the possibility ofvery little information exists for agriculturally

being able to differentiate growth performance based

important finfish. Therefore, our goal in this follow-

on the genetic profile of HSB in the study. Based on

up study was to elucidate how differential dietary

the criteria we used for genetic variation, i.e., small

lysine supplementation of a fishmeal-free dietvs. large growers, we were able to statistically

influences the expression of two muscle genes,

separate differences in myogenin, but not myostatin,

myostatin and myogenin, in HSB with critical roles in

expression based on size. Our work showed thatmyogenesis.

with minimal lysine supplementation, myogenin

A practical diet for HSB was formulated without

expression was significantly reduced in all fish (P =fishmeal and supplemented with Met and Thr and

0.010) as well as in the small growers (P = 0.042) asvarying levels of Lys to form a series of dose–compared to an unsupplemented diet until the “ideal

response diets that were fed to triplicate tanks ofprotein theory level” of 3.51 g Lys / 100g dietjuvenile HSB for 12 weeks as described in Rawles etconsidered by Rawles et al. (2013) was achieved, thenal. (2013). The lysine supplementation feeding trial

myogenin expression increased. Additionally, in all

was executed in tanks at the Harry K. Dupree

fish (P = 0.003) as well the larger growers (P = 0.010),

Stuttgart National Aquaculture Research Center

when lysine was supplemented over what was(HKDSNARC) using juvenile HSB over an 84 - dayconsidered by Rawles et al. (2013) as “ideal” at 3.51 g

period according to Rawles et al. (2013). Briefly, thelysine / 100 g diet, myogenin expression was

test diets were fed to triplicate tanks of juvenile HSB

significantly reduced.

for 12 weeks. During acclimation, fish were fed a

The interplay of myostatin in relation to myogenin

standard commercial diet for maintaining condition

and the effect of balanced diets in HSB found in thiswith minimal growth.

Fish were then pooled,study contributes towards the goal of achieving

individually weighed (118.4 ± 0.9 g; average initial

marker-assisted selection, which has not yet been

weight ± SE) and randomly stocked (35 fish/tank)widely applied in HSB strains, in order to improve

into 27 circular fiberglass tanks (0.63 m ) supplied

fish performance. To a large extent, this is due to thewith flow-through well-water (24 ºC; 4 L / min/ tank)limited availability of molecular markers for genomic

and ample aeration from a regenerative blower. HSB

analysis in HSB strains. Compared to other farm

were fed to satiation once daily and feed intake wasanimals, including rainbow trout, HSB is still adetermined as previously described (Rawles et al.

relatively new commercially produced taxon and has

2013). On the last day of the trial, feed was withheld

not been thoroughly investigated with respect toand all fish were individually weighed and separated

breeds or strains with distinct genotypic or

into three size categories within each treatment dietphenotypic traits.

based on final weight – small, medium, and large.

contributes towards development of fish that haveRandomly selected fish from each tank were used for

improved growth performance when fed fishmeal-

determination of whole body composition, nutrient

free diets by showing that dietary limiting amino acidretention and body composition indices that included

supplementation can influence myogenesis growth

hepatosomatic index (HSI), intraperitoneal fat (IPF)

factors in HSB.

3

ratio, and muscle ratio (MR). Muscle tissues wereisolated from matching locations on each fish andHence, the current work

FULLER, Benjamin H. BECK, Steven D. RAWLES, Bartholomew W. GREEN, Chao LI, Eric PEATMAN,
92 S. Adam
Catherine J. CHILDRESS, T. Gibson GAYLORD, Frederick T. BARROWS, and Matthew E. McENTIRE
Influence of diet on white bass egg fatty acid profile

three weeks to acclimate and were fed Cargill

Nutrition has significant effects on ovarian growth,

AquaFeed 48-18 (protein-lipid) during this time. Fishfecundity, and progeny robustness (Mourente andwere weighed on April 22 to determine initial weight

Odriozola, 1990; Harel et al., 1994; Mazorra et al., 2003).

and each tank was then randomly assigned a diet.

Among the dietary constituents of prepared feeds,

All tanks were fed to satiation once daily. Each tankdietary lipids and their fatty acids are critical for thewas fed its specific diet for 30 days and five fish wereoverall reproductive performance of the female as

randomly selected for samples and weighed on day

well as progeny development and survival during

31. The remaining fish were fed an additional 28

yolk sac resorption (Fernandez-Palacios et al., 1995,

days and five fish were randomly selected for

1997; Navas et al., 1997; Bruce et al., 1999; Mazorra etsamples and weighed on day 60. The six diets usedal., 2003). Although some organisms can synthesize

included: Zeigler Bass Brood 45-15, Bio-Oregon

EPA and DHA de novo to satisfy their EFA

BioBrood 48-20, Cargill AquaFeed 45-12, Cargill

requirements, many fish lack adequate enzymatic

AquaFeed 48-18, Skretting Extruded Steelhead 45-16,

function to produce these fatty acids at a rate

Skretting Classic Brood 46-12.

sufficient to meet nutrient requirements (Lane andKohler, 2007).

Step-wise discriminant analysis (SDA) was

In the case of broodstock, theconducted on the fatty acid profiles of eggs fromrequirement for dietary EFAs is most likely greatest

white bass fed six different commercial diets usingfrom a previtellogenic period to ovulation. Therefore,

the SAS 9.3 program STEPDISC (SAS Institute,

EPA and DHA are often supplemented in diets ofCary, North Carolina). Overall, time had an influence

broodstock for normal growth and development ofin fatty acid content in white bass eggs, with profile

progeny (March, 1993; Sargent et al., 1995).

of fatty acids changing from the 4 week sampling toFeeding live food sources has proven to be an

the 8 week sampling.

Overall, diets grouped byeffective strategy to satisfy the EFA requirements ofmanufacturer regardless of protein or lipid level. A

broodfish. However, an effective formulated feed forfew of the heavy hitters (known to be important inbroodfish would reduce production costs, simplify

larvae) were different among these diets, including

feed management, and eliminate a potential pathogen

DHA, and EPA. Our next step is to get larvalsource in culture facilities.

performance data for comparison. We anticipate full

These feeds may

potentially outperform live foods (nutritionally),

results to be completed and published soon.

ensuring the supply and continuity of viable progeny
to the aquaculture industry. Further knowledge ofGenomic consequences of rearing hybrid striped

nutritional factors responsible for reproductive

bass under hypoxic conditions

success is needed to develop a broodstock diet andHypoxia is a state of oxygen deficiency that is

improve fingerling production for aquaculture

sufficient to cause impairment of organismal function

producers. Therefore, the objective of this study wasor in extreme cases, death. Hypoxia is becoming an

to evaluate the reproductive performance of white

increasingly important environmental concern (Diazbass broodstock fed one of six commercial diets and2001; Wu et al., 2003) and has received extensive

evaluate the relative fatty acid compositions of theirattention in the fisheries sciences, particularly

eggs.

aquaculture. Understanding the tissue-specific andFemale white bass were held in earthen ponds

temporal changes in gene expression in fishes

during the winter and were seined the first week of

exposed to hypoxia could reveal new mechanisms ofApril when water temperatures reached 15 degrees

hypoxia tolerance and shed light on this adaptive

Celsius. Ninety white bass were evenly stocked into

response in vertebrates. In settings of aquaculture,six 600 L fiberglass tanks. The temperature in all six

the intensity and duration of hypoxia depends on atanks was maintained at 16 degrees Celsius with avariety of factors including fish biomass, feeding rate,water chiller. Lids were placed over the tanks andphytoplankton blooms, and installed aeration capacity

artificial lights and a timer were used to mimic(Green et al., 2015). Previously, we characterized thenatural photoperiod. All six tanks of fish were given

effects of hypoxia on performance metrics of hybridHybrid striped bass breeding research program overview

93

striped bass and found that hypoxia led to reduced
feed intake, which resulted in lower nutrient
retention and growth (Green et al., 2015). However,the precise molecular mechanisms contributing tothese disparate phenotypes were unknown.
However, recent advancement in genomic resources
for striped bass (Li et al., 2014; Reading et al., 2012)
and white bass (Li et al., 2014) offer researchers theability to take a closer look directly at many Morone
functional pathways that were previously studied via
model species. Given the importance of hypoxia on
the management and captive rearing of Morone, we
examine here the transcriptional responses of hybrid
striped bass to acute and chronic hypoxia.

Wehighlight unique and shared signatures between
hypoxic treatments and the bioenergetic
consequences of chronic oxygen deprivation on
hepatocellular function. Our findings offer a morecomprehensive view of the cellular and molecularconsequences of hypoxia and reveal new mechanismsof hypoxia tolerance in teleosts.
The fish used in this study were cohorts of thoseFig. 1. The number of genes that were statisticallydifferentially expressed under each condition and the
direction of their expression. Chronic hypoxia (25%oxygen saturation for 90 days) resulted intranscriptional changes to 91 genes (59 upregulated,
32 downregulated); acute hypoxia (25% oxygen
saturation for 6 h) altered 1339 genes (587 upregulated,
752 downregulated), and the movement of fish fromthe chronic hypoxia treatment to normoxia treatment(100% oxygen saturation) changed 39 genes (20upregulated, 19 downregulated).

subjected to hypoxia as previously described by
Green et al. (2015). Fish were held at DO25 andDO100 levels for 90 d after which nine fish from eachtreatment were sacrificed and liver samples werecollected for RNA-seq. Remaining fish from eachtank were removed from their respective treatments
using a dip net and immediately placed in the
opposite DO saturation DO25 → DO100 (restorationof normoxia) and DO100→DO25 (acute hypoxia) for 6h, after which, nine fish per treatment were sacrificed
and livers collected for RNA-seq. For RNA-seq
methods, see Beck et al. (2016).
Differential expression analysis was performed
between 100% DO saturation (DO100 or normoxia)
and 25% DO saturation groups (DO25 or chronic
hypoxia), between DO100 and DO100 → DO25 (acutehypoxia), and between DO25 and DO25 → DO100
groups. A total of 1403 unique genes (based on
assigned identifies from the NR database) showedsignificant differential expression in liver (Fig. 1). In
detail, there were 91 differentially expressed genes
between DO100 and DO25 saturation groups, ameager 39 genes differently expressed between
DO25 saturation and DO25 → DO100 saturationgroups, and the greatest degree of differential

expression was observed in DO100 and DO100 →DO25 saturation comparison, with 587 up-regulated
genes and 752 down-regulated genes (total 1339
genes). Extracellular flux analysis of liver cells
obtained from DO100 and DO25 fish revealed
significant differences in oxygen consumption rates(OCR), with DO25 liver possessing around 25% higher
OCR (Fig. 2A). Mitotracker Red staining of liver cellsuspensions from DO25 and DO100 fish showed that
DO25 liver cells showed heightened fluorescencelevels, indicative of a greater mitochondrial content(Fig. 2B).
Next-generation sequencing platforms havepropelled rates of discovery, particularly in nonmodel species where few genomic and transcriptomic
resources are available. Work by our group showed
previously the phenotypic impacts of chronic hypoxia
on hybrid striped bass feeding behavior and growthtraits (Green et al., 2015). Here, we delve further into
this area to examine the molecular consequences ofoxygen deprivation in an effort to reveal themolecular underpinnings responsible for the overall
poorer performance under hypoxemia.
Previously (using the same experimental cohorts of

FULLER, Benjamin H. BECK, Steven D. RAWLES, Bartholomew W. GREEN, Chao LI, Eric PEATMAN,
94 S. Adam
Catherine J. CHILDRESS, T. Gibson GAYLORD, Frederick T. BARROWS, and Matthew E. McENTIRE
mitochondrial oxidation of lipids as aprotective measure against toxic
metabolites and oxidative stress.
Broadly, hypoxia promotes lipid
storage and inhibits lipid catabolism
through ϐ oxidation (Whitmer et al.,
1978; Bostrom et al., 2006). In contrast,
in aquatic animals, environmental
hypoxia is a common challenge thatmany aquatic organisms experience intheir habitat and responding to hypoxia
requires metabolic reprogramming sothat energy-demanding processes are
regulated to match available energy
reserves (Gracey et al., 2011). Here, we
observed changes — with similarities
and differences to work in mammals
Fig. 2. A) Liver tissue isolated from fish subjected to chronic
hypoxia show greater oxygen consumption rates (OCR; mean ±SEM; five fish per treatment) and B) increased mitochondrial
content (two representative fish from each treatment) as indicatedby the fluorescence intensity derived from staining with themitochondrion-selective dye Mitotracker Red.

and other teleosts — in multiple genes
involved in lipid catabolism and ϐoxidation, particularly following acutehypoxia. Enrichment analysis pointed
us towards a deeper examination of cell
death processes.

Two pathways

r e la t e d t o c e llula r pr e s e r va tion,
apoptosis and autophagy, were clearly

the fish employed here), we observed significantly

influenced by hypoxic insult. In taking together thelarger livers (as indicated by the hepatosomaticgross differences in liver size, the alterations in lipid

index) in HSB reared in normoxia versus hypoxia andutilization, the suppression of apoptosis combined

reported significantly higher levels of whole bodywith the induction of autophagy, our findings suggest

lipid (Green et al., 2015). Qualitatively, the enlarged

that the hepatic tissue of hybrid striped bass may

livers were noticeably pale and soft in texture, while

have entered a state of senescence. This may havefish exposed to hypoxia exhibited livers that wereallowed the animals to liberate, redirect, or shuntdarkened in appearance, firm, and friable (Green etresources to other vital tissues to survive hypoxia.

al., 2015). These observations led us to focus on

The use of RNA-seq allows for the examination of

examining the liver transcriptome in the present

global transcriptional changes in a tissue at astudy. Indeed, the liver plays a central role inparticular snapshot in time. However, it is widely

metabolic homeostasis and is a major site for theunderstood that the level of transcriptional message

synthesis, metabolism, storage and redistribution ofdoes not always correlate with protein. With little toprocessed carbohydrates, proteins and lipids

no resources (i.e., antibodies) available for HSB, we(Bechmann et al., 2012). In tissues such as the heart

utilized extracellular flux analysis to quantify aerobic

and liver, lipids provide a rich source of energy via

respiration in liver tissue obtained from DO100 andoxidative phosphorylation by mitochondria

DO25 fish. Unexpectedly, under conditions of reduced(Jungermann, 1988; Shohet and Garcia, 2007). In

oxygen availability, liver cells from DO25 fish showedmammals, where hypoxic stress is typically viewed

significant increases in basal oxygen consumption

as a pathological state (e.g., fatty liver disease,

rates, a surrogate measure of mitochondrial function.

ischemia), lipid metabolism under low oxygen

Curiously, as compared to our previous work withconditions is reprogrammed to suppress

established fish cell lines (Beck and Fuller, 2012), total

95

Hybrid striped bass breeding research program overview
aerobic respiration was lower and glycolysis was

striped bass were harvested, and equal amounts ofvirtually non-detectable. By using a fluorophore thattissue from each system were pooled prior to RNA

selectively stains mitochondria, we documented an

extraction. The result was two master pools of RNA,

increase in the mass (linked to number/size of

one for each species. Each pool was used for librarymitochondria) of the mitochondrial compartment,

construction and sequencing in a lane of Illumina

which could have accounted for the increase in

HiSeq2000 platform. A total of 262 x 106 total highoxygen consumption by DO25 liver cells. An increase

quality reads were obtained with 135 x 106 reads

in mitochondrial number, a process termed

from striped bass and 127 x 106 reads from white

mitochondrial biogenesis, is a common cellularbass. Using the TRINITY de novo assembly software

response to hypoxia and is thought to improve the(Grabherr et al., 2011), reads were assembled into

efficiency of oxygen consumption and ATP synthesis,

203,587 striped bass unique contigs and 185,531 whiteand in parallel counteract potential cellular damage

bass unique contigs. N50 and average contig sizes

brought about by hypoxia (Kopp et al., 2014).

were 2915 and 1263 bp respectively for striped bass,

Consistent with these observations is a greater thanand 3132 and 1371 bp respectively for white bass

400 fold increase in PGC-1ϐ message in liver tissue(Table 1).

from the acute hypoxia treatment. In contrast totranscripts that were identified for the first time in

mammals, where PGC-1α is regarded as the

striped bass and white bass, respectively. Annotationarchetypal master regulator of mitochondrial

was carried out by BLAST against the UniProt andbiogenesis, recent evidence from teleosts suggests

NR (NCBI non-redundant) databases for both species.

that PGC-1ϐ may exert more control overAt an E-value ≤ 1e-5, 21,186 and 29,624, unigene

mitochondrial gene expression (LeMoine et al., 2008).

matches were obtained against the UniProt and NR

Intriguingly, and related to the above discussion on

databases respectively in striped bass, and 21,001 andlipid metabolism, PGC-1ϐ is also known to play a key

28,906 matches were returned in white bass against

regulatory role in hepatic lipid metabolism with PGC-

the same databases. Of these NR matches, 25,902

1ϐ knockout mice being more susceptible to hepatic(87.4%) in striped bass and 25,484 (88.2%) in white

steatosis (Sonoda et al., 2007) and the transduction ofbass were predicted to have full-length transcriptrodent liver with PGC-1ϐ decreased hepatic lipid

coverage based on TRINITY analysis. Using morewhile plasma triglyceride and cholesterol levels were

stringent criteria, similar results were obtained fromsignificantly elevated (Lin et al., 2005).

Clearly,both species, with 18,630 UniProt and 23,605 NR

further research is needed to better understand theannotated unigenes in striped bass and 18,584

interactions between mitochondrial abundance,

UniProt and 22,354 NR annotated unigenes in white

regulation, and lipid metabolism in hypoxic teleosts.

bass (score ≥ 100, E-value ≤ 1e-20; Table 2).

These included 166,867 and 185,351

In both species most valuably from a management
Development of moronid genomic resources
Genetic information has been restricted to a single

standpoint, the transcriptomes yielded microsatellite
and SNP markers valuable in future downstream

tissue (ovary) transcriptome and a microsatellite
linkage map from striped bass (Reading et al., 2012;
Liu et al., 2012), limiting gene discovery andexpression and functional studies in the two species
and their hybrid. Here we set out to produce wellannotated transcriptomes for both species to advancefuture broad-based RNA-seq studies of gene
expression as well as aid in more targeted studies ofimportant genes and pathways.
Major tissues and organs (brain, liver, spleen, trunk
kidney, ovary, testes, gill, and intestine) from 10
individuals from white bass and 10 individuals from

Table 1. Summary of de novo assembly results ofIllumina RNA-seq data from striped bass and whitebass using Trinity assembler
Striped Bass White Bass
Contigs

203,587

185,531

Largest contig (bp)21,10028,262Large contigs (≥1000bp)

68,39566,891

Large contigs (≥500bp)

98,864

94,485

N50 (bp)

2,915

3,132

Average contig length (bp)

1,263

1,371

FULLER, Benjamin H. BECK, Steven D. RAWLES, Bartholomew W. GREEN, Chao LI, Eric PEATMAN,
96 S. Adam
Catherine J. CHILDRESS, T. Gibson GAYLORD, Frederick T. BARROWS, and Matthew E. McENTIRE
Table 2. Summary of gene identification and annotation of assembled Striped bass and White bass contigs basedon BLAST homology searches against various protein databases (UniProt and nr). Putative gene matches were at
E-value ≤ 1e-5. Hypothetical gene matches denote those BLAST hits with uninformative annotation. Quality
unigene hits denote more stringent parameters, including score ≥ 100, E-value ≤ 1e-20.
Striped bass

White bass

UniProt

nr

UniProtnrContigs with putative gene matches

69,13479,062

68,312

76,884

Annotated contigs ≥1000bp

54,48758,31654,43057,839Annotated contigs ≥500bp

62,602

68,876

62,15867,682

Unigene matches

21,186

29,62421,001

28,906

Hypothetical gene matches
Quality Unigene matches

0

1,901

0

1,858

18,63023,605

18,58422,354analyses. In striped bass, from a total of 32,111

2012: The interaction of hepatic lipid and glucose

microsatellites identified by MSATFINDER

metabolism in liver diseases. J. Hepatol. 56,(Thurston and Field, 2005), 36.05% (n = 11,577) had

952–964.

sufficient flanking regions to allow design of primers.

Beck B. H. and Fuller S. A., 2012: The impact ofThese 11,577 microsatellites were distributed acrossmitochondrial and thermal stress on the

10,055 contigs. Similarly, in white bass, from a total of

bioenergetics and reserve respiratory capacity of

30,408 microsatellites, 34.53% (n = 10,500) had

fish cell lines. J. Aquat. Anim. Health 24, 244–

sufficient flanking regions to allow design of primers.

250.

These 10,500 microsatellites were distributed across

Bostrom P., Magnusson B., Svensson P., Wiklund O.,

9054 contigs. A SNP analysis comparing between

Boren J., Carlsson L. M. S., Stahlman M., Olofsson

species yielded 2220 markers polymorphic in one

S., and Hulten L. M., 2006: Hypoxia converts

species but not the other, including 1661 SNPs

human macrophages into triglyceride-loaded

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myogenin, controlling myogenesis in differentiallyin ischemic and hypoxic hearts. J. Biol. Chem.

growing groups of HSB. Real-time rt-PCR results in

253, 4305–4309.

HSB suggest that the levels of lysine added to theWu R.-S., Zhou B.-S., Randall D. J., Woo N.-Y., and Lam

diet has an impact on myogenin relative to theP.-K., 2003: Aquatic hypoxia is an disrupter andunsupplemented diet, but no effect on myostatin.

impairs fish reproduction.

Moreover, presented data also suggests that theEnviron. Sci.

Technol. 37, 1137–1141.

amount of dietary lysine supplementation influenced

Wullschleger S., Loewith R., and Hall M. N., 2006: TOR

the ratio of myostatin/myogenin expression in HSB

signaling in growth and metabolism. Cell 124,

and that this pattern mimicked that of most of the471-484.

growth, composition of growth and nutrient retention
data from the authors’ previous study and may
Annotated bibliography

therefore be a useful marker for selecting fish for
improved growth performance.

(1) Li C, Beck B. H., Fuller S. A. , and Peatman E., 2014:
Transcriptome annotation and SNP discovery in(3) Fuller S. A., Farmer B. D. and Beck B. H., 2014:

white bass (Morone chrysops) and striped bass

White bass Morone chrysops is less susceptible than(Morone saxatilis). Anim. Genet. 45, 885-887.

its hybrid to experimental infection with

The authors present the first ever multi-tissue

Flavobacterium columnare. Dis. Aquat. Org. 109, 15-

FULLER, Benjamin H. BECK, Steven D. RAWLES, Bartholomew W. GREEN, Chao LI, Eric PEATMAN,
100 S. Adam
Catherine J. CHILDRESS, T. Gibson GAYLORD, Frederick T. BARROWS, and Matthew E. McENTIRE
22.

(4) Beck B. H., Fuller S. A., Peatman E., McEntire M.

The authors present research regardingE., Darwish A. M., and Freeman D.W., 2012: Chronic

Flavobacterium columnare, the causative agent ofexogenous kisspeptin administration accelerates

columnaris disease, susceptibility differences between

gonadal development in basses of the genus Morone.

hybrid striped bass (HSB) and white bass (WB) in aComp. Biochem. Physiol. A Physiol. 162, 265-273.

series of 3 fundamental studies.

In the first

The authors present the effects of chronic

experiment, the authors sought to determine whether

administration of kisspeptins to immature andcolumnaris disease could be developed using a low-mature white bass (WB), striped bass (SB), and hybrid

water flow experimental challenge in HSB using 3

striped bass (HSB) to determine its effects on gonadal

levels of F. columnare (60-, 30-, 10- ml). Each of thesedevelopment in these species. The authors

treatment groups exhibited significantly different

determined that bi-weekly injections (over 7 weeks)survival rates: 0, 3.3, and 13.3%, with higher survival

differentially accelerate puberty, as evidenced byoccurring in treatment groups exposed to lessincreases in the prevalence of spermatozoa in thebacteria. In the second experiment (30 ml), both HSB

testes of juvenile fish. Also, in sexually mature fish,

and WB had a 0% survival rate, but the WB took

kisspeptin treatment led to increased gonad weight,

significantly longer to reach 100% mortality. Finally

gonadosomatic index, and spermatocrit in some whitein Expt 3 (10 ml), no HSB survived, whereas 33% of

and striped bass. Additionally, mature white bass

WB survived. Compared to controls, the authors

treated with kisspeptins showed an advancement in

observed extensive gill damage in HSB treated withoocyte development as determined by histological

10 ml after 24 h, which they hypothesized could haveexamination. Importantly, the gonadal changes

contributed to the higher mortality observed in HSB;

occurred in the absence of any photothermal

an observation not seen on the WB gills. From thesemanipulation or hormone injections. This description

series of experiments, it is clear that HSB are more

was the first report of kisspeptin-mediated pubertal

sensitive to F. columnare, having lower survival andinitiation in fish, and the first evidence that

more extensive histological damage compared to WB

kisspeptins could modulate gonad maturation.

following the bacterial challenge.

水 研 機 構 研 報， 第 45 号，101 － 105， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，101－105，2017

101

Production of Benedenia-resistant Yellowtail (Seriola quinqueradiata)
Families–A Preliminary Approach to the Broodstock Candidates–
Tsutomu NODA＊1, Kazunori YOSHIDA＊1, Takurou HOTTA＊1, Hirotaka MIZUOCHI＊1,
Taro CHUJO＊1, Hideaki AONO＊2, Akiyuki OZAKI＊3, Kazuo ARAKI＊4, Mitsuru YOSHIMOTO＊5,
Hiroki OGAWA＊6, Yasuhiro SHIMA＊7, and Yuichiro FUJINAMI＊1

Abstract: The skin fluke Benedenia seriolae is a parasite specific to Seriola species. It feeds on theepidermal tissues of yellowtail (Seriola quinqueradiata) and causes external injuries that render thefish susceptible to bacterial and viral infection. Infection with this parasite is a serious problem foryellowtail aquaculture because it also leads to a reduction in fish growth. A previous study hasreported the existence of an inherited Benedenia disease-resistance factor in yellowtail. Any
yellowtail families resistant to Benedenia disease have not yet been produced, although the
production of such families for aquaculture would potentially help to reduce not only infectious
diseases but also the labor costs to eradicate Benedenia. In this study, we investigated time serieschanges of the number of Benedenia parasites on each host yellowtail for the purpose of selecting
Benedenia resistant broodstock candidates.
In September 2014, the number of individual parasites on 100 fish that were selected randomly
from 10,000 wild-caught 0-age yellowtail at an aquaculture farm was investigated and it ranged from
1 to 48 individuals / fish. Thus we selected 961 fish that has the lowest 10% parasite susceptibility(3 or fewer Benedenia). The selected fish were then cultured in sea net cages and the number ofparasites on each was counted at 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 individuals / fish over this period, andthe overall mean was 8.9 individuals / fish. One hundred sixty fish with lower than 3 parasites wereselected as broodstock candidates.
We are now using these broodstock candidates as parents to produce F1 yellowtail families for
Benedenia-resistant analysis using DNA marker-assisted-selection breeding methods.
Key words: Yellowtail (Seriola quinqueradiata), Skin fluke (Benedenia seriolae), Breeding, Benedeniaresistant family.

2017年２月28日受理（Received on February 28, 2017）	Goto Laboratory, Seikai National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Nunoura 122-7, Tamanoura,
Goto, Nagasaki 853-0508, Japan＊2
	Seikai National Fisheries Research Institute, Japan Fisheries Research and Education Agency, Taira 1551-8, Nagasaki, Nagasaki 851-2213,
Japan＊3
	National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Nakatsuhamaura 422-1, Minamiise, Watarai, Mie
516-0193, Japan＊4
	Tamaki Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Hiruda 224-1, Tamaki,
Watarai, Mie 519-0423, Japan＊5
	Aquafarm, Maruha Nichiro Corporation, Kamiura Niinameura 3620-7, Saiki, Oita 879-2603, Japan＊6
	Shimaura Aquafarm Branch, Maruha Nichiro Corporation, Shimaura 613-5, Nobeoka, Miyazaki 882-0096, Japan＊7
	Minami-Izu Laboratory, National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, Minami-Izu 183-2, Kamo,
Shizuoka 415-0156, Japan
E-mail: ttmnoda@affrc.go.jp＊1Kazunori YOSHIDA, Takurou HOTTA, Hirotaka MIZUOCHI, Taro CHUJO, Hideaki AONO, Akiyuki OZAKI,
102 Tsutomu NODA,
Kazuo ARAKI, Mitsuru YOSHIMOTO, Hiroki OGAWA, Yasuhiro SHIMA, and Yuichiro FUJINAMI
Introduction

weeks after the treatment. Thus, the farmers haveto treat the cultured fish many times during theMany fish species are farmed in Japan, andaquaculture period and the treatment requires ayellowtail is one of the most important species for

great deal of effort, as well as causing stress to theaquaculture production (Fig. 1).

In yellowtail

fish via both handling and the freshwater treatment.

aquaculture, wild- caught juveniles are farmed to

A previous study of genetic analysis reported amarketable size (Nakada 2008). However, fishing

heritable Benedenia resistance factor in yellowtail

season, abundance and size of wild juveniles are(Ozaki et al. 2013). However, Benedenia-resistantunstable. Additionally, fishing pressure on wild

yellowtail families have not yet been produced. The

juveniles for aquaculture could impact the natural

production of such families would help reduce bothresources of yellowtail. For these reasons, using

risks of infectious disease and efforts to eradicate theartificial seed is anticipated to enable a more stableparasite in aquaculture facilities. In the present

and sustainable aquaculture production.

study, broodstock candidates with a low parasite

Furthermore, farmers require seedlings withcount were selected for breeding value-added

additional value such as disease resistance and rapid“Benedenia-resistant” yellowtail families.

growth during culture (Yoshida et al. 2012).
Materials and methods
Preliminary selection
The preliminary process focused on selecting
yellowtail with the lowest 10% parasite count among
10,000 wild-caught 0-age fish at Shimaura Aquafarm
Branch, Maruha Nichiro Corporation, Miyazaki,
Japan. Because there was no data available on theparasite intensity for the selection, the initial step ofour study was to investigate the infection levels of
the yellowtail.
In September 2014, the frequency distribution of
the parasite number for 100 randomly selected fish
was examined (the procedure of the parasite counts
Fig. 1. Species composition of marine finfish
aquaculture production in Japan 2013. Total
production is approximately 240,000 tons (adaptedfrom the 2013 census of fisheries by Fisheries Agencyof Japan).

is described later).

The parasite count per fishranged from 1 to 48 individuals / fish, and the lowest
10% of them ranged from 1 to 3 individuals / fish (Fig.
2). From these data, we decided to select fish with 3

In yellowtail aquaculture, one of the serious
problems is the parasitic disease caused by the skin
fluke, Benedenia seriolae. It parasitizes the fish bodysurface and feeds on the epidermal tissues. The
parasitization causes external injuries and increases
the risk of viral or bacterial infections, as well as
growth reduction of the host fish. At aquaculture
farms, the most common method to eradicate theparasite is the freshwater bathing treatment.
However, new parasites resurface within a few

Fig. 2. Frequency distribution of the parasite countamong the 100 wild-caught 0-age yellowtail. The
frequencies from 20 to 48 (the highest value)
individuals / fish range from 0 to 2.

Production of Benedenia-resistant Yellowtail

103

or fewer parasites per fish, and 961 fish wereparasites on the fish body surface and the removing

preliminary selected from the sample of 10,000

ones in the bag.

yellowtail at the aquaculture farm. The mean of fork
Results and Discussion

length and body weight of these fish wereapproximately 30.0 cm and 370 g, respectively.

Fig. 3 shows the changes in parasite count for 40
Selection for broodstock candidates
In November 2014, these preliminary selected fishyellowtail with the lowest parasite count and 10
individuals with the highest parasite count in eachwere transferred from the aquaculture farm to

net cage.

floating sea net cages at Goto Laboratory, Seikai

number of parasites per fish had a wide range from

National Fisheries Research Institute, Nagasaki,

0.2 to 39.4 individuals / fish over this period, and theJapan. The mean (± SD) of fork length and bodyoverall mean parasite count was 8.9 individuals / fish.

weight of these fish were 36.5 ± 1.5 cm and 828 ± 109

Based on the results, a total of 160 fish (40 fish withg, respectively. Of all the 961 fish, 800 fish that had

the lowest parasite count were selected from eachno obvious physical damage were selected andcage) were selected for broodstock candidates with

passive integrated transponder (PIT) tags (Biomark

low parasitic susceptibility. The mean (range) ofInc.) were inserted for individual identification. The

parasite count on these candidates was 3.3 (0.2–7.0)

fish were reared in 4 groups of 200 individuals in 4

individuals / fish. Additionally, the average parasite

net cages (A–D).

count for 9 fish was lower than 1.0 individual / fish

The examinations of parasite count on theexperimental fish (detailed in Yoshida et al. 2012)

Among 5 examinations, the averageduring the experimental period, and these fish
showed remarkably less-susceptibility.

were performed at 5 times during November 2014

The purpose of further study is to establish

and July 2015. Because the number of Benedenia

Benedenia-resistant yellowtail families. In 2017, F1

parasites tends to decrease during winter, theyellowtail hatchery juvenile will be produced fromcounting of them was not conducted between

these selected broodstock candidates. Moreover, we

December and March.

At each counting, theare developing DNA markers for marker-assisted

experimental fish were individually captured in a

selection breeding methods. In the F1 generation, wemesh bag and then the bags were placed in awill carry out both trait evaluation and also marker-

freshwater tank for 4 minutes (all Benedenia diedassisted selection by genetic analyses. The goal ofwithin 4 minutes). After that, the fish was identified

this study is fixation of the trait in the F2 generationfrom the PIT tag, and the total number of parasites

and to produce Benedenia-resistant yellowtail families

was obtained by summing the remaining deadfor development of sustainable yellowtail aquaculture

Fig. 3. Time series changes of parasite count per yellowtail by the fish cage. The lowest 40 and the highest 10 fishof parasite susceptibility were drawn. Because the number of Benedenia parasites tends to decrease during winter,counting of them was nod conducted between December and March.

Kazunori YOSHIDA, Takurou HOTTA, Hirotaka MIZUOCHI, Taro CHUJO, Hideaki AONO, Akiyuki OZAKI,
104 Tsutomu NODA,
Kazuo ARAKI, Mitsuru YOSHIMOTO, Hiroki OGAWA, Yasuhiro SHIMA, and Yuichiro FUJINAMI
production.

Aoki J., Kawabata Y., Suzuki J., Akita K., Koyama T.,
Nakagawa M., Hotta T., Tsuzaki T., Okamoto N.,
Acknowledgment

Araki K., and Sakamoto T., 2013: Quantitative Trait
Loci (QTL) Associated with Resistance to a

The authors deeply appreciate the assistance ofMonogenean Parasite (Benedenia seriolae) in

the members of the Goto Laboratory, Seikai National

Yellowtail (Seriola quinqueradiata) through Genome

Fisheries Research Institute for rearing yellowtail.

Wide Analysis. PLoS ONE 8(6): e64987.

This study was supported by grants from theBenedenia infections caused by the monogenean

Agriculture, Forestry and Fisheries Research

fluke ectoparasite Benedenia seriolae seriously impact

Council.

marine finfish aquaculture. Genetic variation in hosthas been inferred to play a significant role inReferences

determining the susceptibility to this parasitic
disease. To evaluate the genetic basis of Benedenia

Mushiake K., Yamazaki H., and Fujimoto H., 2007:

disease resistance in yellowtail (Seriola

Current Situation of Technical Developments inquinqueradiata), a genome-wide and chromosome-Seed Production of Yellowtail (Seriola

wide linkage analyses were initiated using F1

quinqueradiata) in Japan. Proceedings of theyellowtail families (n = 90 per family) based on a highthirty-fourth U.S.-Japan aquaculture panel

density linkage map with 860 microsatellite and 142

symposium (ed. by Stickney R., Iwamoto R., andsingle nucleotide polymorphism (SNP) markers. Two

Rust M.), NOAA Technical Memorandum

major quantitative trait loci (QTL) regions on linkage

NMFS-F/SPO-85, Seattle, pp. 1-4.

groups Squ2 (BDR-1) and Squ20 (BDR-2) were

Nakada M., 2008: Capture-based aquaculture ofidentified. These QTL regions explained 32.9–35.5%

yellowtail. Capture-based aquaculture. Globalof the phenotypic variance. On the other hand, theoverview (ed. By Lovatelli A. and Holthus P. F.),

relationship between QTL for susceptibility to B.

FAO Fisheries Technical Paper 508, FAO, Rome,

seriolae and QTL for fish body size were investigated.

pp. 199–215.

The QTL related to growth was found on another

Ozaki A., Yoshida K., Fuji K., Kubota S., Kai W., Aoki

linkage group (Squ7). As a result, the authors present

J., Kawabata Y., Suzuki J., Akita K., Koyama T.,

first genetic evidence that contributes to detailing

Nakagawa M., Hotta T., Tsuzaki T., Okamoto N.,

phenotypic resistance to Benedenia disease, and the

Araki K., and Sakamoto T., 2013: Quantitative

results will help resolve the mechanism of resistance

Trait Loci (QTL) Associated with Resistance to a

to this important parasitic infection of yellowtail.

Monogenean Parasite (Benedenia seriolae) in
Yellowtail (Seriola quinqueradiata) through(2) Yoshida K., Ozaki A., Nakagawa M., Hotta T., Aoki

Genome Wide Analysis. PLoS ONE 8(6): e64987.

J., Koyama T., Araki K., Okamoto N., Sakamoto T.,

Yoshida K., Ozaki A., Nakagawa M., Hotta T., Aoki J.,

and Tsuzaki T., 2013: Characteristic Evaluation

Koyama T., Araki K., Okamoto N., Sakamoto T.,

Method Relating to Benedenia Disease of Yellowtail

and Tsuzaki T., 2013: Characteristic Evaluation(Seriola quinqueradiata). Proceedings of the forty U.S.-

Method Relating to Benedenia Disease ofJapan aquaculture panel symposium (ed. by Rust M.,

Yellowtail (Seriola quinqueradiata). Proceedings

Olin,P. Bagwill A., and Fujitani M.), NOAA Technicalof the forty U.S.-Japan aquaculture panel

Memorandum NMFS-F/SPO-136, Seattle, pp. 96-99.

symposium (ed. by Rust M., Olin,P. Bagwill

A.

Benedenia is a parasitic disease caused in Seriola

and Fujitani M.), NOAA Technical Memorandum

species by Benedenia seriolae. This parasite can

NMFS-F/SPO-136, Seattle, pp. 96-99.

cause growth reduction and external injuries inyellowtail, increasing the risk of secondary viral or

Annotated Bibliographybacterial infection. The main method of parasite
removal is to soak the fish in a freshwater bath.

(1) Ozaki A., Yoshida K., Fuji K., Kubota S., Kai W.,

However, this method requires a great deal of time,

105

Production of Benedenia-resistant Yellowtail
cost, and effort.

We have been studying DNA

Marker-Assisted Selection (MAS) breeding, to selectfor resistance to Benedenia disease.

that can be released into the wild at a size similar tothat of the wild stock.

Threecomponents ("Reproduction technology", "Character(4) Nakada M., 2008: Capture-based aquaculture ofevaluation", and "DNA analysis") are criticallyyellowtail. Capture-based aquaculture. Global

important to promote MAS breeding success. We

overview (ed. By Lovatelli A. and Holthus P. F.), FAO

focus on one of the key components, "Characteristic

Fisheries Technical Paper 508, FAO, Rome, pp. 199–

evaluation method” relating to Benedenia disease in215.

yellowtail.

The 2004 production of cultured yellowtail (Seriolaspp.) in Japan from 1,288 enterprises was 150,028(3) Mushiake K., Yamazaki H., and Fujimoto H., 2007:

tonnes valued at ¥111.2 billion (US$1.334 billion).

Current Situation of Technical Developments in Seed

Yellowtail mariculture has developed remarkably

Production of Yellowtail (Seriola quinqueradiata) in

due to the abundant supply and low price of wild-

Japan. Proceedings of the thirty-fourth U.S.-Japancaught juveniles (Mojako) and sardines used as theaquaculture panel symposium (ed. by Stickney R.,

main fish feed of fishmeal component. Hatchery

Iwamoto R., and Rust M.), NOAA Technicalproduced yellowtail seed are far more expensive.

Memorandum NMFS-F/SPO-85, Seattle, pp. 1-4.

Other critical elements that supported the growth of

The National Center for Stock Enhancement

yellowtail farming include the existence of abundant(NCSE, formerly Japan sea-farming Association), of

suitable culture sites along the Japanese coast andthe Fisheries Research Agency, introduced the stock

innovative technical developments.

enhancement program for yellowtail (Seriola

The history of yellowtail culture in Japan began

quinqueradiata and Seriola lalandi) in 1977. Technicalover 70 years ago. Before that, fishers cultured

developments in induced spawning as well as larval

undersized fish in ponds and sold them when theyand juvenile rearing techniques have increased thereached marketable size. This utilization of bycatch

population of this species to 1 million juveniles per(undersized fish) was accepted by the public,

year at NCSE.

This project faced three major

particularly as unmarketable fish were often used as

drawbacks: high mortality of larvae, cannibalism, andfertilizer or livestock feed. Currently aquaculture

the smaller size of released juveniles in comparison

production for many species exceeds that landedwith their wild counterparts. The high mortality offrom capture fisheries.

larvae was overcome by utilizing strong aeration

Some commercial culture trials on amberjack haveduring the early larval stage, while cannibalism wasbeen undertaken in Taiwan, Province of China,

controlled by grading juveniles by size selection. The

Mexico and Vietnam, but no successes have been

two-month delay in the spawning season of reared

achieved with raising yellowtail.

broodstock (the usual spawning season is late April toconstraints include diseases and low production costs

early May), which caused the smaller size of released

in tropical areas. In contrast, the culture of Seriola

juveniles, was solved by developments in advanced

spp. is promising due to their strong vitality andspawning techniques.

rapid growth, and may well expand at the global

Photoperiod and watertemperature manipulations were used to produce
eggs in February, thus producing yellowtail juveniles

The mainlevel through hatchery-produced juveniles.

水 研 機 構 研 報， 第 45 号，107 － 112， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，107－112，2017

107

Coho Salmon Broodstock Development: A Case Study of the Domsea
Coho Salmon (1977 to 2015)
Robert N. IWAMOTO＊1, Richard TOWNER＊2, James MYERS＊3, Greg HUDSON＊1,
and Patricia MUNSELL＊1

Abstract: There are few long-term studies on Pacific salmon that inform on the potential for gains
by systematic selective breeding. As a consequence, there is limited data on the potential for
inbreeding losses because of constrained population sizes and matings among closely related
individuals over a prolonged period of time. In 1977, Domsea Farms Inc., the University ofWashington, 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 at that time regarding
the potential for genetic improvement in coho salmon, the program was initially designed with twocentral goals: 1) collect basic information (heritabilities, genetic and phenotypic correlations) on thepotential for genetic improvement in 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 and operations, the program has
remained remarkably consistent over the past 38 years or 19 generations of selection. We havedemonstrated that selection for improved growth to the smolt stage (7.1-11.3% per generation) andadult phases (43-53 g per generation) can be achieved. Overall, the growth 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. While
traditional genetic approaches have been demonstrably successful for this program, it is anticipatedthat further consideration and application of molecular approaches will help further characterize
and advance this broodstock program for coho salmon.
Key words: Coho salmon, Oncorhynchus kisutch, Domsea, selection gains, inbreeding

Introduction

there is less known about the effects of long-term
intensive selection and the resulting inbreeding on

Over the past 50 years or so, the application ofclosed populations because of the lack of well-genetic selection theory to improve strains of fish anddocumented, multi-generational selection programs.

shellfish has been an active and, in many cases,

This paper presents the results of a 19-generation

successful endeavor (see Quinton et al. 2005-Atlantic

long and still on-going selection program on a closed-

salmon; Dunham and Smitherman 1984-catfish; Tave

population of coho salmon (Oncorhynchus kisutch). It

1999-Tilapia). As a result, the genetic bases for trait

outlines the general facets of the broodstock

improvement are now well characterized for many(commonly known as the Domsea coho) and somecommercially important aquatic species. However,major results including trait characterization and2017年２月28日受理（Received on February 28, 2017）	Riverence LLC, Rochester, WA 98579, U. S. A,＊2	GenTec Consulting, Payette, ID 83661, U. S. A.
＊3
	NOAA NMFS Northwest Fisheries Science Center, Seattle, WA 98112, U. S. A.
E-mail: riwamoto@reverence..com
＊1

108

Robert N. IWAMOTO, Richard TOWNER, James MYERS, Greg HUDSON, and Patricia MUNSELL

improvement as well as inbreeding effects to date.
Materials and methods
The Domsea coho genetics program was initiatedin 1977 as a cooperative effort among Domsea Farms(a then subsidiary of Union Carbide, Inc.), theUniversity of Washington, and the Washington Sea
Grant Program. The initial population was founded
in 1974 with gametes from the Wallace River
Hatchery strain, Skykomish River Basin, in western
Washington State.

The population had been

maintained in captive culture via random mating
prior the selection program’s start three years later.
The initial goals were to develop a population on a
two-year spawning cycle and to characterize thegenetic bases for several commercially important
traits as well as to apply that information to develop
a selection program.
Domsea Farms, in the mid to late 1970s, was thefirst commercial net-pen operation in Puget Sound,WA, and its goal was to produce pan-sized (500 g)
coho salmon. Fast growth in fresh and seawater andFig. 1. Selection program for Domsea coho salmon
broodstock development The program has been
virtually unchanged for 19 generations with the
exception of the change from partial seawater to total
freshwater freshwater rearing.

improved smoltification and subsequent seawater
survival were the primary traits of selection interest.
Since then, other traits of interest have included
fecundity, egg size, and size at maturity.
A breeding design involving 40 full- and half-sib
families initially and later just 40 full-sib families was
initially used to evaluate growth performance andestimate heritabilities and genetic correlations forand among traits. The selection strategy included
between and within-family selection using a selection
index and a mating scheme to minimize inbreedingas much as possible. The program utilized multiple
selection events at major life history/rearing
transition points (Fig. 1). Each year individuals fromthe top 10 families were crossed in a circular mating
pattern to create six full-sib families within each of
the top 10 family crosses*1. The 60 families were thenreduced to 40 families after incubation based on
assessments of egg size, fecundity, and survival toponding. Each family was reared separately until＊1＊2large enough to externally identify with a freeze
brand, then families were combined and transferred
to marine net-pens. Fish were sampled during the
freshwater growth period, at time of transfer tosaltwater and at two other periods during saltwater
rearing, at 3.5 and 8.0 months post-transfer to assess
saltwater survival and growth. The resulting data
were then used in the selection index to reduce thenumber of families to 10* 2 .

After transfer tofreshwater for final maturation, the 6-10 largest males
and females from each of the selected families weremated using a circular mating design to avoid matingclosely-related individuals.
In 1986-1987, because of high pre-spawningmortality for adults maturing in marine net-pens; thesurvival, maturation, and reproductive success ofadults held in freshwater throughout their entire
lives were evaluated. As a result of those trials, thebroodstock program was then shifted, and has

Family crosses were adjusted to avoid first-cousin crosses.
	In some years the 10 families were selected from the top 15 performing families, due to inbreeding concerns or poor survival post-index
selection.

109

Domsea coho broodstock development
remained ever since, to an all freshwater program.

compared with the select line to assess changes to

The selection strategy and mating design has

the selected lines.

remained the same except that the saltwater holding
Results and discussion

period was then eliminated.
Because of the two-year generational cycle, evenand odd-year lines were established. In 1992, theGenetic estimateseven-year line was lost due to a hatchery accident, so

The first quantitative genetic parameters for coho

about half of the odd-year line were subsequently

salmon traits were reported by Iwamoto et al. (1982),

photoperiod-manipulated to spawn two years later,

Saxton et al. (1984), and Iwamoto et al. (1990) from thethus re-creating an even-year spawning group. The

Domsea coho program (see Table 1 for a summary).

de novo even-year line has been maintained as aOverall these genetic estimates for freshwater

separate line ever since.

growth, smoltification and initial seawater survival,

Pedigrees for both even- and odd-year lines haveseawater growth, carcass-related, and reproductive

been maintained from the start, and in combination

traits for the Domsea coho salmon indicated that awith population numbers and microsatellite, allozyme,

systematic selection program would be successful

and serum protein (transferrin) data, have been used

(Hershberger et al., 1990).

to estimate inbreeding levels for each generation.

Selection gains for weight and length at 7, 11, andAt the initiation of the program, control lines were

14 months post-fertilization were predicted using themaintained to assess selection efficacy. However,

heritability estimates and a range of selection

within two generations, logistics of the program andintensities (Table 2). The range of predicted selection

the space constraints forced the termination of thegains was quite high. For example, the predicted

control lines. Since then, the performance of fish

improvement for 7-month weight after the firstfrom the founding hatchery population has been

generation of selection varied from 1.4% to 46%.

Table 1. Heritability estimates (standard error in parentheses) for various traits at different generations. Estimatesare based on sire (S), pooled sire and dam (S + D), full-sib (FS), or pooled full-sibs/double first cousin (FS + DFC)
components. Other abbreviations: SW = saltwater; FW = freshwater.
Generations under selection
Component

Trait

1

h2S

8 m SW Weight

0.19 (0.11)

2

3

5

0.40 (0.15)

0.30 (0.07)

0.21 (0.09)

0.29 (0.08)

0.22 (0.09)

0.26 (0.07)

0.20 (0.07)

0.30 (0.07)

0.24 (0.07)

14

0.62 (0.21)
h2S+D

8 m SW Weight

0.20 (0.06)
0.33 (0.10)

h2FS

h2FS+DFC

8 m SW Weight

8 m SW Weighth2FS

24 m FW Weight

0.422 (0.146)

h2FS

24m FW Fecundity

0.237 (0.108)h FS

24 m FW Egg Size

0.432 (0.135)

h2FS

Carotenoid

0.50 (0.16)

0.30 (0.14)

h2FS

Lipid

0.18 (0.13)

0.19 (0.23)

2

110

Robert N. IWAMOTO, Richard TOWNER, James MYERS, Greg HUDSON, and Patricia MUNSELL

Table 2. Prediction of selection gains based on different selection intensities
Trait
7 Month
11 Month
14 Month%

Min Estimated%WeightMean
14.46 g6.61 ga

46

0.2 g

1.4

Length

101.45 mm

11.63 mma

11

0.6 mm0.6

Weight136.71 g

27.79 g

20

3.3 g

2.4

Length210.42 mm

28.97 mmb

14

0.7 mm0.3

Weight374.17 g

72.00 gc

19

7.9 g

2.1

Length305.37 mm

21.76 mm

7

3.2 mm1.0

aBased on selecting 60 individuals out of 10,000bBased on selecting 60 individuals out of 7,000
Based on selecting 60 individuals out of 3,000cd

Max EstimatebcBased on index results and selecting 10 families out of 40

Improvements
The 7-month weight of the odd-year line changed
from 11.2 g in 1977 to 24.7 g in 2011, while the evenyear line increased from 11.2 g in 1977 to 34.1 g in2012 (Fig. 2). The increase for the odd-year line
represented a 7.1% per generation increase, while theincrease for the even-year line was equivalent to an
11.3% per generation increase.

While the total

increase could not be attributed to directed selection
alone, it does indicate that there has been substantial
improvement for that trait. This is despite changes
in level of care and husbandry over the past 38 years
due to changes in ownership of the company. The
fluctuations in care and husbandry are partiallyreflected by the yearly variability in performanceFig. 2. Change in 7-month weight after 14 generationsof selection

reflected in Fig. 2.
Although adult weight was not a selected trait
until fairly late in the program, the fairly high genetic
correlation between spawn weight and 7-monthweight, led to increases in spawn weight.

Theaverage spawn weight of the odd-year females in2011 was 2568 g compared with 1669 g for the first
generation (Fig. 3). This was an increase of 53 g per
generation or an overall increase of 54% over 17
generations.
Because of the changes in ownership, and thedifferences in husbandry and rearing environment
over the many generations, it is difficult to precisely
estimate the improvement in growth due to genetics
or other causes. The best estimate for this line ofcoho salmon is that growth rate is improving between
3% and 8% per generation.

Fig. 3. Change in female weight at spawn over thecourse of 16 and 17 generations for the odd- and evenyear broodstocks.

111

Domsea coho broodstock development
Inbreeding

inbreeding.

Despite the circular mating scheme, inbreeding
References

could not be avoided but merely delayed because of
the intense selection the closed populations were
subjected to every generation. Over the life of the

Bondari K. and Dunham R. A., 1987: Effects ofprogram, inbreeding has been estimated in various

inbreeding on economic traits of channel catfish.

ways. For example, Myers et al. (2001) estimated that

Theor. Appl. Genet. 74(1), 1-9.

after 9 and 10 generations of selection for the even-

Dunham R. A. and Smitherman R. O., 1984: Ancestry

and odd-year lines respectively, inbreeding had

and breeding of catfish in the United States.

increased by 13 - 48% depending on the method of

Alabama Agricultural Experiment Station

estimation.

Circular 273. Auburn University, Auburn, AL

Overall, via allozyme analysis, theauthors determined that the two lines had undergone

Hershberger W. K., Myers J. M., Iwamoto R. N.,

little change in average heterozygosity but significant

McAuley W. C., and Saxton A. M., 1990: Genetic

changes in loss of variability at several loci. Pedigree-changes in the growth of coho salmon

based inbreeding estimates show a progressive(Oncorhynchus kisutch) in marine net-pens,increase in inbreeding of about 1.5% per generation

produced by ten years of selection. Aquaculture(Fig. 4). The inbreeding estimates for the even-year

85, 187-197.

line shows a striking decrease in inbreeding within

Iwamoto R. N., Myers J. M., and Hershberger, W. K.,

the last two generations as a result of deliberate

1990: Heritability and genetic correlations foroutcrossing with the founder strain.

flesh coloration in pen-reared coho salmon.

There have been no obvious indications ofAquaculture 86, 181-190.

inbreeding depression despite the relatively highIwamoto R. N., Saxton A. M., and Hershberger W. K.,

levels of inbreeding. If the current level of inbreeding

1982: Genetic estimates for length and weight ofis 25% or higher, it would be equivalent to that of full-coho salmon (Oncorhynchus kisutch) during

sib matings – levels at which other studies havefreshwater rearing. J. Hered. 73, 187-191.

indicated can negatively affect reproductive success

Myers J. M., Heggelund P. O., Hudson G., andand early growth and survival of fish (Bondari andIwamoto R. N., 2001: Genetics and broodstock

Dunham, 1987; Wang et al., 2002). Because of the

management of coho salmon. In: Lee, Cheng-absence of a control group, the effects of inbreeding

Sheng and Donaldson, E. M. Reproductive

on overall growth and survival could not be assessed.

biotechnology in finfish aquaculture.

It is possible that had inbreeding levels been lower
over the course of the program, larger improvements
per generation might have been observed.

Aquaculture 197, 43-62.
Myers J. M., Iwamoto R. N., Teel D., Van Doornink D.,
and Hershberger W. K., 1999: Coho salmon
broodstock developmet---1977-1998. Ten

Conclusion

generations of systematic selective breeding.
Bull. Natl. Res. Inst. Aquac. Suppl. 1, 63-70.

The Domsea coho broodstock program has andQuinton C. D., McMillan I., and Glebe B. D., 2005:

continues to provide important data on the efficacy ofDevelopment of an Atlantic salmon (Salmo salar)a long-term genetic selection program to improvegenetic improvement program: Genetic

commercially important traits. More importantly, it

parameters of harvest body weight and carcass

provides an opportunity to address the question ofquality traits estimated with animal models.

the effects inbreeding may have on a sustained basis.

Aquaculture 247, 211-217.

As the program matures, it will be informative to

Saxton A. M., Hershberger W. K., and Iwamoto R. N.,

incorporate genomics theory into the current

1984: Smoltification in the net-pen culture of cohoquantitative genetics-based program to further

salmon: quantitative genetic analysis. Trans.

characterize the genetic basis for commercially

Am. Fish. Soc. 113, 339-347.

important traits and concomitant effects ofTave D., 1999: Inbreeding and brood stock112

Robert N. IWAMOTO, Richard TOWNER, James MYERS, Greg HUDSON, and Patricia MUNSELL
management. FAO Fisheries Technical Paper#392, 123p.

Wang S., Hard J. J., and Utter F., 2002: Salmonid

The authors examine reproductive traits and therelationship between environment (feeding ration)and genetics (family) in the Domsea coho salmon

inbreeding: a review. Rev. Fish Biol. Fish. 11,

stock after 15 generations of selection.

201-319.

suggested that phenotypic improvements could be

Results

achieved through changes in ration and/or direct
Annotated bibliography

selection.

(1) a. Iwamoto R. N., Saxton A. M., and Hershberger(3) Myers J. M., Iwamoto R. N., Teel D., Van Doornink

W. K., 1982: Genetic estimates for length and weightD., and Hershberger W. K., 1999: Coho salmonof coho salmon (Oncorhynchus kisutch) during

broodstock developmet – 1977-1998. Ten generations

freshwater rearing. J. Hered. 73, 187-191.

of systematic selective breeding. Bull. Natl. Res.

b. Saxton A. M., Hershberger W. K., and Iwamoto

Inst. Aquac. Suppl. 1, 63-70.

R. N., 1984: Smoltification in the net-pen culture of

The authors summarize the concept of the Domsea

coho salmon: quantitative genetic analysis. Trans.

coho salmon broodstock program from the selection

Am. Fish. Soc. 113, 339-347.

scheme to the circular mating design. They alsoc. Hershberger W. K., Myers J. M., Iwamoto R. N.,

present allozyme comparisons of the founder strain

McAuley W. C., and Saxton A. M., 1990: Genetic

and the broodstock after 10 generations of selectionchanges in the growth of coho salmon (Oncorhynchus

and indicate that significant genetic changes had

kisutch) in marine net-pens, produced by ten years of

occurred in the broodstock as a result of genetic

selection, Aquaculture 85, 187-197.

selection and drift.

d. Iwamoto R. N., Myers J. M., and Hershberger W.
K., 1990: Heritability and genetic correlations for flesh(4) Myers J. M., Heggelund P. O., Hudson G., andcoloration in pen-reared coho salmon. Aquaculture 86,

Iwamoto R. N., 2001: Genetics and broodstock

181-190.

management of coho salmon. In: Lee, Cheng-Sheng

The papers cited above provide much of the earlyand Donaldson, E. M. (Eds.) Reproductive

quantitative genetic parameters for coho salmon

biotechnology in finfish aquaculture, Aquaculture

traits. Genetic estimates for freshwater growth,197, 43-62.

smoltification and initial saltwater survival, seawaterThis paper summarizes the information collected

growth, and flesh coloration for the Domsea cohoon the Domsea coho salmon broodstock program

salmon were derived from full- and half-sib analyses.

after 13 generations of implementation. It discusses

In almost every case, the magnitude of the derived

the results of sib trials in marine net pens and two

heritabilities and genetic correlations indicated that a

freshwater environments and consequently thesystematic selection program would be successful.

importance of considering genotype-environment
interactions in broodstock programs for specific(2) Myers J. M., Park L. K., Neely K., Swanson P., Elzapplications. It tracked theoretical (pedigree data)A., and Hard J. J., 2011: Feeding ration, genetics, andand genetic changes (allozyme data and individual

reproductive traits in female coho salmon: Is bigger

traits) in the broodstock population.

better? J. World Aquac. Soc. 42, 812-823.

水 研 機 構 研 報， 第 45 号，113 － 121， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，113－121，2017

113

Development of Improved Catfish Germplasm at the Warmwater
Aquaculture Research Unit, DSDA - ARS
Brian BOSWORTH＊ and Geoffrey WALDBIESER＊

Abstract: Farm-raised catfish is the largest aquaculture enterprise in the U.S. in terms of bothproduction volume and value. The USDA-ARS Warmwater Aquaculture Research Unit (WARU)
mission includes development and release to farmers of catfish germplasm improved for economicallyimportant traits. Historically, the channel catfish (Ictalurus punctatus) comprised nearly all U.S.
farm-raised catfish production, but in the last 10 years, production of the F1 hybrid between thechannel catfish and blue catfish (Ictalurus furcatus) has increased to about 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
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 haveevaluated over 21,000 animals from 750 full-sib families produced by 408 sires and 624 dams between
2008 and 2015. Heritabilities for harvest weight and residual carcass weight are 0.29 and 0.36,
respectively, and fish are selected based on an index that puts equal weight on individual breedingvalues for growth and family average breeding values for residual carcass weight. Improvements
in growth have been 8 - 10% per generation and are in agreement with expected improvements.
Increases in residual carcass weight have been less substantial (-2 g per generation) than those for
harvest weight due, at least in part, to the necessity for selection on less accurate family average
breeding values for carcass weight.
The hybrid catfish improvement program has focused on evaluation, identification and selectionof blue catfish sires that produce superior performing hybrid progeny. We initiated a program tocollect several blue catfish populations and evaluate performance of their purebred and hybrid
progeny. Initial evaluations suggest the majority of genetic variance for growth in hybrid progeny
is additive in nature, and populations and individual sires that produce purebred blue catfish
progeny with faster growth also produce hybrid progeny with faster growth. We have ongoingexperiments to estimate heritabilities and genetic correlations in the purebred blue and hybrid
catfish, which will give us direction in selecting purebred blue catfish for improved hybrid catfish
performance. We use DNA markers to identify parentage and establish pedigrees in our populations
and are developing a SNP chip to be used for genomic selection to improve our breeding value
accuracy and rate of improvement, particularly for carcass yield. We are evaluating other traits forinclusion in our selection index, and 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.
Key words: Channel catfish, blue catfish, genetic improvement

2017年２月28日受理（Received on February 28, 2017）	Warmwater Aquaculture Research Unit, USDA-ARS, P.O. Box 38, Stoneville, MS 38776, U.S.A
E-mail: brian.bosworth@ars.usda.gov

＊1

114

Brian BOSWORTH and Geoffrey WALDBIESER
Introduction
Materials and Methods

Catfish farming is the largest aquaculture
enterprise in the U.S. in terms of both production

Purebred channel catfish

volume and value (National Agricultural Statistic

In 2006, 10 to 12 spawns (full-sib families) were

Service 2012). Approximately 150 million kg of catfish

collected from eight commercial farms where farm

were produced and processed in the U.S. in 2015.

owners indicated fish were from unique base

Most of the production occurs in the southeastern

populations. Spawns were hatched and raised in

U.S. and the vast majority of product is consumed

separate family tanks. Fish were fed commercial

domestically. Prior to 2005, nearly all production

diets of appropriate size and composition for theirconsisted of purebred channel catfish, Ictalurusdevelopmental stage. Fish densities in tanks werepunctatus, but in the last 10 years, production of

periodically reduced and equalized in all tanks. Whenhybrid catfish (F1 hybrid between female channel

fish averaged greater than 40 g, 100 fish per full-sib

catfish and male blue catfish, Ictalurus furcatus) has

family were tagged with individually coded passive

increased dramatically and represented about 50% of

integrated transponders (PIT) tags (BioMark, ID,

catfish production in 2015.

USA).

Although catfish

Fish were then transferred to replicate

aquaculture represents the majority of U.S.

earthen ponds and families reared communally. Fishaquaculture production, current production is about

were fed a 32% protein commercial catfish diet tohalf of what it was at its peak in 2003. The reduced

apparent satiation once daily until the majority of the

production of catfish in the U.S. is related to increasedfish were of market weight (0.5 to 1.0 kg). Fish were

production costs (particularly feed), competition fromthen harvested, and measured for total weight andlower-priced imported fillets (Pangasius and Tilapia)

gender. The largest 3 to 6 males and 4 to 7 females

targeted at similar markets, and the subsequentfrom each full-sib family were retained as broodfish

conversion of catfish ponds to more profitable row-

and an additional 80 mature broodfish of unknown

crop production. The USDA-ARS Warmwater

age were obtained from each of two additional

Aquaculture Research Unit (WARU) mission includes

commercial farms and PIT tagged.

development and release of improved catfish

pedigree file was established with an additive

germplasm to U.S. farmers that will improve

relationship of 0.5 among individuals within full-sib

production efficiency and help U.S. farmers remainfamilies and assuming all other fish were unrelated.

competitive in a global seafood market.

Fish derived from this base population are referred

An initial

The process of genetic selection to develop

to as the Delta Select strain of channel catfish. Blood

improved germplasm for release to the industry

samples were collected from all broodfish (835requires production and performance evaluation of

females and 638 males) for DNA isolation and thenpurebred blue catfish, purebred channel catfish andbroodfish were stocked in the spring of 2008 in

hybrid catfish. Mating designs used for genetic merit

earthen ponds at 800 kg/ha and allowed to mate at

estimation require collection of performance data on

random. Spawns were collected from ponds every 2

traits targeted for selection in large, pedigreed

to 3 days and moved to the hatchery from mid-April

populations. Development of improved germplasm isthrough early August. Fry were hatched and treateda recurrent process of broodfish selection, offspring

as described above and a sample of 8 to 10 fry were

production and evaluation, genetic merit estimationcollected from each spawn for DNA isolation.

and selection of the next generation of broodfish.

Microsatellite genotypes of fry from each spawn andBreeding projects at WARU are focused on selection

potential parents were compared to determine

to improve performance of purebred channel catfish

parentage of spawns (Waldbieser and Bosworth,and selection of purebred blue and channel catfish to2013).

improve hybrid catfish performance. The WARU’sstocked communally in earthen ponds and grown tocurrent catfish breeding program and future

market weight as described previously. Fish weredirections are summarized in this paper.

harvested in late October to November when water

Sixty to 75 fish per family were tagged,

Development of improved catfish germplasm

115

temperatures cooled and feeding activity declined.

because animals measured for carcass weight wereFish were harvested by seining the ponds, thenselected based on a size range required to fit theanesthetized with 200 ppm MS-222 and gender, PIT

processing equipment and therefore were not a

tag number and weight to the nearest 0.5 g wererandom sample, which would have biased therecorded for each fish. A sample of 4 to 5 males andcorrelation estimate. Instead, the genetic correlation

4 to 5 females from each full-sib family in the weight

between harvest weight and carcass weight was

range typically processed at commercial facilities (0.4

estimated as the correlation between full-sib family

to 1.0 kg) were electrically stunned, decapitatedaverage breeding values for harvest weight and(Baader 166 heading machine, Baader North America

residual carcass weight.

Corporation, Auburn, WA, USA), eviscerated by hand
and the carcasses were weighed.

Breeding values for harvest weight and residualcarcass weight were estimated with MTDFREML

Broodfish used in the 2008 spawning season wereand approximately the top 10% of fish from each

held over winter, inventoried, weighed and restocked

year-class were selected as broodfish based on an

into spawning ponds spring of 2009. The percentage

index placing equal weight on individual’s breedingof male and female channel catfish that are mature

value for harvest weight and full-sib family value forand spawn is only about 25% at two years old but

residual carcass weight. Response to selection forincreases to over 50% at three years old. Therefore,

harvest weight and residual carcass weight was

to increase the number of broodfish that spawn andevaluated by estimating the correlation between mid-maintain a higher effective population size, selectedparent breeding value (average breeding value of abroodfish were typically spawned as two year olds

sire and dam) and the phenotypic means of theirand again as three year olds. This process has beencorresponding full-sib progeny family means after

repeated with selected broodfish being spawned inadjustment for relevant fixed effects (year, pond, sex).

2011 and 2012, and 2014 and 2015.

Genetic trends (changes in mean breeding value of

Phenotypic variance, additive genetic variance,
heritablilities and breeding values were estimated forthe population over time) were also estimated for
harvest weight and residual carcass weight.

each trait (harvest weight and carcass weight) withlinear single-trait animal models using MTDFREML

Blue and hybrid catfish(Boldman et al., 1995). The model for harvest weight

There is little commercial production of blue

included fixed effects of pond, year, and gender; age

catfish and little data relevant to genetic effects ofwithin year*gender as a linear covariate; and animal

blue catfish on purebred or hybrid catfish

additive genetic and common environment

performance.

(confounded effects of full-sib family and fingerling

gather blue catfish germplasm from diverse sources

rearing tank) as random effects. Carcass yield (theand evaluate the effects of these populations on

percentage of whole weight that is comprised of

harvest weight and carcass yield in their purebred

carcass weight) is a trait of high value to catfish

and hybrid progeny. Because blue catfish mature atprocessors. However, because of statistical issues

a late age (typically five years or older) and shipping

related to estimation of variance components forlarge fish is costly and difficult, we obtained fish fromratios, carcass data was analyzed as residual carcass

some sources as larvae, some as juveniles and someweight (carcass weight adjusted to a common wholeas mature adults. Our initial evaluations of blue

animal weight by using whole weight as a linear

catfish focused on comparisons on the population

covariate). The model for residual carcass weightlevel rather than evaluations of individual males

included fixed effects of pond, year, gender and day

within populations due to the fact that obtainingof processing; whole weight within year*gender as asperm from blue catfish males to produce hybrids

linear covariate; and random effects of animal

requires killing the male and storage of fresh sperm

additive genetic and common environment. Genetic

is limited to about five days.

Therefore, our initial goal was tocorrelations between harvest weight and carcass

We conducted a series of studies comparing effects

weight were not analyzed using a multi-trait modelof blue catfish populations on purebred blue catfish

116

Brian BOSWORTH and Geoffrey WALDBIESER

and hybrid catfish progeny growth. Typically, blue

populations tested.

catfish are pond- spawned, reared in family tanks,
Results

PIT-tagged and then stocked in ponds and rearedcommunally as described previously for channel
catfish. Hybrids are produced by hormone (LHRHa

Channel catfish

or pituitary extract) induced ovulation of female

A total of 21,055 progeny from 750 full-sib families

channel catfish and fertilization with blue catfish

produced by 624 dams and 408 sires have beensperm obtained by maceration of testes (Bosworth etmeasured for harvest weight; 4,060 progeny from 585

al., 2005).

families produced by 494 dams and 342 sires haveHybrid catfish larvae are reared in familytanks, PIT-tagged and evaluated for performance inbeen measured for carcass weight (Table 1).

earthen ponds as described previously.

heritability estimates for harvest weight and residualThe

Data from five growth trials are presented. The

carcass weight are 0.29 (± 0.03) and 0.35 (± 0.05),

first trial (2009 year-class) compared effects of 4 to 5

respectively. Values for phenotypic, additive genetic

sires from each of two blue catfish populations onand common environmental variance andharvest weight of their hybrid catfish progeny; theheritabilities for harvest weight and residual carcass

second trial (2010 year-class) compared effects of 7 toweight are listed in Table 2. The correlation between

10 sires from each of five blue catfish populations on

full-sib family mean breeding values for harvest

harvest weight of their hybrid catfish progeny; theweight and carcass yield was -0.11. Correlations

third trial (2012 year-class) compared effects of 5 to 15

among mid-parent breeding values and corresponding

full-sib families from each of three blue catfish

full-sib family means were 0.50 (p < 0.0001) for

populations on harvest weight of their purebred blue

harvest weight and 0.41 (p < 0.0001) for residual

catfish progeny; the fourth trial (2014) comparedcarcass weight.

effects of 3 to 15 full-sib families from each of four

harvest weight were 0 g for the base population, 78 g

blue catfish populations on fingerling weight of theirfor the 2011 and 2012 year-classes, and 140 g for the

purebred blue catfish progeny; and the fifth trial

2014 year-class; mean breeding values for residualcompared effects of 9 to 27 full-sib families from eachcarcass weight were 0 g for the base population, 2.2 gof three blue catfish populations on fingerling weight

for the 2011 and 2012 year-classes, and 4.8 g for theof their purebred blue catfish progeny. Two trials

2014 year-class.

The mean breeding values for(2014 and 2015 year-classes) are ongoing and,
therefore, only weights of fingerlings at stocking arepresented.

Blue and hybrid catfish
Blue catfish population had a significant effect on

Mean harvest weight of hybrids and purebred

purebred blue catfish and hybrid catfish progeny

blues produced from blue catfish populations were

growth in each of the five trials presented (Table 3).

compared within each trial using the Mixed

Purebred and hybrid catfish progeny produced from

Procedure of SAS (SAS Institute, Cary, NC, USA).

Rio Grande population parents consistently had theModels for harvest weight included pond and sex as

highest progeny harvest weight. The overall mean

fixed effects, age as a covariate and full-sib familycorrelations across studies between mean harvest

within population as a random effect. Models forweight for hybrid and purebred progeny produced

fingerling weight included age as a covariate and full-by the various populations tested was 0.98 (p < 0.001),

sib family within population as a random effect. Full-indicating the effect of blue catfish population on

sib family was used as the error term in comparisonsprogeny growth is consistent across hybrid andof population means. Correlations between mean

purebred progeny.

weight of purebreds and hybrids produced using
each population were estimated for each trial and the

Discussion

mean of these correlations was determined andprovided an estimate of the relationship between

Heritabilities for harvest weight (0.29) and residual

purebred and hybrid growth of the various

carcass weight (0.35) in the Delta Select strain of

117

Development of improved catfish germplasm

Table 1. Number of full-sib families, sire and dams and means (+ SD) and number of fish measured for harvest
weight and carcass yield for 2008, 2009, 2011, 2012 and 2014 year-classes of Delta Select channel catfish
Year-class

Full-sibfamilies

Sires1

Dams1

161

107

2008
2009

Harvest Weight

Carcass Yield %

Mean

SD

n

Mean

SD

n

149

777.4

284.9

4,762

58.3

1.7

829

186

113

181

614.7

225.3

5,686

58.0

1.8

1,352

3

2011

170

89

161

702.2

263.7

1,982

--

--

--

2012

104

67

90

814.6

281.0

4,484

64.4

1.7

924

2014

113

73

109

881.4

287.5

4,141

64.3

1.7

955

Harvest weight

750

408

624

Carcass yield

585

342

494

Total n21,0554,060

	The total number of sires and dams is less than the sum of sires and dams across years because some sire and dams
spawned in consecutive years.
2	Carcass data were analyzed as residuals carcass weight after covariate adjustment to a common whole weight, but values
presented in Table 1 are carcass yield (100*carcass weight/whole weight) to be more meaningful to the reader. 2008 and2009 carcass yield data were calculated based on skin-off carcass weights, 2012 and 2014 carcass yield data were
calculated based on skin-on carcass weight.
3
	A severe outbreak of proliferative gill disease resulted in substantial mortalities in 2011 and, therefore, no fish were
processed.
1

Table 2. Phenotypic, additive genetic and common environmental (tank and full-sib family) variance andheritabilities (+ SE) for harvest weight and residual carcass weight in the Delta Select strain of channel catfish
Variance Component

Trait

Phenotypic

Additive
Genetic

CommonEnvironment

58,302

16,965

4,797

0.29

0.03

40.5

6.5

0.35

0.05

Harvest weight g
Residual Carcass weight g

114.4

Heritability

SE

Table 3. Effect of blue catfish sire population on least square means1 for harvest and fingerling weight of purebredand hybrid catfish offspring

1

Year-Class

Hybrid/Purebred

Trait

2009

Hybrid2010

Blue Catfish Population

Rio Grande

D&B

MS River

MO RiverKentuckySE

HarvestWeight g---

362.0a------278.3b

23.1

HybridHarvestWeight g

572.2a525.0a,b

506.7b

459.6c442.0c26.2

2012

Purebred

HarvestWeight g

489.2a309.9b286.2b

---

---

50.5

2014

PurebredFingerling
Weight g

45.2a

38.9a,b

31.5b

16.1c

---

5.5

2015

PurebredFingerling
Weight g

143.8a

95.4b

91.3b

---

---

8.1

Means within row are significantly different at P < 0.05

118

Brian BOSWORTH and Geoffrey WALDBIESER

channel catfish are similar to those for growth andpopulations each mated to two blue catfish females

carcass yield observed in other farmed fish species

and 2 to 3 channel catfish females in a series of(Navarro et al., 2009; Nguyen et al., 2010) andfactorial matings. Offspring from these matings are

terrestrial livestock (Hermesch et al., 2000; Aslam etbeing evaluated for growth and carcass weight andal., 2011). The positive correlation between mid-

the data will be used to estimate heritabilities ofparent estimated breeding values (EBVs) and meanthese traits in purebred blue catfish and hybrid

harvest weight of their full-sib progeny (r = 0.5) andcatfish, genetic correlations among traits in purebred

increased genetic trend over time indicate that theblue and hybrid catfish, and relative importance of

response to selection for harvest weight is inadditive and dominance genetic variation for traits.

agreement with expectations. Data from the first

This information will provide details on the optimal

two generations of selection demonstrated an

approach to selecting purebred catfish forincrease of about 8 to 10% in average breeding values

improvement of hybrid catfish.

for harvest weight each generation, similar to

Current germplasm development has focused on

response reported for farmed carp (Dong et al., 2015),

improving catfish growth and meat yield because

tilapia (Hamzah et al., 2014), trout (Kause et al., 2005)

these traits are economically important to catfish

and salmon (Gjedrem, 2000). Response to selection forfarmers and processors and because they can becarcass weight was positive but less than expected.

measured accurately and relatively inexpensively on

The lower response to selection for carcass weightthe large numbers of animals required for accurate

relative to harvest weight is due, at least in part, toestimation of heritabilities and breeding values.

the necessity to select on full-sib family mean EBVs

However, we have been collecting data on

for carcass weight, which are less accurate than the

reproductive traits, disease resistance and meat

individual EBVs used to select for harvest weight.

quality and may include additional traits in future

However, the correlation between mid-parent EBVs

selection indices, if the heritabilities and economic

and offspring carcass weight (r = 0.41) and increased

values of the traits indicate that it would benefit

genetic trend over generations suggest positive gain

catfish farmers and processors.

in carcass weight is being made.

Even small

The use of microsatellite DNA polymorphisms toincreases in carcass yield have a large effect ofidentify parentage in channel, blue and hybrid catfish

profitability of catfish processors.

populations has played an important role in our

The high correlation between mean growth of blue

genetic evaluations by providing pedigree information

catfish and hybrid catfish progeny produced by therequired for heritability and breeding value

same blue catfish sire populations, along with earlier

estimation (Waldbieser and Bosworth, 2015).

published data demonstrating much higher variation

However, we are now planning to expand use offor general combining ability than for specific

DNA technology in our breeding program bycombining ability for effects of blue catfish sires on

evaluating the use of SNP markers for genomic

hybrid progeny growth (Bosworth and Waldbieser,

selection in the Delta Select channel catfish population2014), suggest that much of the genetic influence ofand in evaluations related to improvement of hybrid

blue catfish on growth in hybrid progeny is additive.

catfish performance. We are near completion of a 50

This may be due to genes acting in an additive

K SNP chip (Affymetrix, Santa Clara, CA, USA) based

genetic manner or common dominance deviations

on markers that segregate in the Delta Selectbetween the blue catfish populations and channel

population and will be evaluating approaches to using

catfish we have evaluated.

genomic selection to improve accuracy of breeding

The current datasuggests that purebred blue catfish population

value estimates.

performance is predictive of hybrid catfish

particularly beneficial for improving accuracy of

performance and that we can substantially improve

breeding value estimates and the rate of genetic gain

hybrid growth simply by selecting for increased

for traits like carcass yield, which have highpurebred blue catfish growth. We have a large on-

heritability and large economic value but cannot begoing project with 120 blue catfish sires from fourmeasured on live animals (Daetwyler et al., 2012).

Genomic selection should be119

Development of improved catfish germplasm
Ultimately, one of our primary missions at theblue catfish germplasm is tremendous. Calculations

WARU is to release improved catfish germplasm tobased on discussion with commercial hybrid hatchery

U.S. catfish farmers for commercial production. The

operators suggest 3,000 to 4,000 male blue catfishrole of development and release of commercial

would suffice for current levels of hybrid catfish fry

germplasm is a unique situation for a research

production.

agency and requires considerable planning to allow

doubles, that is still less than 10,000 males a year,

equitable distribution of germplasm, evaluation ofwhich is substantially less than the male offspring

performance and determination of impact after

produced from a single large blue catfish full-sib

release. We are currently evaluating Delta Selectfamily. Therefore, identification and development ofchannel catfish from our third generation of selection

improved blue catfish germplasm, with subsequent

and are discussing options for a commercial release

rapid expansion to commercial industry production,with industry stakeholders. The data collected to

has the potential to have a rapid and dramatic

date indicate that the selected population should havepositive benefit on hybrid catfish performance.

Even if hybrid catfish production

substantially faster growth and higher carcass yield
References

than the base population, which represented fishfrom hatcheries providing greater than 50% of
industry production at the time the base population
was formed.

Aslam M.L., Bastiaansen J., Crooijmans R., Ducro B. J.,

However, at this time, no direct

Vereijken A., and Groenen M., 2011: Genetic

comparisons of performance of the Delta Select andvariances, heritabilities and maternal effects onchannel catfish currently being used by the industry

body weight, breast meat yield, meat quality

have been conducted. Attempting to replicate thetraits and the shape of the growth curve in

multitude of management strategies employed byturkey birds. BMC Genet. 12, 14.

farmers (stocking densities, feeding strategies,

Boldman K. G., Kriese L. A., Van Vleck L. D., Van

aeration management, etc.) and compare the Delta

Tassell C. P., and Kachman S. D., 1995: A manual

Select and industry fish in a research setting is likely

for the use of MTDFREML. A set of programs toimpossible. Therefore, a scenario in which the fishobtain estimates of variances and covariances

are initially released to a small number of producers[Draft]. USDA-ARS, Clay Center, NE.

willing to provide accurate feedback on commercial

Bosworth B. G., Small B. C., Steeby J., and Avery J.,

performance, including growth, survival, feed

2005: Producing hybrid catfish fry: workshop

conversion and processing yield, prior to large scalemanual. Mississippi State Extension Service,release may be the best approach. If performance

Mississippi State University, Stoneville, MS,data from the initial release is favorable, the highHybrid Catfish Manual, 115pp. (Technicalreproductive capacity of catfish would allow rapid

Manual).

expansion and additional widespread release of the

Bosworth B. G. and Waldbieser G. C., 2014: General

Delta Selects to commercial producers. Releases

and specific combining ability of male blue

could be made every couple of generations to

catfish (Ictalurus furcatus) and female channel

distribute fish with additional improvements tocatfish (I. punctatus) for growth and carcass yield

farmers if the initial release is favorable.

of their F1 hybrid progeny. Aquaculture 420-

The blue catfish breeding program is not as421, 147-153.

advanced as the channel catfish program for various

Daetwyler H.D., Swan A. A., van der Werf J. H. J., andreasons, including a lack of domesticated populations,Hayes B.J., 2012: Accuracy of pedigree andthe time involved in testing blue catfish due to the

genomic predictions of carcass and novel meatlater age at maturity of blue catfish compared toquality traits in multi-breed sheep data assessedchannel catfish, and the requirement to kill blueby cross-validation. Genet. Sel. Evol. 44, 33.

catfish males to obtain sperm for hybrid catfish

Dong Z., Nguyen N. H., and Zhu W., 2015: Genetic

production. However, the potential for industry

evaluation of a selective breeding program for

impact through development and release of improvedcommon carp Cyprinus carpio conducted from

120

Brian BOSWORTH and Geoffrey WALDBIESER
2004 to 2014. BMC Genet. 16, 94.

Annotated bibliography

Gjedrem T., 2000: Genetic improvement of cold-water
fish species. Aquac. Res. 31, 25-33.
Hamzah A., Ponzoni R. W., Nguyen N. H., Khaw H.-L.,
Yee H.-Y., and Nor S. A. M., 2014: Genetic(1) Lush J. L., 1947: Family Merit and Individual Meritas Bases for Selection. Part I. Am. Nat. 81, 241-261
and 81, 362-379.

evaluation of the Genetically Improved Farmed

No abstract available. These two papers detail

Tilapia (GIFT) strain over ten generations ofLush’s development of the use of information on

selection in Malaysia. Tropical Agric. Sci. 37,

individual performance and performance of relatives

411–429.

(family merit) to improve accuracy of breeding value

Hermesch S., Luxford B. G., and Graser H. U., 2000:

estimates and form the basis for development of

Genetic parameters for lean meat yield, meat

selection index theory and development of BLUP

quality, reproduction and feed efficiency traits

based breeding programs.

for Australian pigs: 1. Description of traits andheritability estimates. Livest. Prod. Sci. 65, 239-

(2) Henderson C. R., 1975: Use of all relatives in248.

intraherd prediction of breeding values andHu E., Bosworth B. G., Baxter J., and Tiersch T. R.,

producing abilities. J. Dairy Sci. 58, 1910–1916.

2014: On-site evaluation of commercial-scale

Commonly used cow evaluation methods apply

hybrid catfish production using cryopreserved

principles of the selection index to herdmate

blue catfish sperm. Aquaculture 426-427, 88-95.

deviations on the cow and close relatives. In contrast,

Kause A., Ritola O., Paananen T., Wahlroos H., andbest linear unbiased prediction adjusts records by

Mantysaari E., 2005: Genetic trends in growth,

best linear unbiased estimates of all fixed effects insexual maturity and skeletal deformations, andthe model and simultaneously weights those adjusted

rate of inbreeding in a breeding programme forrecords by selection index principles. It would be

rainbow trout (Oncorhynchus mykiss).

advantageous to utilize all known relationships

Aquaculture 247, 177-187.

among animals in the herd in the latter method, but

Navarro A., Zamorano M. J., Hildebrandt S., Ginés R.,

computations have been too laborious, requiring theAguilera C., and Afonso J. M., 2009: Estimates of

inverse of the numerator relationship matrix. By aheritabilities and genetic correlations for growthmethod of writing this inverse rapidly without

and carcass traits in gilthead seabream Sparus

computing the relationship matrix itself, all

aur at us L . ), u n d er in d u st rial c on d it io ns .

relationships can now be used in intraherd cow

Aquaculture 289, 225-230.

evaluation. Further, tests of progeny by artificial

National Agricultural Statistic Service, 2014: Catfish
Production Report.

insemination on sires used in the herd can beincorporated.

Nguyen N. H., Ponzoni R. W., Abu-Bakar K. R.,
Hamzah A., Khaw H.-L., and Yee H.-Y., 2010:(3) Wei M. and van der Werf J. H. J., 1994: Maximizing

Correlated response in fillet weight and yield togenetic response in crossbreds using purebred andselection for increased harvest weight incrossbred information. Anim. Prod. Sci. 59, 401-413.

genetically improved farmed tilapia (GIFT

A combined crossbred and purebred selection

strain),Oreochromis niloticus. Aquaculture 305,

(CCPS) method, i.e. using crossbred and purebred

1-5.

information, was proposed to achieve genetic

Waldbieser G. C. and Bosworth B. G., 2013: A

response in crossbred animals. Selection index theory

standardized microsatellite marker panel for

was applied to establish a CCPS index. The CCPS

parentage and kinship analyses in channel

was compared with pure-line selection (PLS) andcatfish, Ictalurus punctatus. Anim. Genet. 44,

crossbred selection (CS) methods. The genetic

476-479.

correlation between purebred and crossbred
performance (rpc) and crossbred heritability (hc2) are
crucial factors in the comparison. The CCPS is always

Development of improved catfish germplasm

121

better than PLS or CS when a fixed number ofbetween the markers. Using least squares, allpurebred progeny is tested. With a fixed total

haplotype effects could not be estimatednumber of purebred and crossbred tested progeny,

simultaneously. When only the biggest effects were

CCPS is only worse than PLS for very high values ofincluded, they were overestimated and the accuracyrpc (> 0.8). Superiority of CCPS over PLS increasesof predicting genetic values of the offspring of theand over CS decreases with decreasing r pc. The

recorded animals was only 0.32. Best linear unbiased

larger hc is relative to purebred heritability,the moreprediction of haplotype effects assumed equal

response CS and CCPS will achieve. The robustness

variances associated to each 1-cM chromosomal

2of CCPS against inappropriate assumptions on rpc andsegment, which yielded an accuracy of 0.73, although

hc2 values was investigated. The expected response isthis assumption was far from true. Bayesian methods

always an overestimate, and the actual response isthat assumed a prior distribution of the variancesmaller than the optimal response when r pc is

associated with each chromosome segment increased

assumed one but the true r pc is smaller. The

this accuracy to 0.85, even when the prior was notdifference between actual and optimal response

correct. It was concluded that selection on genetic

increases as rpc decreases, but it is small for large rpc

values predicted from markers could substantiallyvalues (e.g. < 3% for rpc > 0.7). The expected response

increase the rate of genetic gain in animals andis smaller than the actual response when rpc is large

plants, especially if combined with reproductive

and hc > hp . Finally, the actual response to CCPS istechniques to shorten the generation interval.

22

larger than the optimal response to PLS for positive
values for rpc. The main conclusions are: (1) CCPS

(5) Legarra A. O.F., Christensen O.F., Aguilar I., andmethod is optimal for obtaining genetic response in

Misztal I., 2014: Single Step, a general approach forcrossbreds and (2) CCPS with inappropriate

genomic selection. Livest. Prod. Sci. 166, 54–65.

assumptions on rpc and hc values (e.g. recognizing

Genomic evaluation methods assume that thecrossbreds as purebreds) achieves more genetic

reference population is genotyped and phenotyped.

response than PLS for common values of rpc andThis is most often false and the generation of pseudo-crossbred heritability.

phenotypes is uncertain and inaccurate. However,2markers obey transmission rules and therefore the(4) Meuwissen T. H. E., Hayes B. J., and Goddard M.

covariances of marker genotypes across individuals

E., 2001: Prediction of total genetic value using

can be modelled using pedigree relationships. Basedgenome-wide dense marker maps. Genetics 157, 1819-

on this, an extension of the genomic relationship

1829.

matrix can be constructed in which genomic

Recent advances in molecular genetic techniques

relationships are propagated to all individuals,

will make dense marker maps available andresulting in a combined relationship matrix, which

genotyping many individuals for these markers

can be used in a BLUP procedure called the Single

feasible. Here, we attempted to estimate the effects ofStep Genomic BLUP. This procedure provides so far

z50,000 marker haplotypes simultaneously from athe most comprehensive option for genomic

limited number of phenotypic records. A genome ofevaluation. Several extensions, options and details are

1000 cM was simulated with a marker spacing of 1

described: compatibility of genomic and pedigree

cM. The markers surrounding every 1-cM region

relationships, Bayesian regressions, multiple trait

were combined into marker haplotypes. Due to finite

models, computational aspects, etc. Many details

population size (Ne 5 100), the marker haplotypes

scattered through a series of papers are put together

were in linkage disequilibrium with the QTL locatedinto this paper.

水 研 機 構 研 報， 第 45 号，123 － 123， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，123－123，2017

123

Abstracts of Poster Presentations
List of Presentations
1:	Whole genome re-sequencing of fugu populations.

minute rotifer, Colurella cf. adriatica. (Stenly(Sho HOSOYA, Fisheries Laboratory, University

WULLUR, Faculty of Fisheries and Marineof Tokyo)

Science, Sam Ratulangi University, Manado –2:	Incipient transition of a sex-determining geneIndonesia)

among closely related species of fugu. (Kiyoshi

12:	Production of myostatin gene-knockout Japanese

KIKUCHI, Fisheries Laboratory, University ofanchovies (Eugraulis Japonicus) using TALEN-

Tokyo)

based genome editing. (Keishi SAKAGUCHI,

3:	Estimation of breeding value in model fish,

Fisheries Research Institute of Karatsu,

guppy (Poecilia reticulata) and its application for

Department of Joint Research, Faculity of

selective breeding in aquaculture. (Masamichi
NAKAJIMA, Tohoku University)
4:	The life table demography and population
growth of the rotifer Brachionus angularis Gosse,

Agriculture, Kyushu University)
13:	Isolation and Screening of Novel Probiotic Lactic
Acid Bacteria for Aquaculture. (Nguyen Thi Hue
LINH, University of Miyazaki)

from Kenya; the influence of temperature and14:	Effect of Protease Addition to EP diet on thefood density. (Erick Ochieng OGELLO, Nagasaki

Growth of amberjack, Seriola dumerili. (Yousuke

University)

TAOKA, University of Miyazaki)

5:	E f f e c t o f d i s s o l v e d o r g a n i c m a t t e r o n

15:	Amino Acid Profile of Thraustochytrids Cells

electrochemical removal of ammonia inand Potential of Application to Aquafeed. (Kenya

recirculating aquaculture systems. (Satoshi

HORII, University of Miyazaki)

TADA, Nagasaki University)

16:	Diurnal changes in frequency of the burst

6:	Effect of tetrodotoxin-containing diet on feeding,swimming behavior of adult Pacific bluefin tunadigestion and growth of tiger puffer, Takifugu(Thunnus orientalis) in a land-based tank. (Akiko

rubripes juveniles. (Kogen OKITA, Nagasaki

TSUJITA, Seikai National Fisheries Research

University)

Institute, FRA)

7:	Temperature tolerance in two clonal strains of

17:	Spawning frequency of Pacific bluefin tunamangrove killifish Kryptolebias marmoratus.

Thunnus orientalis in a land-based tank. (Ayako(Marina YAMADA, Nagasaki University)

SUZUKI, Seikai National Fisheries Research

8:	Effects of protozoa Euplotes sp. coexistence on

Institute, FRA)

the population growth of minute monogonont

18:	Effect of timing of restricted feeding on sexualrotifer Proales similis. (Naoshi WAKIMURA,

maturation in the yellowtail, Seriola

Nagasaki University)

quinqueradiata. (Kentaro HIGUCHI, Seikai

9:	Distribution of larval and juvenile greater

National Fisheries Research Institute, FRA)

amberjack (Seriola dumerili) around the Penghu

19:	Nitrogen excretion in Pacific bluefin tuna.

islands, Taiwan. (Takamasa HASEGAWA,(Toshinori TAKASHI, Seikai National Fisheries

Nagasaki University)

Research Institute, FRA)

10:	Effect of starvation on mixis induction in

20:	A high density genetic linkage map for yellowtail

offspring and its genetic mechanism of the(Seriola quinqueradiata) containing 6,275 EST-

monogonont rotifer Brachionus manjavacas.

based SNPs. (Akiyuki OZAKI, National Research(Shohei KAMIZONO, Nagasaki University)

Institute of Aquaculture, FRA)

11:	Body siza, Culture and fish larval ingestion on a

水 研 機 構 研 報， 第 45 号，125 － 141， 平 成 29 年
Bull. Jap. Fish. Res. Edu. Agen. No. 45，125－141，2017

125

Abstracts of Poster Presentations
1:	W h o l e g e n o m e r e - s e q u e n c i n g o f f u g u
populations

choosing marker loci for the management of broodstocks and conservation of wild populations. In thisstudy, we resequenced wild individuals of fugu from

Sho HOSOYA , Satoshi＊

TASUMI , and Kiyoshi＊

KIKUCHI＊

Wakasa Bay (Japan Sea) and Mikawa Bay (Pacific
Ocean) to compare the genetic diversity within andbetween populations. Two libraries each containing＊

Fisheries Laboratory, University of Tokyo,

ten individuals from either of the populations were

Hamamatsu, Bentenjim 2971-4 Japan

constructed for paired-end sequencing (2 x 101bp) on

E-mail: ahosoya@mail.ecc.u-tokyo.ac.jp

the Illumina Hiseq2000. We obtained 43.2M reads per
sample yielding coverage of 11.4 per genome, on

Key words

average.

Fugu, Takifugu rubripes, conservation, genetic

reference genome (Fugu v.5) and called single-

diversity, single nucleotide polymorphism, Fst,

nucleotide polymorphisms (SNPs) using BWA,

linkage disequilibrium, genomic selection

Samtools and GATK software. The number of SNPs

We mapped these reads on the fugu

detected per individual was about 700 thousand and
Abstract

the SNP frequency was about 480bp per SNP.
Missing SNPs because of the shallower depth were

Historically, the prevalence of intraspecificestimated as 2 to 2.5 per cent of the total SNPs fordiversity, both neutral and adaptive, in many marine

each sample. Multidimensional scaling plot clearlyspecies has been unclear.

However, the recent

separated the two populations, and individuals fromadvent of new technologies, in particular, high-Ise Bay were genetically closer than those fromthroughput DNA sequencing is making it feasible to

Wakasa Bay. However, the global Fst (= 0.0057) was

evaluate genetic variability at the whole-genome

small and no outlier locus was detected by BayeScan

level. Fugu (tiger pufferfish Takifugu rubripes) is one

software. These results suggest that the geneticof the important cultured marine fish species in East

divergence between the two populations is shallow.

Asia.

The yield of cultured fugu accounted for

Linkage disequilibrium analysis was done using

approximately 5% of total marine fish aquaculture in

PLINK software. The two populations were similar

Japan, and ranked 3 -4 by value. In addition, thisin the LD state. We detected putative 3,000 LD

species is becoming an important target for stock

blocks from each population but 90 per cent of themenhancement because of severe declines in the wild

were smaller than 1kb. The mean r2 value was 0.48

populations. In recent years, approximately two

between two SNPs at 100bp distance whilst that was

million hatchery-born seedlings have been released

less than 0.20 at 1kb distance. This rapid LD decay

each year. Tagging and releasing experiments haveindicates these populations have maintained atsuggested that fugu shows homing behavior to therelatively healthy states until recently. The expectednatal site for spawning following long-range

SNP size for the implementation of genomic selection

migration, and by extension, that distinct local

program for fugu breeding was 300k.

rdthpopulations may exist. As expected, recent study
using 21 microsatellite loci revealed a shallow

Annotated Bibliography

divergence between two wild populations from Japan
Sea and Pacific Ocean. Nonetheless, the detailed

Katamachi D., Ikeda M., Sato T., Suzuki S., Kikuchi K,

degrees of population and adaptive structuringand Ojima D. 2014: Development of a multiplex PCR

remain unclear.

Moreover there is virtually no

assay for population genetic analysis of the tiger

information regarding the extent and magnitude ofpuffer Takifugu rubripes using 16 microsatellite DNA

linkage disequilibrium (LD) that play essential roles inloci. Aquaculture Sci., 62, 55－63

126
The tiger puffer Takifugu rubripes is a marine fish

often specific to a few stock population of the focal

species economically important to East Asia,

species, greatly limiting use of such markers for theparticularly Japan. To evaluate the genetic variability

management of aquaculture species. To gain more

and population structure of the tiger puffer in detail,insights into the transition between SD systems in

we generated a multiplex PCR assay of microsatellite

teleosts, we study closely-related species of fugu

DNA loci, a fast and cost-effective technique thatbelonging to genus Takifugu.

allows high-throughput genotyping. In this study, we

undergone an adaptive radiation in the last 2-5 million

report the development of four multiplex PCR assays

years, resulting in about 20 extant species including

for this species using 16 microsatellite DNA locifugu (tiger pufferfish). Fugu is one of the most

located on independent chromosomes. We ensured

economically important food fish in Japan and also isquality control throughout all steps of the multiplex

the first fish with a fully sequenced genome.

PCR assay development, i.e., exclusion of loci detected

Previously, we have shown that sex in fugu appears

with stuttering, allele dropouts, or null alleles. We

to be determined by a missense single nucleotide

evaluated this set of microsatellite DNA loci forpolymorphism (SNP) in the Amhr2 gene. In thispolymorphisms using 113 fishes collected from threestudy, we have taken advantage of this finding anddifferent locations in the sea around Japan. This

the rich genomic resources of fugu to explore the

combination of loci will prove useful for future

genetic basis of sex determination in closely-related

investigations of the fine-scale population genetic

species of fugu. We found that while sex in the

structure of this species.

majority of Takifugu species is likely determined byThis genus has

the SNP in the Amhr2 gene, it is clearly not the casein a few species. To confirm this, we performed2:

Incipient transition of a sex-determining genegenome-wide linkage analysis and identified novel SD

among closely related species of fuguloci distinct from the Amhr2 locus in these species.
Interestingly, the transition of the SD system appears

Risa IEDA , Sho HOSOYA , Satoshi TASUMI ,to be in progress at least in one species, as a small

Shigenori SUZUKI , and Kiyoshi KIKUCHI

percentage of males still retains the “sex-determining

＊1

＊1

＊2

＊1

＊1,3

SNP” on the Amhr2 gene. These results indicate that
＊1,3

Fisheries Laboratory, University of Tokyo,

Hamamatsu, Bentenjim 2971-4 Japan＊2

Minami-Izu Laboratory, National Research Institutefugu and its closely-related species can be an
excellent model group for investigating thetransitions between alternative master SD genes.

of Aquaculture, FRA, Shizuoka, Minami-izu 415-0156
Japan
E-mail: akikuchi@mail.ecc.u-tokyo.ac.jp＊33:

E stimation of breeding value in model fish,guppy (Poecilia reticulata) and its application

Abstract
Sex determination in teleosts fish is often genetic,
where segregation of sex-determining loci assign thephenotypic sex.

for selective breeding in aquaculture
Masamichi NAKAJIMA＊, Toyoko NAKAJIMA＊, andSyunsuke OBINATA＊

In these species, sex-linked
Laboratory of Marin Life Science and Genetics,

polymorphic markers can be used for sex＊

identification, potentially providing important

Graduate School of Agricultural Science, Tohoku

information regarding the management of cultured

University, Sendai, Miyagi 981-8555, Japan

species and conservation of wild species. However,E-mail: mnkjm＠bios.tohoku.ac.jp

the different master sex-determining (SD) genes indifferent fish lineages appear to have evolved

Key words

independently and have been frequently replaced by

Guppy（Poecilia reticulate）, selective breeding, bodynew genes. Therefore, the sex-linked markers are

size, breeding value127

Abstracts of Poster
such as Bluefin tuna and Japanese eel.
Abstract
Selective breeding is one of the most important

4:	The life table demography and population

methods for the genetic improvement in not only

growth of the rotifer Brachionus angularis

livestock animals, but also aquatic organisms. Many

Gosse, from Kenya; the influence of temperature

varieties and strains were produced in aquaticand food density

organisms; however the most succeeded example of
selective breeding in aquatic organisms is ornamental

Erick Ochieng OGELLO＊1, 2, Hee-Jin KIM＊1, Koushirou

fish. In the case of quantitative traits, the example of

SUGA＊1, and Atsushi HAGIWARA＊1

succeeded selective breeding is scare. In the case of
Graduate School of Fisheries & Environmental

the selective breeding based on phenotypic value,＊1

many cases showed inconsistent result. This is due

Sciences, Nagasaki University, 1-14 Bunkyo, Nagasaki

to its inability to remove environmental and852-8521, Japandominance effect. Therefore the method to evaluate＊2

the accurate abilities of parent is necessary. The(KMFRI), Kegati Aquaculture Research Station, P.O.

breeding value was contrived from suchBox 3259 - 4200 Kisii, Kenya

circumstances. Though the breeding value possesses

E-mail: bb53414801@cc.nagasaki-u.ac.jp

Kenya Marine & Fisheries Research Institutesuch importance, the applications of the breedingvalue in the aquaculture are not so much. The

Key words

reasons why the application in aquaculture is little

Rotifera, alga, life table parameters, fecundity,

are 1) the large number of offspring can obtain fromBrachionus angularis

small number of parental fish; 2) short life cycle oftarget species caused difficulties to estimate breeding

Abstract

value in parental fish and their offspring. Although,it is hard to obtain the breeding value, the role of

The nature of reproduction of the Kenyan strain ofbreeding value is very important for the efficient

Brachionus angularis was investigated using

selection. From the above mentioned reason, it isindividual and small batch culture approaches. First,

necessary to identify the breeding value for thethe Kenyan rotifer was identified using morphologicaleffective selective breeding in aquaculture. In thisand molecular techniques. The life-table demography

study, the breeding values were estimated fromand the population growth studies were conducted atselection experiment for body size in the guppy,

three temperatures (i.e. 20, 25 and 30°
C) using

Poecilia reticulata, and examined the efficiency of the

Chlorella vulgaris at three densities (i.e. 2.5x10 5,

selection between used phenotype and breeding

2.5x106 and 2.5x107 cells mL-1). The lorica length (85.6

value. Comparison of breeding value in parental and± 3.1 µm) and width (75.4 ± 3.6 µm) of the Kenyan

offspring indicated significantly positive correlation.

sample were smaller than those of similar species

Positive correlation also observed between breedingcited in the literature.

values of parental and body size of their offspring.

grouped the Kenyan sample together with

Selection based on breeding value showed 5% larger

Brachionus caudatus and Brachionus angularis.

in body size compared with selection using

However, with additional morphological data e.g.

phenotype.

Selection based on breeding value

presence of two median occipital spines with either

showed lesser increment in coefficient of inbreeding

reduced or lacking sub-median spines, Brachionus

compared to the selection based on phenotype.

angularis was identified as the most likely match for

These results suggest that the breeding values are

the Kenyan sample. The rotifers were most fecund

effective to the evaluating parental fish and useful for(2.11 ± 0.07 offspring female-1 day -1) and reproductive

selective breeding. It is expected that the application

C with 2.5×106(8.43 ± 0.24 offspring female -1) at 25°of breeding value in the industrially important fish,

cells mL-1 of C. vulgaris. The highest intrinsic rate of

The phylogenetic tree

128
Abstract

natural increase (0.74 ± 0.02 d-1), specific population
growth rate (0.49 ± 0.01), longest life expectancy athatching (12.41 ± 0.28 d) and shortest generation time

Improvement of water treatment systems in closed(2.87 ± 0.03 d) were also observed at 25°
C with 2.5×10

6recirculating aquaculture systems (RAS) is necessary

cells mL-1 of C. vulgaris. However, the duration offor reducing total volume of culture water, which

hatching to first egg spawning was shortest (2.86 ±determine the size of the whole system as well as

0.21 h) at 30°
C with 2.5×10 and longest (8.83 ± 0.39 h)

running costs.

at 20°
C with 2.5×105 cells mL-1 of C. vulgaris. In theoxidation for removal of ammonia in culture seawater

7

New RAS using electrochemicalbatch cultures, the highest population density (255.7

are being developed by Nagasaki Prefectural± 12.6 ind mL–1) and lowest (122.0 ± 3.6 ind mL–1)

Institute of Fisheries. In this system, hypochlorous

C withwere realized at 25°
C with 2.5×10 and at 20°

acid, which produced by electrolysis of seawater,

2.5×10 cells mL of C. vulgaris on day 8 respectively.

oxidizes the bromide ions, yielding hypobromous

There was earlier population density peaks at higher

acid. Both hypochlorous and hypobromous acids

food densities (2.5×10 cells mL

of C. vulgaris)

react with ammonium ion and oxidize it to nitrogen

regardless of temperature. In conclusion, the Kenyan

gas. Since these free chlorine and free bromine alsostrain of B. angularis seems to have favorable

react with dissolved organic matter (DOM), efficiency

morphological and reproductive features makingof electrochemical ammonia removal could bethem suitable for aquaculture activities. The life

affected by quantity and reactivity of DOM in culture

table demography of this strain is optimal at 25°
Cseawater. In this study, removal of total ammonia

6

5

-1

7

-1

with 2.5×10 cells mL of C. vulgaris. The results ofnitrogen (TAN) and DOM by additions of

this study are relevant for improvement of the

hypochlorous acid was investigated using culture

freshwater aquaculture activities. Further studies onseawater of two RAS for tiger puffer Takifugu

the population growth of the rotifer are recommended

rubripes (RAS-1) and kelp grouper Epinephelus

using other different food types.

bruneus (RAS-2). The RAS-1 comprised of a culture

6-1tank (20 m3), a solid settler, a circulation pump, a foam
fractionator, an electrolysis unit, a reaction tank (for5:	E f f e c t o f d i s s o l v e d o r g a n i c m a t t e r o nammonia removal), and activated charcoal tank (forelectrochemical removal of ammonia inremoval of residual chlorine). The RAS-2 has arecirculating aquaculture systems

biofiltration tank in addition to the units provided in
the RAS-1.

Satoshi TADA and Shigenobu TAKEDA＊＊The culture seawater was filteredthrough Whatman GF/F filter and dispensed into
seventeen replicate 250-ml amber glass bottles.

Graduate School of Fisheries and Environmental

Additions of chlorine (Sodium hypochlorite solution)

Sciences, Nagasaki University

were conducted to achieve 17 steps chlorine doses ofE-mail: tdsts.goopy@gmail.com

0~16 (or 70) mg/L as Cl2. After 20 min contact period＊

at 25ºC, free available residual chlorine and combined
Key words

available residual chlorine in the sample were

Recirculating aquaculture system, electrochemical

analyzed by the DPD method. TAN was measured

oxidation, ammonia, dissolved organic matter,using an autoanalyzer. DOM was determined as

seawaterhumic-like and protein-like fluorophores based on thethree-dimensional excitation emission matrix
spectroscopy. Concentration of TAN in the culture
seawater consistently decreased along with theincrease in chlorine dose and then disappeared (whatis called breakpoint). In the bottles with higher levelsof chlorine dose, free available residual chlorine was
detected according to the excess amount of chlorine

129

Abstracts of Poster
dose. Concentrations of combined available residual

6:	Effect of tetrodotoxin-containing diet on

chlorine were very low but small increase wasfeeding, digestion and growth of tiger puffer

observed around the breakpoint. Humic-like andTakifugu ruburipes juveniles

protein-like fluorophores showed large decrease by
the addition of 1 mg/L Cl2 (the lowest chlorine dose)

Kogen OKITA＊, Tomohiro TAKATANI＊, Osamu

and then kept relatively constant concentrations until

ARAKAWA＊, Atsushi HAGIWARA＊, andthe breakpoint. In the bottled added with excess

Yoshitaka SAKAKURA＊

amount of chlorine (higher than the breakpoint),
Graduate School of Fisheries and Environmental

further decreases in humic-like and protein-like＊fluorophores were observed. These results suggest

Sciences, Nagasaki Universitythat DOM consists of highly reactive fraction andE-mail: jj20140049@cc.nagasaki-u.ac.jp

semi-labile fraction, and the former could reduce
efficiency of electrochemical ammonia removal in the

Key words

culture seawater. Compare to the chlorine demand

Tiger puffer, Takifugu rubripes, tetrodotoxin, feeding

to achieve the breakpoint in DOM-free artificial

trial, appetite

seawater, the culture seawater from RAS-2 required
17~42% more chlorine to oxidize TAN, but the

Abstract

difference between the artificial seawater and RAS-1
culture seawater was not clear. Skin mucus of kelp

Marine pufferfish contain tetrodotoxin (TTX), angrouper seems to be one of the sources of highly

extremely potent neurotoxin. We recently clarified

reactive DOM in RAS-2.

that a tiger puffer Takifugu rubripes juvenile detects
TTX by olfactory organ, and actively ingests andAnnotated Bibliography

accumulates TTX not only in liver and skin but alsoin central nervous system. We further revealed that

Ohwaki H., S. Yamamoto, A. Okamoto, and Y.

gene expression of some appetite peptides in the

Kurokawa. 2011. Development of a large-scale closed

brain of hatchery-reared non-toxic fish changed byrecirculating aquaculture system for saltwater fish.

TTX-sensing and TTX-administration.

Report of Industrial Technology Center of Nagasaki,present study, feeding trial using non-toxic and toxic

40, 52-55 (in Japanese)diets was conducted with hatchery-reared T.

In theFor the constraction of a large-scale closed

rubripes juveniles in order to examine the relation

recirculating system for aquaculture of saltwater fish

between the appetite and TTX ingestion. A total ofon land, we have been developing a new seawater

120 non-toxic cultured juveniles (mean body weight

treatment unit using hypochlorous acid produced byof 3.6 g) were randomly divided into two groups

electrolysis of seawater with platinum modified

where one group was fed with non-toxic commercial

titanium electrode, and a closed recirculating

diets and the other was fed with TTX-containing

aquaculture system with the electrochemical water

diets (5.3 MU/g feed). Fish were maintained in 3

treatment. Acidic water, which is a byproduct oftanks (20 fish/200 l) for each group with flow throughelectrolysis of seawater, can be used for the removal

system (2 l/min) and vigorous aeration. The fishof dissolved CO2 that accumulated in the culture

were fed with non-toxic and toxic diets to apparent

water.

Our computer simulation model could

satiation three times a day at 08:00, 13:00 and 17:00

reproduce the flow pattern in the electrolysis unit

hours for 28 days. All fish survived until the last day

and the estimated pH value of the out-flow waterof feeding trial. There were no significant differences

agreed well with the observation. We found thatin the growth performance between fish fed non-dissolved CO 2 in the culture water can be easily

toxic diets and toxic diets: total length (85.8±1.2 vsremoved by bubbling of the acidic water.

85.1±2.7 mm), standard length (81.1±1.0 vs 80.1±1.5mm), degree of loss of caudal fin (77.7±0.6 vs75.5±5.5%), body weight (18.4±1.7 vs 17.6±1.1 g), feed

130
intake (11.7±0.6 vs 11.3±0.8 g), assimilation rate

the growth rate. Since Panama and Belize show

(91.1±1.6 vs 86.3±3.0%), weight gain (14.9±1.6 vsdifferent climate, we hypothesized that PAN-RS,

14.0±1.1 g), feed efficiency (127.3±7.1 vs 123.7±5.3%).

PDHy and DAN show different temperature

Total amount of administered and accumulated TTX

tolerance.

in the fish fed toxic diets are 59.9±4.1 MU/fish andWe used two clonal strains (PAN-RS and DAN) and

33.6±2.0 MU/fish (56.4±7.1% of administered),

hybrid strains (PDHyI, II, III, and IV). Fish were kept

respectively. These results indicate that TTX at thisin plastic containers filled with 60 mL of 17 ppt

high dose may not have function as feeding stimulant

artificial brackish water under 25 ˚C and photoperiod

for T. rubripes juveniles, but that feeding activity andof 14L:10D. “Upper and lower thermal acclimation

growth of the juveniles are not inhibited by potent

limits” were quantified for each strain using chronic

neurotoxin. We will further perform quantitative

thermal tolerance methodology (Fangue et al. 2006).

analysis and investigate the immunohistochemical

All fish were kept under 25 ˚C for one week, and 20

localization of appetite peptides in the brain of TTX-

fish from each strain were subjected to either

sensed and TTX-administered hatchery-reared T.

increasing or decreasing water temperatures of 0.5 ˚Crubripes juveniles.

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

7:	Temperature tolerance in two clonal strains oftemperature at which 50% morbidity was observed.

mangrove killifish, Kryptolebias marmoratus

The respective chronic thermal maximum wassignificantly higher in DAN (36.6 ˚C) than PAN-RS

Marina YAMADA＊ and Yoshitaka SAKAKURA＊(35.7 ˚C), (Log-rank test, p<0.01). The respective
chronic thermal maximum was 30.7 ˚C, 33.6 ˚C, 33.7 ˚CGraduate School of Fisheries and Environmental

and 32.5 ˚C for PDHyI, PDHyII, PDHyIII and PDHyIV,

Sciences, Nagasaki University, Japan.

respectively. PDHyI showed the lowest respective

E-mail: bb53115032@cc.nagasaki-u.ac.jp

chronic thermal maximum among all strains.

＊

Respective chronic thermal maximum of each PDHy
Key words

strain was lower than their parents. The respective

Mangrove killifish, Kryptolebias marmoratus, thermal

chronic thermal minimum was 9.4 ˚C for PAN-RS andtolerance, hybrid

9.6 ˚C for DAN, with no significant difference (Logrank test, p=0.59).
Abstract

Mangrove killifish Kryptolebias marmoratus is the

8:	Effects of coexistence of protozoa Euplotes sp.

only known self-fertilizing vertebrate. This uniquecoexistence on the population growth of minutespecies broadly distributes in coastal mangrove

monogonont rotifer Proales similis

habitats from southern Brazil through the Caribbean
Islands and Central America to North Florida. They

Naoshi WAKIMURA＊, Yoshitaka SAKAKURA＊, andare capable of synchronous self-fertilization,

Atsushi HAGIWARA＊

producing homozygous clones as a consequence. Our
Graduate School of Fisheries and Environmentallaboratory has two clonal strains, PAN-RS and DAN,＊

which were originally collected from near Bocas delSciences, Nagasaki University, Japan.

Toro, Republic of Panama and Dangriga, Belize,

E-mail: bb53115036@cc.nagasaki-u.ac.jp

respectively. PDHy strain which is the hybrid of
PAN-RS and DAN was produced by artificial

Key words

insemination (Nakamura et al.

Proales similis, Euplotes sp., monoculture, mixed-2008).

The

descendants of PDHy are divided into 4 strains,
PDHyI, PDHyII, PDHyIII and PDHyIV according toculture131

Abstracts of Poster
Abstract

9:	Distribution of larval and juvenile greateramberjack (Seriola dumerili) around thePenghu Islands, Taiwan

The rotifer Brachionus rotundiformis (SS-type) is
commonly used as starter food for rearing smallHowever, several

Takamasa HASEGAWA＊1, Hsin-Ming YEH＊2, June-

tropical marine fishes have much smaller mouth gap,

Ru CHEN＊3 , Ryo KAWABE＊4 , and Yoshitaka

hence cannot feed on the B. rotundiformis. To solve

SAKAKURA＊1

mouthed marine fish larvae.

this problem, the minute monogonont rotifer Proales
Graduate School of Fisheries and Environmental

similis is preferred due to its smaller size than B.

＊1rotundiformis. However, the culture of P. similis is

Science, Nagasaki University, Japaneasy to be collapsed due to inability to withstand the＊2handling stresses and environmental changes in the

Fisheries Research Institute, Council of Agriculture,

Coastal and Offshore Resources Research Center,

culture medium. In this study, we investigated the

Taiwaninteraction between P. similis and the protozoa＊3

Euplotes sp.

in the rotifer culture water.

We

Fishery Research Institute, Council of Agriculture,

TaiwanInstitute for East China Sea Research, Nagasaki

independently cultured P. similis, Euplotes sp. and a＊4

combination of P. similis and Euplotes sp. in 144 ml

University, Japan

glass jar containing 50 ml of seawater (22 ppt) at an

E-mail: bb53512002@cc.nagasaki-u.ac.jp

initial density of 1 ind./ml at 28°
C in darkness for 14
days. All the treatments were triplicated and daily

Key wards

fed with Nannochloropsis oculata at 8.0 x 10⁵ cells/ml

Greater amberjack, Seriola dumerili, early life,

without water exchange. A similar experiment was

spawning ground, otolith

conducted separately with Chlorella vulgaris as foodat 2.9 x 10⁵ cells/ml daily without water exchange.

Abstract

When fed with N. oculata, population density
increased in Euplotes sp. in the monoculture and

The greater amberjack Seriola dumerili (family

mixed-cultures. However, the population growth ofCarangidae) widely distributes around the world.

P. similis in the mixed-culture decreased after 8 days

Because of its commercial importance, rapid growthand P. similis disappeared after 14 days of culture.

and good adaptation to captivity, S. dumerili is a very

There was a significant difference in the population

important species for aquaculture in Japan. Juveniles

density of either P. similis or Euplotes sp. betweenof S. dumerili associate with floating objects such as

the monoculture and mixed-culture on day 14. When

drifting seaweeds. However, there is very limited

cultured with C. vulgaris, population density of P.

knowledge about larval and early-juvenile stages ofsimilis decreased from day 6, while that of Euplotes

this species in the wild. In order to investigate thesp. reached its highest peak on the same day. P.

early life history of S. dumerili, firstly we validated

similis dissapeared completely on day 14. Thesethe otolith daily increments using artificially-raised

results suggest that the presence of Euplotes sp. infish (11-51 days after hatching). Then, field surveys

the P. similis cultures significantly suppressed thewere made by R/V Hai-an, Fishery Research

population growth of P. similis presumably due toInstitute, Council of Agriculture, Taiwan around thestressful interactions. Furthermore, the competition

Penghu Islands, Taiwan, from May to August 2015.

between P. similis and Euplotes sp. for bacteria may

Frontal zone and drifting seaweeds were visuallyalso have existed. Even though we did not observe aobserved during survey, and drifting seaweeds were

behavioral interaction of Euplotes sp. and P. similis,

scooped together with associated fishes by a hand net

the swimming speed of the Euplotes sp. was higher(Φ45 cm, 3 mm mesh). Surface tows of plankton net

than the P. similis.

(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

132
Abstract

profile of water temperature and salinity were
measured by a CTD (SBE-19 plus, Sea-Bird
Electronics Inc.). Fish species were identified andShort time starvation of neonates hatched fromzooplankton abundance (mg DW・m ) and species

rotifer resting eggs induces active mixis up to thecomposition (%) were calculated. We also measured

10th generation. However, this phenomenon remains

four S. dumerili samples caught in 2014 deposited at

unexplained whether it can last for further

Penghu Marin Biology Research Center, Fisherygenerations. Also, the mode of heredity and the

Research Institute, Council of Agriculture, Taiwan.

acquired parental characteristics inherited by the

Increments of sagittal otolith of reared fish showed

subsequent generations are not clear. In this study,

the same number as their age in days after hatching

we used the rotifer Brachionus manjavacas and(ANCOVA, df=1, p=0.32). Relationships between

Brachionus plicatilis to investigate the heredity ofotolith diameter (ydia, μm) and age (xday, days), andmixis induction in rotifers up to the 80th generation

between otolith diameter (ydia, μm) and TL (xTL, mm),and the inheritance of the acquired traits byday

were described as following equations: ydia=0.0003・x1.93

methylation of DNA. The maternal rotifers hatched(r=0.991), and y dia =0.1937・x TL -0.1018 (r=0.981),

from resting eggs were starved for 12 hours before

respectively. We caught a total of four larval anddetermination of the acquired characteristics. The

juvenile S. dumerili by surface towing, but not fromcontrol group was not starved. A set of 8 individual

drifting seaweeds. Total length of S. dumerili ranged

rotifers were randomly selected from starved andfrom 7.4 to 42.5 mm, and age ranged from 18 to 56

control group. Rotifers were individually cultured indays after hatching. All S. dumerili were caught in

0.2 ml of sea water (22 ppt.) in 8 well microplate at 1

open water and in frontal zone, however we could not

individual per well untill the 80th generation. The

detect significant differences in zooplankton

rotifers were daily provided with Nannochloropsis

abundance between frontal zone and other station

oculata suspension at 6.0 × 106 cells/ml. The mixis(t-test, p=0.87).

Thecostraca was significantly

induction in each generation was determined. We

abundant in the stations away from the frontal zones

determine the genetic factors that changed the mixis(t-test, p<0.01). In 2014, larval and juvenile S. dumeriliinduction rate of the parent and future generations.

were caught in January and May, and body length

First, we designed the primer using partial baseranged from 5.8 mm in NL to 54.2 mm in TL. Oursequence of the methyltransferase, which was thestudy indicates that S. dumerili spawns from April to

DNA methylase which EST analysis of B. plicatilis

June in 2015, and frontal zone in open water isperformed PCR as template cDNA of B. manjavacas.

nursery ground for S. dumerili around the Penghu

The mixis induction of the offspring from starved

Islands, Taiwan.

parents increased until 38th generation. In addition,-3

mixis induction during accumulated generations
peaked at the 17 th generation.
10:	Effect of starvation on mixis induction in

We observed

repetitions of increase and decrease of beyond theoffspring and genetic mechanism of the

17 th generation.

monogonont rotifer Brachionus manjavacas

fragment of the predicted size was confirmed

The amplification of the DNA

through PCR analysis using the primer designed＊

Shohei KAMIZONO , Koushirou SUGA , Yoshitaka

from the DNA methyltranferase (DNMT) gene of B.

SAKAKURA＊, and Atsushi HAGIWARA＊

plicatilis. This phenomenon could be explained by＊

the epigenetic inheritance involving methylation of
Graduate School of Fisheries and Environmental

the DNA. It was estimated that the DNMT gene

Sciences, Nagasaki University, Bunkyo 1-14, Nagasaki

fragment by BLAST analysis.

852-8521, Japan．

investigating this by comparing the DNMT geneE-mail: bb53415001@cc.nagasaki-u.ac.jp

expression level among generations.

＊

We have been

133

Abstracts of Poster
11:	Body size, culture and fish larval ingestion on aminute rotifer Colurella cf. adriatica

was cultured at salinity of 20 ppt and placed in acontrolled room temperature at 25±1℃.

Water

volume of the culture was 4 ml (using 3x4, multiwell
Stenly WULLUR , Petrus LETSOIN , Rina P.

plate) and the initial density of the rotifer was 1 ind./

ASTUTI＊3, and Atsushi HAGIWARA＊4

ml. Observation was made daily by counting the＊1＊2

numbers of rotifer in each well until the density＊1

Faculty of Fisheries and Marine Science, Sam

Ratulangi University, Manado - Indonesia＊2State Polytechnic of Fisheries. Tual, Southeast

Maluku – Indonesia＊3

Larval ingestion on the rotifer wasinvestigated in Gondol Research Institute for
Mariculture, Bali-Indonesia. Approximately 10 ind./leggs of humpback grouper (Cromileptes altivelis)

Gondol Research Institue for Mariculture, Bali –Indonesia＊4decline.

were transferred to four 200-l larval rearing tank.
The first two tanks were fed with 10 ind./ml of

Graduate School of Fisheries Science androtifer C. cf. adriatica from day 2 till day 5 after

Environmental Studies, Nagasaki University, Japan

hatching, while the other two tanks were left without

E-mail: miracle_given@yahoo.com

any addition of food. All surviving larvae were
harvested on day 5 and the numbers of remaining

Key words

larvae were counted. Gut content of the surviving

Minute rotifer, Colurella cf. adriatica, body size,

larvae was analyzed to see the presence of rotifer.

population growth, fish larvae, ingestion

Body length and width of C. cf. adriatica were
distributed from 82.8-103.2 and 46.8-61.7 μm,

Abstract

respectively. The mean body length (95.9±3.8 μm;
mean± standard deviation) and width (46.8-61.7 μm)

Current procedure of rearing small mouthedof C. cf. adriatica were significantly smaller/narrower

marine fish larvae is using the Super Small (SS) typethan B. rotundiformis (175.2±9.2 and 123.5±7.7 μm,of rotifer Brachionus rotundiformis as starter foodrespectively) (t-test, p<0.05). Rotifer C. cf. adriatica

during first days of larval first feeding. The B.

grew well in all N. oculata treatments. The rotifer

rotundiformis, however, is ineffective or evenattained highest population densities on day 16

unsuitable for larvae of several marine tropical fish

(774±167 ind./ml) and 18 (656±139 ind./ml) at N.

with even smaller mouth size including, Napoleon fish

oculata densities of 6 and 9x106 cells/ml, while it was(Cheilinus undulatus), groupers (genus Epinephelus),

reached on day 26 (646±85 ind./ml) and 34 (560±58

angelfishes (family Pomachantidae). In present study,

ind./ml) at N. oculata densities of 3 and 12x106 cells/

we examined the feasibility of a minute rotifer C. cf.

ml, respectively. Humpback grouper larvae show

adriatica as live food by measuring its body size,

higher survival (1.5%) on C. cf. adriatica treatment

analyzing population growth and fish larval ingestion

than control (0.3%) (t-test, p<0.05). By analyzing gut

on the rotifer.

content of the remaining larvae, it was found that

Rotifer C. cf. adriaticawas isolated using a plankton

individual of rotifer C. cf. adriatica presence in gut ofnet (45 μm mesh size) from an estuary in Mangket,

the larvae indicating that larvae of humpback

North Minahasa, North Sulawesi, Indonesia. Water

grouper ingested the rotifer.

temperature and salinity at the time of sampling
were 28±1 °C and 30±1 ppt, respectively. Sixty
adults of the rotifer were measured for its body

12:	Production of myostatin gene-knockout

length and width. As comparison, body length andJapanese anchovies (Engraulis japonicus)

width of a local strain B. rotundiformis were alsousing TALEN-based genome editing

measured.

Population growth of Colurella cf.

adriatica was assessed by culturing the rotifer under

Keishi SAKAGUCHI＊1, Kanako NAKASHIMA＊2,

four densities (3, 6, 9, 12 x 10 cells/ml) of

Hajime KITANO＊1, Naoki NAGANO＊1, and Michiya

Nanochloropsis oculata as food source. The rotifer

MATSUYAMA＊2

6

134
gene inhibits skeletal muscle growth even in fish.
Fisheries Research Institute of Karatsu,

Thus, to produce a fish breeding model generated by

Department of Joint Research, Faculty of Agriculture,

genome editing, we performed targeted gene

Kyushu University

disruption of the MSTN gene in Japanese anchovies＊1＊2

Laboratory of Marine Biology, Faculty ofusing TALEN technology. We constructed three

Agriculture, Kyushu University

TALEN pairs targeting the first intron of the MSTN

E-mail: keishi_s@agr.kyushu-u.ac.jp

gene and the in vitro transcribed RNAs of the pairs
were injected into the yolk of embryos at the one-cell

Key words

stage. As a result, mutant F0 embryos were obtained

Japanese anchovy, Engraulis japonicus, myostatin,with a very high insertion and/or deletion (indel)knockout, TALEN, genome editing

mutation rate (~96.9%), and thus the F0 founders
were mated with each other to produce MSTN-

Abstract

knockout anchovies at the F1 generation. To ourknowledge, this is the most advanced study for

Genome editing techniques such as zinc finger

genome editing in marine fishes. The rearing of F1-

nucleases (ZFNs), transcription activator-like effector

individuals and their genotyping is now in progress.

nucleases (TALENs), and clustered regularlyinterspaced short palindromic repeats (CRISPR) haveattracted considerable attention in recent years since

13:	Isolation and screening of novel probiotic lactic
acid bacteria for aquaculture

these technologies can mediate targeted and efficient
genetic modifications (knockout, knock-in, and genemodification) in various organisms. Thus, targeted

Nguyen Thi Hue LINH＊1, Masato OTANI＊2, andgenome editing, which enables researchers to tailor

Yousuke TAOKA＊3

genomic loci of interest, is one of the most promising
approaches for plant, animal, and fish breeding.

＊1

Interdisciplinary Graduate School of Agriculture

Nevertheless, the application of the techniques toand Engineering, University of Miyazaki

teleost fishes have been limited almost exclusively to＊2Graduate School of Agriculture, University of

popular experimental fish models, such as zebrafish

Miyazaki(Danio rerio) and Japanese medaka (Oryzias latipes).

＊3

There are no literature reports on the application of

Sciences, Faculty of Agriculture, University ofthese techniques to marine fish, which includes many

Miyazaki

important species for the fisheries industry. In thisE-mail: nb14005@student.miyazaki-u.ac.jp

Department of Marine Biology and Environmental

context, we regard the Japanese anchovy (Engraulisjaponicus) as a most suitable candidate for genome-

Key words

editing experiments in marine fish, because they

Probiotics, lactic acid bacteria, Lactobacillus sp.,

have the following advantages: 1) easy rearing andpathogen, antagonistic activity, tolerance in

breeding in a small-scale fish tank.■2) year-roundgastrointestinal juice

and multi-year spawning under photoperiod andtemperature control.■3) quick growth into mature

Abstract

individuals, which produce another generation ofeggs, about three months.

Myostatin (MSTN),

Most of probiotics used in aquaculture arepreviously referred to as growth differentiationconsidered as alternative therapies for the use offactor 8 (GDF8), is a negative regulator of skeletal

antibiotics to prevent diseases in aquatic animal.

muscle growth.

In mammals, MSTN-deficientAmong of probiotic bacteria, lactic acid bacteria areanimals resulted in an increase of skeletal muscle

one of potential candidate due to several strains havemass with both hyperplasia and hypertrophy.

been isolated from fish gut as beneficial microflora

Likewise, recent studies revealed that the MSTN

and acted antagonistic against Gram-negative fish

135

Abstracts of Poster
pathogens.■Therefore, in this study we conducted to

14:	Effect of protease addition to EP diet on thegrowth of amberjack, Seriola dumeriliisolate lactic acid bacteria from fermented food ascandidate strains which may be used to reduce theantibiotics using for sustainable aquaculture.■The

Yousuke TAOKA＊1, Takayuki MINAMI＊2, Nguyen

probiotic properties of isolates were surveyed.■Lactic

Thi Hue LINH＊3, and Terumi KOGA＊4acid bacteria were isolated from samples such as
Department of Marine Biology and Environmental

fermented foods by an agar plating method using＊1

GYP and MRS media. The antagonistic activity test

Sciences, Faculty of Agriculture, University ofagainst fish pathogens Lactococcus, Streptococcus andMiyazaki

Edwardsiella was carried out according to the＊2

Miyazaki Prefectural Fisheries Research Institutemethod of the double layer agar method.

＊3

Interdisciplinary Graduate School of AgricultureThe

tolerant ability of isolates on NaCl (0%, 3%, 5%, 10%),and Engineering, University of Miyazaki

pH (from 2 to 9), artificial gastric juice (at pH range＊42-4) with pepsin and intestine juice (at pH 8) with gall

E-mail: yousuketao@cc.miyazaki-u.ac.jp

TOA Pharmaceutical CO., Ltd.

powder were evaluated. Isolates were identified
based on the sequences of 16 S rRNA gene (~700bp).

Key words

Totally 55 strains of lactic acid bacteria were isolated

Seriola dumerili, protease, digestibility, growth rate,from rice bran and several kinds of fermented

feed, low temperature, aquaculture

vegetables.

In antagonistic test, three isolates
Abstract

showed positive results against three strains of
Edwardsiella tarda, three strains of Streptococcus
disgnactie, three strains of S. iniae and three strains

In aquaculture, the late growth of the cultured fishof Lactococcus garvie. These three strains of GYP 31,

in winter season is serious problem for economical

GYP 69 and GYP 4-20 were identified as Lactobacillus

aquaculture. The extruder pellet (EP) has been used

sp.. The relative growth of GYP 31 strain at pH

as an composed feed for the culture fish because EP

range 5-8 was from 100% to 80%, at pH 2-4 and pH 9

can be produced efficiently. However, the EP is very

were below 20%; while GYP 69 strain and GYP 4-20

hard to decompose it by digestive enzymes as

strain only grew well at pH 5, pH 6 with 100% of thecompared with raw diet. The activities of digestive

relative growth. Three strains grew well at NaCl

enzymes in the gastrointestinal tracts of the cultured

concentration from 0-5%, 3-5% and 0-3% with thefish significantly decrease due to the low temperature

relative growth from 80% to 100%, respectively. In

during winter season. Therefore, it is very difficult

tolerance test on acid and artificial gastrointestinalthat the digestive enzymes with low activities

juices, GYP 31 strain expressed the sustained ability

decompose the EP diet efficiently. To dissolve thisand survival itself better than GYP 69 strain and GYP

problem, in this study, the application of proteolytic

4-20 strain. GYP 31 strain showed the highest viable

enzymes from microorganisms was examined tocount as 1x10 , 1.4x10 , 5.6x10 cfu/ml in acid solution

accelerate the digestion of the EP diet for the(at pH 3,5-4), artificial gastric juice at (pH 4) andcultured fish. Acid and alkaline proteases fromintestine juice (at pH 8), respectively, although themicroorganisms was used. The effect of pH andviable count was lower than those in the control

temperature on enzyme reaction was alsogroup (2.2x109cfu/ml at pH 7). From these results,

investigated. Alkaline and acid protease were added

Lactobacillus sp.. GYP 31 strain is considered as ato the EP diets in a 0.1M Tris-HCl buffer and 0.1M

potential probiotic candidate in aquaculture due to its

glycine-HCl buffer, respectively.

ability competition with pathogens and high tolerance

mixtures were incubated at 15, 20, 25, 30 and 35°
C for

in the gastrointestinal tract of fish.

180 min.

8

8

7

The reaction

The degradation of the EP diet wasevaluated by weighing the solid bodies. The nitrogenconcentration of the centrifugal supernatant in thereaction mixtures were determined according to the136
Kjeldahl method. The alkaline proteases showed

Aquaculture, feeds, fishmeal, protein source,

stable activities at 20-35°
C, and the activity at 15°Cthraustochytrid, amino acid composition, byproducts

significantly decreased by 44%. The decompositionof the EP diet was enhanced by addition of both

Abstract

proteases at 20°
C. These results indicated that theaddition of proteases from microorganisms is

Fishmeal is used as protein sources for aquaculture

effective to enhance the decomposition of the EP dietfeeds. However, the price is drastically increased toat low temperature. Amberjack, S. dumerli was fed

around 170,000/ ton in 2013 during recent 10 years

with EP diet with or without alikaline proteasebecause of the decrease of fish resources, anchovy.

(control group) for 60 days. After 60 days of rearing,This is serious problem for sustainable aquaculture.

the average fish body weight was higher in the group

Therefore replacement of fishmeal to anotherwith alikaline protease as compared with that in the

resource is urgently needed. Thraustochytrids arecontrol group.

These results showed that it ismarine protists and classified to one of Stramenopiles.

possible that the addition of protease enhance the

They are widely distributed in marine environment

growth rate of S. dumerili in winter season.

and accumulate large number of lipids in cell bodies.
Therefore, thraustochytrids have attracted strong

Annotated Bibliographyinterests for production of valuable lipids as single
cell oils (SCOs) such as biodiesel and omega-3 fatty

Satoh K. 2005. Study on improvement of composed

acids. In the process of lipid extraction from thediet for yellowtail culture. Bulletin of Oita Institute ofcultured cells, some solids (extract residue) are

Marine and Fisheries Science, 6, 19-77 (in Japansese)

produced as byproducts. It is considered that thisTo date, Japanese aquaculture, mainly for yellowtail

extract residue except for lipids is mainly consists ofSeriola quinqueradiata, has been developed using

protein. From the viewpoints of industrial application

raw-fish as their feedstuff, such as sardine andof thrautochytrid cells as protein sources, we havemackerel obtained from adjacent sea, that used to beplanned to use the byproducts as resources instead ofabound and available with low cost. The reduction offishmeal for aquafeeds. In this study, we evaluatedsardine resources definitely caused the nessesity of

the recovery rate of protein in a thrausotchytrid,

composed diet in yellowtail culture. Therefore, theAurantiochytrium limacinum strain mh0186 known

studies examined to improve and to improveas a docosahexaenoic acid (DHA) producer under theaccommodate the composed diet for yellowtails as tolipid extraction process. For selection of adequate

feedstuff, protein digestibility, feed additives, andstrains for protein production, thraustochytrids werefeeding regime.

isolated from marine environment. A. limacinumstrain mh0186 was cultured in a GY broth. The
cultured cells were collected by centrifugation and

15:	Amino acid profile of thraustochytrids cells andpotential of application to aquafeed

lyophilized for proximate analysis. The content ofprotein, lipid and ash in the lyophilized cells wasdetermined according to Kjeldahl method, Folch

Kenya HORII＊1, Masahiro HAYASHI＊1, and Yousuke

method and heat-ashing method, respectively. The

TAOKA

composition of amino acid and fatty acid were＊2

analyzed by liquid chromatography-mas spectrophy
Department of Marine Biology and Environment(UF-Aminostation, Shimazu Co. Ltd., Japan) and gas

Sciences, Faculty of Agriculture, University ofchromatography (GC-2014, Shimazu Co. Ltd., Japan),Miyazaki

respectively. In the process of lipid extraction, the＊1,2

E-mail: gd12020@student.miyazaki-u.ac.jp ,extract residue was collected and re-lyophilized. The

yousuketao@cc.miyazaki-u.ac.jp

protein content and amino acid composition were※1

※2analyzed by same method described as above.
Key words

Seawater, sands, leaves, seaweed were collected for

137

Abstracts of Poster
the isolation of thraustochytrids from coastal area in

artificially-reared fingerlings is needed to ensure the

Miyazaki, Kumamoto and Oita, Kyushu, Japan.

sustainability of aquaculture through a reduction on

Thraustochytrids were isolated on a B12 Culture

the reliance on wild captured juveniles. Therefore,

Agar “Nissui” plate medium by pine pollen-baiting

we constructed two large indoor land-based PBT

method. The isolates were cultured in a GY broth,broodstock tanks at Seikai National Fisheries

and the cultured cells were collected by centrifugation

Research Institute, FRA, Japan, and are examining

for analysis of the composition of amino acid and fatty

the environmental cues such as water temperatureacid. Isolates were identified at genus level based on

and photoperiod which are essential for successful

the 18S rRNA sequence analysis. The content ofand stable spawning of PBT.

crude protein lipid and ash per g of the cultured cellmortality of adult PBTs occur in the tank due toof mh0186 strain were 333 mg, 440 mg and 42 mg. On

collisions with the tank wall. It is considered that thethe one hand, the protein content of the extract

collision deaths are associated with the burst

residue obtained from 1g of the cultured cells was 226

swimming behavior. In this study, to clarify themg (recover rate, 68%).

In both samples of theprocess of the collision death, we examined diurnal

cultured cells and the extra residue, glycine, leucine,changes in frequency of the burst swimming

isoleucine, glutamate and arginine were mainly

behavior of three-year-old PBTs in the land-based

detected. One hundred twenty thraustochytrids

tanks. Nineteen three-year-old PBTs were reared instrains were isolated. In some isolates including A.

the land-based tanks (20 m in diameter, 6 m in depth)

limacinum strains SR21 and mh0186, T.aureum

and their swimming behaviors were recorded using a

ATCC34304, Schizochytrium aggregatum ATCC28209,

video camera for six days.

glycine, leucine glutamate, arginine and werecompartmentalized into six periods defined as dawn

detected as major amino acids.

Strain Tak2

(7:00 to 9:00), daytime-I (11:00 to 13:00), daytime-II

specifically accumulate 25% cystathionine to total(15:00 to 16:00), dusk (16:00 to 18:00), night-I (20:00 toamino acids.

22:00) and night II (0:00 to 2:00) according to changes

However, heavy

A day wasin illumination. We counted the frequency of the
burst swimming behavior at each period from the
16:	Diurnal changes in frequency of the burst

recorded video imagery. As a result, the frequency

swimming behavior of adult Pacific bluefinof the burst swimming behavior at dawn wastuna (Thunnus orientalis) in a land-based tanksignificantly higher than that in other time periods.
Notably, the burst swimming behaviors were

Akiko TSUJITA , Toshinori TAKASHI , Kentaro

frequently observed in 30 min just after illumination

HIGUCHI＊, Tatsuru KADOTA＊, Koichiro GEN＊,(from 32 to 85 lux) during dawn. Additionally, theAyako SUZUKI , Junpei KONISHI , Keiichi

frequency of the burst swimming behavior of

MUSHIAKE , and Masakazu OKA

multiple PBTs in the tank like “a panic” were often

＊

＊

＊

＊

＊

＊

observed during dawn, whereas solitary burst＊

Research Center for Tuna Aquaculture, Seikaiswimming behaviors were observed at the other time

National Fisheries Research Institute, FRA

periods. These results suggest that collision deaths

E-mail: tsujiaki@affrc.go.jp

of PBT in the tank were caused by burst swimming
behavior associated with sudden increases of light

Key words

intensity during dawn.

Pacific bluefin tuna, collision death, burst swimming
behavior, diurnal change, broodstock tank17:	Interval and spawning frequency of Pacific
Abstract

bluefin tuna Thunnus orientalis in a landbased tankIn aquaculture of Pacific bluefin tuna, Thunnus
orientalis (PBT), development of a stable system of

A y a k o S U Z U K I ＊1 , K o i c h i r o G E N ＊1 , T a t s u r u138
KADOTA＊1, Kenji SAITOH＊2, Takuma SUGAYA＊3,

observed to have 3 haplotypes. These haplotypes

Masakazu OKA＊1, and Keiichi MUSHIAKE＊1

were named A, B and C, respectively. Among thebroodstock fish, haplotype B was detected at high＊1

Research Center for Tuna Aquaculture, Seikai

National Fisheries Research Institute, FRA＊2

Research Center for Aquatic Genomics, National

Research Institute of Fisheries Science, FRA

frequencies, secondly haplotype C, and thirdly
haplotype A.

The number of each broodstockindividuals was 32, 3, and 1, respectively. Eachhaplotype of eggs and hatched larvae was detected

Research Center for Marine Invertebrates, Nationalon 14 days, 1 day and 8 days, respectively, during the

Research Institute of Fisheries and Environment of

15 sampling days. Haplotype B occurred in the 3

Inland Sea, FRA

days of consecutive spawning. This is consistentE-mail: ayakos@affrc.go.jp

with genetic or histological observations that other＊3

wild Thunnus species spawn multiple times and on
Key words

consecutive days. These results demonstrate that

Pacific bluefin tuna (Thunnus orientalis), spawning

PBT in a land-based tank may have the potential tofrequency, mtDNA D-loop region, haplotype

spawn consecutively and multiple times.

Abstract
18:	Effect of timing of restricted feeding on sexual
maturation in the yellowtail, Seriola

Recently, the exploitation of natural stocks ofPacific bluefin tuna Thunnus orientalis (PBT) has

quinqueradiata

increased to dangerous levels though increased
fishing pressure. The scarcity of this species and its

Kentaro HIGUCHI＊1,2, Kazunori YOSHIDA＊1, Koichirohigh commercial value, together with its very highGEN＊1, Toshinori TAKASHI＊1, Kiyoshi SOYAMA＊2,

growth rates, makes it a potential candidate forand Keiichi MUSHIAKE＊1

marine aquaculture.

Therefore, the artificial
Seikai National Fisheries Research Institute, FRA,

propagation and seed production techniques for PBT＊1

represent objective for sustainable utilization of this1551-8 Taira-machi, Nagasaki, Nagasaki 851-2213,

resource. Fundamental information on the spawningJapan

ecology of PBT is essential for the development of＊2

production techniques. However, this information is

University, 1551-7 Taira-machi, Nagasaki, Nagasaki

still largely lacking. In this study, we investigated851-2213, Japan

spawning frequency of PBT by comparing

E-mail: higuken@affrc.go.jp

Institute for East China Sea Research, Nagasaki

mitochondrial DNA D-loop region haplotypes ofbroodstock fish, with those of fertilized eggs and

Key words

hatched larvae. Broodstock PBT, fertilized egg andYellowtail (Seriola quinqueradiata), sexual maturation,

hatched larval samples were obtained from a land-

restricted feeding, broodstock management

based tank at Research Center for Tuna Aquaculture,
Seikai National Fisheries Research Institute in

Abstract

Nagasaki, Japan. Spawning activity in a land-basedtank was observed over a 98 day period during May

In aquaculture of large sized marine fish such as

16 to August 28 in 2014. The sampling of fertilized

bluefin tuna and yellowtail, extensive amounts of fish

eggs and hatched larvae was conducted for a total of

feed are needed for the broodstock management

15 days within the 98 days. A total of 15 samples

because of their large body size. In order to save on

were collected for an initial consecutive 3 day period

the feeding cost for their broodstock management,

and then at approximately 1 week intervals fromdevelopment of a restricted feeding technique

May 16 to August 28 in 2014. The 36 broodstock

without affecting reproductive performance isindividuals and 659 eggs and hatched larvae wererequired. However, available information about the139

Abstracts of Poster
effect of food supply on reproduction is limited in fish.

(Thunnus orientalis) measured in the land-based

In this study, we examined the effect of restrictedtankfeeding during the gonad immature and vitellogenic
phases on sexual maturation in females of the

Toshinori TAKASHI＊1, Takeshi EBA＊2, Tadashi

yellowtail, Seriola quinqueradiata.

Two-year-old

I M A I ＊3 , T e t s u o M O R I T A ＊3 , Y o s h i h i s acultured yellowtail females, which the average body

YAMAMOTO＊3, Hiroyuki MATSUNARI＊4, Junpei

weight was 5.0 kg, were divided randomly into three

KONISHI＊1, Kazuki KUMON＊2 , Koichiro GEN＊1, andsea cages on November 2012 and reared until the

Masakazu OKA＊1

next spawning period (April 2013) under natural
conditions at the Goto station, Seikai National
Fisheries Research Institute, FRA, Japan.

The

＊1

Seikai National Fisheries Research Institute, FRA,

8-1551 Taira, Nagasaki, Nagasaki 851-2213, Japan
Amami Laboratory, Seikai National Fisheries

feeding regimes in each cage were defined as follows:＊2

control group fed to satiation three times a week

Research Institute, FRA, 9555 Hyousakiyamahara,throughout the experimental period, two restricted

Oshima Setouchi, Kagoshima 894-2414, Japan

groups fed 30% of the amount of feed given to the＊3control group (100%) during the immature (fromFisheries and Environment of Inland Sea, FRA, 234

November to January) or vitellogenic phase (fromYashima-higashi-machi, Takamatsu, Kagawa 761-

Yashima Laboratory, National Research Institute of

February to April), respectively. At the end of the0111, Japan

experimental period, the average body weights were＊4

6.6 kg in the control group, 5.8 kg in the restricted422-1 Nakatsuhamaura, Minamiise, Mie, 516-0193,

group during the immature phase and 5.9 kg in theJapan

restricted group during the vitellogenic phase, which

E-mail: ttakasi@fra.affrc.go.jp

National Research Institute of Aquaculture, FRA,

shows that the restricted feeding reduced
approximately 50% of somatic growth throughout the

Key words

experimental period.

Pacific bluefin tuna (Thunnus orientalis), nitrogen,

Interestingly, the gonad

weights in the restricted feeding group during theammonia excretion rate, ration size

vitellogenic phase were low as compared with thecontrol group and the restricted feeding group

Abstract

during the immature phase. Histological observations
revealed that females in all groups had oocytes thatAmmonia excretion in marine teleosts accounts forcompleted the accumulation of yolk globules at the

70 to 90% of their total nitrogen excretion. Ammonia

spawning period. However, the mean diameter ofis toxic to fish and is a major factor limiting fish

most advanced ovarian follicles in the restrictedbiomass and stocking density in intensive culture

feeding group during the vitellogenic phase wassystems and aquariums. Quantification of ammonium

significantly smaller as compared with the othernitrogen is important for estimating stocking

groups. Furthermore, plasma estradiol-17β levels inbiomass/density, water flow and size of the biological

the restricted feeding group during the vitellogenic

filter in the culture system. Several studies havephase were significantly lower as compared with thesuggested that ammonia excretion is affected byother groups at the spawning period. These results

several factors such as species, body weight, water

indicate that the restricted feeding during thetemperature and ration size. Although several tunavitellogenic phase alters the gonadal development inspecies such as Pacific bluefin tuna (PBT) andrelationship with the plasma estradiol-17β levels inyellowfin tuna have been reared in aquariums andthe yellowtail females.

research facilities including Seikai National Fisheries
Research Institute, there is a lack of studies aboutammonia excretion in tunas.

In this study, we

19:	Evaluation of nitrogen excretion in young,investigated the effects of body weight and ration

immature and adult Pacific bluefin tunalevel on nitrogen excretion for PBT in captivity.

140
Young (0.67 ± 0.14 kg, n = 50), immature (14.4 ± 1.88

20:	A high density genetic linkage map for

kg, n = 2) and adult (42.9 ± 6.5 kg, n = 2) PBTs were

yellowtail (Seriola quinqueradiata) containing

introduced to experimental land-based tanks from6,275 EST-based SNPs

net cage or rearing tank, and thereafter they wereacclimated to running sea water conditions.

Akiyuki OZAKI＊1, Jun-ya AOKI＊1, Kazuo ARAKI＊1,

Experimental tank size varied according to fish size

Hironori USUKI＊1, Kazunori YOSHIDA＊2, Tsutomu(20 kl for young fish; 65 kl for immature fish and 150

NODA＊2, Takuro HOTTA＊2, Hirotaka MIZUOCHI＊2,

kl for adult fish). The PBTs were fed raw fish or

Taro CHUJO＊2, and Yasuhiro SHIMA＊1,2

artificial feed until the experiments. Before the
National Research Institute of Aquaculture, FRA,

fasting and postprandial experiments, the fish were＊1

deprived of food for 48 hours. Rearing water was

422-1, Nakatsuhamaura, Minamiise-cho, Watarai-gun,sampled every 2 h for the first 12 h, and at 4 hMie, 516-0193, Japan

intervals from 12 to 24 h.

＊2

In the postprandial

Seikai National Fisheries Research Institute, FRA,

experiment, the experimental fish were fed bait fish

122-7, Nunoura, Tamanoura-machi, Goto-shi, Nagasaki,(chub mackerel, Scomber japonicas, or sandlance,

853-0508, Japan

Ammodytes personatus) or artificial feed. Fish feces

E-mail: aozaki@affrc.go.jp

were collected from the tank bottom after 24 h.
Determination of fecal nitrogen and concentration of

Keywords

ammonium, nitrite and nitrate nitrogen was carried

Yellowtail（Seriola quinqueradiata;）,EST-based SNPs,

out on each fecal and water samples. Weight-specificgenetic linkage map, quantitative trait loci (QTL),

ammonia excretion rates of fasted fishes showed an

Affymetrix

inverse relationship with body weight (W). The
relationship for total ammonia nitrogen (TAN) was:

Abstract

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

The marine products industry has developed asmuch as it is for red seabream (unpublished data).

majority of the fishery, which are captured andAlthough postprandial ammonium excretion rate was

directly using of aquatic resources. Only recently thed ),breeding are considered as important researchlinear regression analysis indicated that TAN

because available of aquatic resources are restrictedexcretion rates increased with ration size: TAN (mggradually. The expectation of aquaculture researchd ) = 332.4・R−179.6 (r =0.87). There was nois getting higher in response to the prediction of

in relation to the ration size (R, mg feed-N W

−1

N W

−1

−1

−1

2

significant difference in the rates between PBT fed

aquatic resources depletion.

the bait fish and the artificial feed. Postprandial fecal

improvement of economic traits are needed, it hopes

nitrogen was positively correlated with ration size.

apply to superior fish breeding, because artificial

Fecal nitrogen was excreted at a level of 0.9-1.9% forjuvenile have a possibility to improve the phenotype

the baitfish and 0.3-1.3% for the artificial feed.

for suited to aquaculture condition in every

Comparing the results of PBT with other fish, thesegeneration. We are researching practical application

values were lower than that of red seabream (12.8%)

about selection of economic important traits fromand pufferfish (16.1%). This study highlights the neednatural genetic resources using yellow tail (Seriola

for effective evaluation of nitrogen loading and water

quinqueradiata) as target species. High density SNP

quality management in PBT rearing facilities andarrays have become the tool of choice for QTL

aquaculture grounds.

mapping, Genome-wide association studies, marker-

Also the genetic

assisted selection (MAS) and genomic selection (GS).
More recently, high-density linkage maps generated
by SNP array data have proven to be crucial for theaccurate assembly of scaffolds and contigs in wholegenome sequencing efforts. Earlier mapping studies

141

Abstracts of Poster
have identified QTL for important commercial traits

application package of LINKMFEX version 2.3. This

including parasite disease resistance, and combining

application can separate originated alleles from malethe resources of a high density genetic map withor female. In order to avoid the error of genotyping,genome sequence data will facilitate the fine mapping

the accuracy of genotypes in their progenies wasof these loci and the identification of candidate genes.

checked from parental male and female alleles.

In this study, Affymetrix SNP array was used to

Genotype data were converted to a backcross format

genotype 460 samples collected across five families

as though the grandparent genotype was unknown.

from wild population in coastal waters of Goto Fukue-

Pairwise analysis was performed, and markers wereisland. To establish EST (expressed sequence tag)sorted in linkage group at a minimum LOD threshold-based SNP array, a cDNA library was generatedof 5.0.

from pooled RNA samples extracted from 11 tissues

retrospectively by examining the assortment offrom a single individual. Sequencing on Roche/454

alleles among linked markers. A total of 6,275 EST-GS FLX platform generated 1,353,405reads. The

based SNPs were mapped to 24 linkage groups. The

sequencing of SNP identification produced 570,846

total distance covered by the male and female maps

raw reads derived from the full-length library andwere 1,230cM and 1,031cM. This map is currently

456,482 raw reads derived from the 3’-anchoredbeing used to map QTL for a number of commerciallylibrary derived from 5 hundred juveniles. Quality –important traits, and will be used to improve thebased variant calling using CLC Genomics

assembly of the yellowtail genome. It is possible toWorkbench detected 9,356 biallelic putative SNPs in

rapidly develop domesticated strains having

6,025 contigs, with a minor allele frequency (MAF)

commercially important traits in yellowtail>25%. A Linkage analysis was performed using

aquaculture.

Linkage phases were determined