Preface
The 41st Joint Meeting of the UJNR Aquaculture Panel was conducted from October 2013
(October 9 th ‑ 13th at Sapporo and Hakodate, Hokkaido) through December 2013 (December
10th ‑ 13th at Yokohama, Kanagawa and Miyako, Iwate). Through the long history of UJNR,
Aquaculture Panel has contributed to development of aquaculture researches of both countries
by means of various cooperative activities, i.e. the exchange of scientists, the exchange ofliteratures and promoting joint research projects. It is understood that the Aquaculture Panel is
one of the most active UJNR Panels by both countries. Further positive action is expected to the
UJNR Aquaculture Panel for a solution of the varied problems presenting in aquaculture of bothcountries.
The present special issue of Bulletin of Fisheries Research Agency is the proceedings of the 41stUJNR Aquaculture Panel Symposium “Advanced Aquaculture Technologies”. This Symposium
was a venue to exchange original, high-quality research on new and developing technologies thatgive promise to improve or revolutionize aquaculture, based on the key bottleneck issues identiﬁed
at the 39th Scientiﬁc Symposium “The Present and Future of the Aquaculture Industry”.
Further development of aquaculture is highly desired as a production source of importantfoods for human being all over the world. Stable supplies of safe and economical feed ingredientsare requisite for the sustainable development of aquaculture. Nevertheless, in recent years we
recognized some serious problems, which threatened the use of important feed ingredientslike fish meal and oil. It is my great pleasure that the present UJNR proceedings containing
high quality papers of the selected American and Japanese aquaculture scientists will help
integrating aquaculture and fisheries technologies to optimize value from coastal resources,
zoning for aquaculture, use of biotechnology in aquaculture and eﬀects on natural population, andimprovement of public perception.
Finally I would like to express my sincere gratitude to the colleagues involved in the UJNR
Aquaculture Panel for their eﬀorts to prepare and organize the symposium. I also would like todeeply thank the editorial board members for publishing the proceedings.

Fuminari Ito
Chair of UJNR Aquaculture Panel
Executive Director
Incorporated Administrative Agency,
Fisheries Research Agency

CONTENTS
Preface
Contents
Program

Overview of Aquaculture in the US and Japan
1. The Fisheries Agencyʼs policy directions for aquaculture
Kazumasa Ikuta (Counselor, Resource Enhancement Promotion Department
Japan Fisheries Agency, Ministry of Agriculture Forestry and Fisheries)………………………………………………………………………………………………………………

19

2. Aquaculture in the US: The ﬁrst annual national report on aquaculture
Marie Fujitani (NOAA Oﬃce of Aquaculture) ………………………………………………………

21

3. The challenge of reconstructing coho salmon aquaculture after the Great East Japan
Earthquake and Tsunami in 2011
Ikutaro Shimizu (National Research Institute of Fisheries Science, FRA) …………………………

33

Fish Diet and Nutrition
4. Development of a new type of ﬁsh diet, non-ﬁsh meal extruded-pellet
Noriko Ishida (National Research Institute of Fisheries Science, FRA) ……………………………

39

5. The importance of taurine and n-3 fatty acids in cobia nutrition
Aaron Watson (University of Maryland) ………………………………………………………………

51

6. Availability of ﬁsheries by-product materials with cadmium removal treatment as a feed
ingredient for ﬁngerling black rockﬁsh
Nobukazu Satoh (Fisheries Research Department Mariculture Fisheries Research Institute,
Hokkaido Research Organization) ………………………………………………………………………

61

7. Development and characterization of several open formula reference diets for marine ﬁsh larvae
Ken Webb (NOAA, Northwest Fisheries Science Center) …………………………………………

67

8. Eﬀect of feed ingredients on digestive enzyme secretion in ﬁsh
Koji Murashita (National Research Institute of Aquaculture, FRA) ………………………………
9. Cholecystokinin and trypsin responses of larval red drum (69) in response to algae,

live prey, and inert particles
Michael B. Rust (NOAA) …………………………………………………………………………………

75

Management, Social and Economic Issues of the Aquaculture Industry
10. An analysis of the causality between the market price of imported ﬁshmeal and market price ofmarine farmed ﬁsh
Yoshifumi Takahashi (Tohoku National Fisheries Research Institute, FRA) ……………………

79

11. Good aquaculture practices to reduce the use of chemotherapeutic agents, minimize bacterial
resistance, and control product quality
Stanley Serﬂing (Food and Drug Administration Oﬃce of Food Safety) …………………………

83

12. Modeling intraspeciﬁc genetic eﬀects for management of aquaculture programs
Jason Volk (ICF International) …………………………………………………………………………

89

Techniques of Aquaculture Production
13. Speciﬁc monovalent and divalent ion supplementation for culture of marine species
Chris Green (Louisiana State University Agricultural Center) ……………………………………

97

14. Genetically modiﬁed salmon in aquaculture: well regulated and safe
Paul G. Olin (UC San Diego/ Scripps Institution of Oceanography) ……………………………… 109
15. Reproductive dysfunction in cultured sableﬁsh ()

Jose Guzman (NOAA) …………………………………………………………………………………… 111
Invertebrate Aquaculture I: Echinoderm
16. Induced spawning in the sea cucumber

by neuropeptide, cubifrin

Keisuke Yamano (National Research Institute of Aquaculture, FRA) …………………………… 121
17. Mass production of artiﬁcial seed of the Japanese common sea cucumber() in Hokkaido, Japan
Yuichi Sakai (Fisheries Research Department Mariculture Fisheries Research Institute,
Hokkaido Research Organization) ……………………………………………………………………… 129

18. Fluorochrome marking of out-planted green sea urchins,,

for sea ranching and restocking programs in the Gulf of Maine, USA
Stephen Eddy (University of Maine, Center for Cooperative Aquaculture Research) ………… 135
19. Improving the food quality of sea urchin gonads by suppressing gametogenesis
Tatsuya Unuma (Hokkaido National Fisheries Research Institute, FRA) ………………………… 145
20. Maturation control of the short-spined sea urchin,, by lowtemperature rearing using deep-sea water, with the aim of extending the market season
Takaaki Kayaba (Kushiro Fisheries Research Institute, Hokkaido Research Organization) …… 155
Invertebrate Aquaculture II: Mollusc
21. Long-term outcomes in the tech transfer of scallop spat collection techniques,from Aomori Prefecture, Japan to Maine, USA
Dana Morse (Maine Sea Grant) ………………………………………………………………………… 167
22. Oyster culture in Hokkaido, Japan
Natsuki Hasegawa (National Research Institute of Aquaculture, FRA) …………………………… 173

The 41st Scientiﬁc Symposium of the UJNR Aquaculture Panel

October 9th

10th, 2013

Advanced Aquaculture Technologies
ProgramWednesday, October 9th, 2013

Registration

13：00‑17：00

Welcome and opening remarks（Masaaki Fukuda, Director,
Hokkaido National Fisheries Research Institute, FRA）……………………… 13：30‑13：45
Aim of the Symposium（Katsuma Hanafusa, Japan Panel Chair）……………… 13：45‑14：00
Session Ⅰ. Overview of Aquaculture in the US and Japan（Moderators: P. Olin & Y. Machiguchi）
1. The Fisheries Agencyʼs policy directions for aquaculture
Kazumasa Ikuta（Counselor, Resource Enhancement Promotion Department
Japan Fisheries Agency, Ministry of Agriculture Forestry and Fisheries）…………………………………………………………………………… 14：00‑14：20
2. Aquaculture in the US: The ﬁrst annual national report on aquaculture
Marie Fujitani（NOAA Oﬃce of Aquaculture） ……………………… 14：20‑14：40
3. The challenge of reconstructing coho salmon aquaculture after theGreat East Japan Earthquake and Tsunami in 2011
Ikutaro Shimizu（National Research Institute of Fisheries Science, FRA）…………………………………………………………………………… 14：40‑15：00
Discussion ……………………………………………………………………………… 15：00‑15：10
Break …………………………………………………………………………………… 15：10‑15：20
Session Ⅱ. Fish Diet and Nutrition（Moderators: K. Webb & K. Murashita）
4. Development of a new type of ﬁsh diet, non-ﬁsh meal extruded-pellet
Noriko Ishida（National Research Institute of Fisheries Science, FRA）…………………………………………………………………………… 15：20‑15：40
5. The importance of taurine and n-3 fatty acids in cobia nutrition
Aaron Watson（University of Maryland）……………………………… 15：40‑16：00
6. Availability of ﬁsheries by-product materials with cadmium removal
treatment as a feed ingredient for ﬁngerling black rockﬁsh
Nobukazu Satoh（Fisheries Research Department Mariculture
Fisheries Research Institute, Hokkaido Research Organization）…… 16：00‑16：20
7. Development and characterization of several open formula reference
diets for marine ﬁsh larvae
Ken Webb（NOAA, Northwest Fisheries Science Center） ………… 16：20‑16：40

8.

Eﬀects of diets supplemented with herb essential oils on ectoparasitic
protozoan infections on chum salmon（）fry
Shinya Mizuno（Salmon and Freshwater Fisheries Research Institute,
Hokkaido Research Organization） ……………………………………… 16：40‑17：00

Discussion ……………………………………………………………………………… 17：00‑17：20
Thursday, October 10th, 2013

Registration

9：00‑ 9：20

Session Ⅱ. Fish Diet and Nutrition; continued（Moderators: A. Watson & N. Satoh）
9. Eﬀect of feed ingredients on digestive enzyme secretion in ﬁsh
Koji Murashita（National Research Institute of Aquaculture, FRA）… 9：20‑ 9：40
10. Cholecystokinin and trypsin responses of larval red drum（）
in response to algae, live prey, and inert particles
Michael B. Rust（NOAA）（Presented by Ken Webb） ……………… 9：40‑10：00
Discussion ……………………………………………………………………………… 10：00‑10：10
Session Ⅲ. Management, Social and Economic Issues of the Aquaculture Industry（Moderators: M. Fujitani & I. Shimizu）
11. An analysis of the causality between the market price of imported ﬁshmeal andmarket price of marine farmed ﬁsh
Yoshifumi Takahashi（Tohoku National Fisheries Research Institute, FRA）…………………………………………………………………………… 10：10‑10：30
12. Good aquaculture practices to reduce the use of chemotherapeutic agents,
minimize bacterial resistance, and control product quality
Stanley Serﬂing（Food and Drug Administration Oﬃce of Food Safety）（Presented by M. Fujitani） ……………………………………………… 10：30‑10：50
13. Modeling intraspeciﬁc genetic eﬀects for management of aquaculture programs
Jason Volk（ICF International） ………………………………………… 10：50‑11：10
Discussion ……………………………………………………………………………… 11：10‑11：20
Group Photo Session ………………………………………………………………… 11：20‑11：40
Lunch Break…………………………………………………………………………… 11：40‑13：00
Session Ⅳ. Techniques of Aquaculture Production（Moderators: J. Volk & K. Okuzawa）
14. Speciﬁc monovalent and divalent ion supplementation for culture of marine species
Chris Green（Louisiana State University Agricultural Center） …… 13：00‑13：20
15. Genetically modiﬁed salmon in aquaculture: well regulated and safe
Paul G. Olin（UC San Diego/ Scripps Institution of Oceanography）… 13：20‑13：40
16. Reproductive dysfunction in cultured sableﬁsh（）
Jose Guzman（NOAA） …………………………………………………… 13：40‑14：00

Discussion ……………………………………………………………………………… 14：00‑14：10
Session Ⅴ. Invertebrate Aquaculture I: Echinoderm（Moderators: S. Eddy & T. Unuma）
17. Induced spawning in the sea cucumber
byneuropeptide, cubifrin
Keisuke Yamano（National Research Institute of Aquaculture, FRA）…………………………………………………………………………… 14：10‑14：30
18. Mass production of artiﬁcial seed of the Japanese common sea cucumber（）in Hokkaido, Japan
Yuichi Sakai（Fisheries Research Department Mariculture Fisheries
Research Institute, Hokkaido Research Organization） ……………… 14：30‑14：50
Break …………………………………………………………………………………… 14：50‑15：00（Moderators: D. Morse & K. Yamano）
19. Fluorochrome marking of out-planted green sea urchins,,
for sea ranching and restocking programs in the Gulf of Maine, USA
Stephen Eddy（University of Maine, Center for Cooperative
Aquaculture Research）…………………………………………………… 15：00‑15：20
20. Improving the food quality of sea urchin gonads by suppressing gametogenesis
Tatsuya Unuma（Hokkaido National Fisheries Research Institute, FRA）…………………………………………………………………………… 15：20‑15：40
21. Maturation control of the short-spined sea urchin,,by low temperature rearing using deep-sea water, with the aim of extending
the market season
Takaaki Kayaba（Kushiro Fisheries Research Institute,
Hokkaido Research Organization） ……………………………………… 15：40‑16：00
Session Ⅵ. Invertebrate Aquaculture II: Mollusc（Moderators: C. Green & Y. Sakai）
22. Long-term outcomes in the tech transfer of scallop spat collection techniques, from
Aomori Prefecture, Japan to Maine, USA
Dana Morse（Maine Sea Grant） ………………………………………… 16：00‑16：20
23. Relationship between spat density and environmental factors in cultured Japanese
Scallop,in Funka Bay, Japan.
Katsuhisa Baba（Hakodate Fisheries Research Institute,
Hokkaido Research Organization） ……………………………………… 16：20‑16：40
24. Oyster culture in Hokkaido, Japan
Natsuki Hasegawa（National Research Institute of Aquaculture, FRA）…………………………………………………………………………… 16：40‑17：00
Discussion ……………………………………………………………………………… 17：00‑17：10
Closing remarks（Paul Olin, U.S.A. Panel Chair） ……………………………… 17：10‑17：20

水研センター研報，第40号，19，平成27年
Bull. Fish. Res. Agen. No. 40，19，2015

19

The Fisheries Agencyʼs Policy Directions for Aquaculture
Kazumasa IKUTA＊1

Abstract: Aquaculture is an important industry in Japan, and production levels occupy
approximately 20% in terms of yield, and 30% in terms of market value, of the countryʼs totalﬁsheries production. However, the economic aspects of aquaculture have been precarious due tothe low price of certain products and increasing costs of production. In addition, the guarantee offood safety, minimization of environmental impact, and management of natural stock populations
are highly necessitated in order to achieve the sustainability of the industry.
In order to address these problems, the Fisheries Agency held a series of committee meetings
during 2013 in order to develop ideal projections for the aquaculture industry. Thereafter, policy
directions for aquaculture were put forth as follows:
1) Measures to improve unstable business practices of aquaculture should be implemented; thisincludes the expansion of mutual-aid systems, conversion of feed resources to alternative
usages of protein, maintenance of carrying capacity, improvement of systems to compensate
for increases in feed costs, and the promotion of planned production including the enhanced
exportation of products.
2) Measures to improve production techniques should be implemented; this includes achieving
improved food safety based on production systems that have high traceability, risk
management and the usage of vaccines, conservation of the environment in and aroundaquaculture grounds, a shift from the use of wild ﬁsh seedlings and natural feed resources tothe use of artiﬁcially-produced seedlings and feeds, the development of enclosed recirculating
aquaculture systems, and R&D for advanced aquaculture technologies that will reduce
costs and enhance productivity such as offshore aquaculture systems, enhanced breeding
methodologies, and raft aquaculture systems for new bivalve species.

2015年 1 月30日受理（Received on January 30, 2015）＊1
Counselor, Resource Enhancement Promotion Department, Japan Fisheries Agency, Ministry of Agriculture Forestry and Fisheries
E-mail: ikutak@aﬀrc.go.jp

水研センター研報，第40号，21−31，平成27年
Bull. Fish. Res. Agen. No. 40，21−31，2015

21

First Annual Report on Aquaculture and Policy in the United States ofAmerica for the 2013 UJNR PanelMarie L. Fujitani＊, Robert C. Jones＊, and Mike B. Rust＊

Abstract: The ﬁrst U.S. National Report on Aquaculture for the 41st U.S. Japan Natural Resources
Panel on Aquaculture overviews major policy items, current trends in aquaculture production, andother important developments aﬀecting contemporary U.S. aquaculture.
Policy- April 2013 saw the release of the National Ocean Policy Implementation Plan (NOPIP), a document to translate President Obamaʼs 2010 National Ocean Policy (Executive Order
13547 --Stewardship of the Ocean, Our Coasts, and the Great Lakes) to specific federal actions.
Aquaculture featured prominently in the Implementation Plan. The Joint Sub-committee on
Aquaculture (JSA) was re-named the Interagency Working Group on Aquaculture (IWG-A),
and was tasked with identifying and supporting milestones in the NOP-IP. The NOP-IP also
supported the National Shellﬁsh Initiative to increase shellﬁsh production and restoration in U.S.
waters. NOAA and the Gulf of Mexico Fishery Management Council are working on a Fishery
Management Plan to permit aquaculture in federal waters in the Gulf of Mexico. The U.S.
currently does not have a framework to permit aquaculture in the EEZ, and this would be a ﬁrst
for the U.S. if it goes into eﬀect.
Developments- For the ﬁrst time in some years, in 2013 many new sites for shellﬁsh aquaculture
have been permitted. This is attributed to increased interest in shellﬁsh aquaculture, particularlyin the northwest and northeast, federal interagency and state eﬀorts on behalf of the Washington
Shellfish Initiative, and state-level successes at streamlining permitting. In September 2013,
the California Shellfish Initiative was launched. A program is underway to permit and restore
Hawaiian ﬁshponds, a form of traditional Hawaiian aquaculture, for cultural heritage, subsistence,and possibly commercial production.
Production Trends- Aquaculture production in the U.S. is largely composed of catﬁsh, crawﬁsh,
trout, salmon, oysters, mussels, clams, tilapia, and shrimp. For the 15-year period from 1996‑2011,
the value of U.S. aquaculture showed 3% average year-over-year growth, though total volume
fell by an average of 0.5%. Mariculture (salmon and shellﬁsh), on the other hand, showed robust
4% by volume and 5% by value year-over- year average growth in the same period. 2011 is themost current year published aquaculture statistics are available. From 2010 to 2011, total U.S.
aquaculture went down ‑18.8% by volume (to 277,335 metric tons) but grew 4.2% by value (to1.34billion dollars).

From 2010 to 2011 mariculture decreased by ‑12.4% by volume (to 35,739

metric tons) while growing 0.3% by value (to 314 million dollars). However, in the previous year
from 2009‑2010, mariculture grew 21.6% by volume (to 40,823 metric tons) and 28.5% by value (to312 million dollars). Most of the growth in value for marine aquaculture was from salmon () and oyster (mainlyand) production.

Keywords: aquaculture policy, aquaculture production, annual report

2015年 1 月30日受理（Received on January 30, 2015）＊
NOAA Oﬃce of Aquaculture, 1315 East-West Highway, Silver Spring, MD 20910
E-mail: NMFS.aquaculture.science@noaa.gov

22Marie L. Fujitani, Robert C. Jones, and Mike B. Rust
Backgroundand business opportunities.
The National Ocean Policy Implementation Plan:

The U.S. imports over 90 percent of its seafood,In 2010 President Obama signed the Nationalhalf of which is a product of foreign aquaculture.

Ocean Policy (NOP; Executive Order 13547

While aquaculture produces 50 percent of all seafood--Stewardship of the Ocean, Our Coasts, and theeaten worldwide, in 2011 U.S. domestic aquaculture

Great Lakes, http://www.whitehouse.gov/the-production was only 9% of total domestic landings.

press-office/executive-order-stewardship-ocean-

Total domestic aquaculture in the U.S. provided

our-coasts-and-great-lakes).

around 5% of the seafood supply; mariculture was

National Ocean Policy Implementation Plan (NOP-

the source of less than 1% of U.S. seafood.

IP, http://www.whitehouse.gov/administration/eop/

In April 2013 theoceans/implementationplan) was released, which
Policycharges federal agencies to work together and takespecific actions to address ocean issues of national

The Department of Commerce, NOAA Fisheries,

importance. Aquaculture is featured prominently inand Aquaculture: Marine aquaculture is an

the Implementation Plan.

important part of the missions of the Department

The aquaculture-specific actions in the NOP-IP

of Commerce (DOC) and the National Oceanic

relate to increasing efficiencies in the aquacultureand Atmospheric Administration (NOAA). The

permitting processes, coordinating agency

DOC sees aquaculture as a way to create jobs andparticipation, and providing jobs and economic

economic activity. A federal agency under the DOC,

value by protecting and restoring coastal wetlands,

NOAA sees aquaculture as a critical component tocoral reefs, and other natural systems. Along withmeeting increasing global demand for seafood andgoals for commercial aquaculture, restoration is also

maintaining healthy ecosystems.

specifically mentioned in the NOP-IP, to improve

The NOAA Office of Aquaculture is within
the National Marine Fisheries Service (NMFS), a

coastal and estuarine restoration efforts throughbetter monitoring, coordination, and planning.

branch of NOAA. NMFS is involved in aquaculture
from both science and policy perspectives: scienceto foster efficiency and sustainability and policy
to enable marine aquaculture while ensuring
environmental responsibility. Marine aquaculture isan increasingly important part of the NOAA NMFS
mission. Marine aquaculture is providing a growing
amount of seafood to U.S. consumers, and supportscommercial and recreational fisheries and helps
restore species and habitat.
Aquaculture is garnering increasing attention
from Commerce, members of Congress, and theprivate business sector for its ability to provide
economic opportunities (especially in fishingcommunities) and potential to grow more seafood

Agency Abbreviations:
NOAA: National Oceanic and Atmospheric Administration
USDA: U.S. Department of Agriculture
ARS: Agricultural Research Service
NIFA: National Institute of Food and Agriculture
APHIS: Animal and Plant Health Inspection Service
EPA: Environmental Protection Agency
USCG: U.S. Coast Guard
DOC: Department of Commerce
DOI: Department of the Interior
DOL: Department of LaborUSFWS: U.S. Fish and Wildlife Service
FERC: Federal Energy Regulatory Commission
DOE: Department of Energy
HHS: Department of Health and Human Services
FDA: Food and Drug Administration
ACOE: The U.S. Army Corps of Engineers

domestically in a safe, sustainable way. In 2011,
NOAA and the Department of Commerce released
complimentary aquaculture policies (http://www.
nmfs.noaa.gov/aquaculture/policy/2011̲policies̲
homepage.html) supporting aquaculture to enable

Develop and implement permitting regulatory

domestic production of seafood, maintain and restore

efficiencies for aquaculture. [NOAA, USDA,

healthy marine ecosystems, and create employment

EPA, USACE, USCG, DOI (USFWS)]

2013 US Annual Report on Aquaculture for UJNR

23

Establish an interagency aquaculture initiative

council. The purpose of the working group is tothat supports jobs and innovation through theincrease the overall eﬀectiveness and productivity of

National Science and Technology CouncilʼsFederal aquaculture research, technology transfer,

Interagency Working Group on Aquaculture

and technology assistance programs. Efficient,and other partnerships. [DOC, NOAA, USDA

coordinated permitting processes will allow ocean(ARS, NIFA)]

industries, including commercial shellfish andThrough leveraging existing research priorities,

finfish aquaculture, to save time and money andprovide scientific information on the environ-encourage economic growth without compromisingmental health effects of finfish aquaculture

federal agency responsibilities to protect health,to streamline permitting and improve watersafety, and the environment. Improved interagency

quality monitoring. [Aquaculture Regulatory

coordination and reduced redundancy will decrease

Task Force]

administrative waste, the burden on federal agencies,

Through the National Shellﬁsh Initiative develop

and the regulatory burden for industry.

pilot projects to identify ways to both maxi-mize

Under the IWG-A, an Aquaculture Regulatory

the environmental sustainability and ecosystem

Task Force (hereafter ʻtask forceʼ) was created inbenefits (e.g., nutrient filtration, carbon

2013under the auspices of the National Aquaculture

sequestration, ﬁsh habitat) and the commercial

act of 1980, and will address speciﬁc milestones andvalue of shellﬁsh aquaculture. This would help

activities in support of the Implementation Plan

develop a comprehensive plan to sustainablyof the National Ocean Policy in consultation andincrease shellﬁsh production and restore popula-partnership with the National Ocean Council andtions in U.S. waters. [NOAA, USDA (ARS,

the IWG-A. The task force consists of the following

NIFA)]

federal agencies:

The USDA: ARS, NIFA, and

APHIS; DOC: NOAA; HHS: FDA; DOI: USFWS;
Identify and make available best management

EPA; and the ACOE. The task force also includes

practices to inform and improve Federal

the following organizations in the Executive Officepermitting processes for aquaculture. [NOAA,

of the President: National Ocean Council;Office of

USDA, EPA, USACE, USCG, DOI (USFWS)]Management and Budget; and Oﬃce of Science andDevelop an analysis of the contribution andTechnology Policy. The task force will operate from

impacts (including job creation) of emerging

2013‑2015 unless renewed.

uses̶including renewable energy, aquaculture,

In 2013 the task force assembled a draft charterand biotechnology̶on the economies of theto support aquaculture milestones in the NOP-IP, ascommunities and regions dependent on marine

well as a draft work plan identifying speciﬁc actions

and coastal resources. [NOAA, DOE, DOI,

towards task force goals. Progress has been made on

FERC, DOL, DOC]

facilitating inter-agency communication, coordination
to streamline multi-agency permitting processes, andDevelop and incorporate adaptation strategies

increasing the ease of access to information about

for coastal and ocean species and habitats into

the permitting process.

future planning and management processes,

The Gulf of Mexico Fishery Management Plan: The

such as ﬁsheries, protected species, coral reefs,

U.S. currently does not have a framework to permit

or shellﬁsh aquaculture. [NOAA, DOI, EPA]

long-term aquaculture projects in the Exclusive
Economic Zone (federal waters), 3 to 200 miles (5The Inter-Agency Working Group on Aquaculture

to 322 kilometers) oﬀshore. This is despite the huge

(IWG-A): The Inter-agency Working Group on

potential for offshore aquaculture in the U.S. A

Aquaculture (IWG-A; formerly called the Joint

recent publication from the Food and Agricultural

Sub-committee on Aquaculture) is an inter-agency

Organization (FAO) of the United Nations (Kapetskygroup under the auspices of the Life Sciences Sub-., 2013) listed the US as number one in the worldcommittee of the National Science and Technology

for oﬀshore marine production potential. Japan also

24Marie L. Fujitani, Robert C. Jones, and Mike B. Rustranked highly (ﬁfth) in this category. The rankings

Shellfish Initiative Inter-agency Working Group towere obtained by accounting for the area in eachcoordinate and streamline the permitting process incountryʼs Exclusive Economic Zone with appropriatethat state led to 7 new shellﬁsh site permits, the ﬁrst

depth, currents, temperature, and distance to ports

issued by the ACOE in six years.

for ﬁnﬁsh (e.g. salmon and cobia) and shellﬁsh (e.g.
mussels) production.

The Washington State Shellﬁsh Initiative also led
to the Ocean Acidiﬁcation Blue Ribbon panel report

An ongoing development for U.S. offshoreand new funding from the State of Washington

aquaculture is the Fishery Management Plan forfor shellfish ocean acidification research. The Blue

Regulating Offshore Marine Aquaculture in the

Ribbon Panel gave a summary of ocean acidiﬁcation

Gulf of Mexico (Aquaculture FMP). The Gulf ofresearch, and a summary of recommendations for

Mexico Fishery Management Council proposedshellﬁsh hatcheries and growers. Ocean acidiﬁcation

regulations for an Aquaculture FMP, the ﬁrst timeis of major concern on the west coast, particularlythat a regional fishery management council has

in the Pacific northwest. In previous years before

approved a comprehensive regulatory program forocean acidification was identified as the culprit

offshore aquaculture in U.S. federal waters. NOAA

and mitigation measures were employed, someis currently reviewing the proposed regulations that

hatcheries experienced up to 80% larval mortality.

would implement the Aquaculture FMP. The FisheryResearch led to the finding that acidic water fromManagement Plan authorizes NOAA Fisheries to

upwelling put stress on larval shellfish, rendering

issue permits to culture species managed by thethem unable to build shells in a critical point in their

Gulf of Mexico Fishery Management Council, with

development. Hatcheries now have sensors and only

the exception of shrimp and corals. We expect ﬁnal

draw seawater when the pH level is ʻsafeʼ for larvae.

regulations to be eﬀective sometime in 2014.

However, this does not help with shellfish in thewild, and some bays have not seen a natural set in

Recent Key Developments in U.S. Aquaculture

almost ten years. The Washington Shellﬁsh initiative
also led to funding construction of a hatchery in

National and Regional Shellfish Initiatives: The

Manchester, WA, for native Olympia oyster seed.

National Shellfish Initiative is a policy created

In September 2013, the California Shellfish

in conjunction with the 2011 DOC and NOAA

Initiative was launched with a kickoff meeting

Aquaculture Policies. The goal of the initiative isinvolving agencies and stakeholders to identify

to support increases in shellfish production and

key actions for expanded shellfish farming andrestoration in U.S. waters. In 2013 the Office of

restoration to support and conserve Californiaʼs

Aquaculture and aquaculture regional coordinators

unique ecosystem. Over 70 people attended. One

supported a number of actions for the National

outcome of this meeting was coordination between

Shellﬁsh Initiative in partnership with other NOAANMFS staff and the Humboldt Harbor District on

offices, industry, the restoration community, andshellfish aquaculture in Humboldt Bay, California.

state and tribal partners in California, Washington

The Harbor District is pursuing an innovative

State, the Northeast, and Hawaii.

approach for permitting, where the Harbor District

The Washington State Shellﬁsh Initiative, throughowns a regional permit for the entire bay, bears

the actions of the Washington State Shellfish

the cost of the permitting process, and then issues

Initiative Inter-agency Working Group, completedleases to individual growers. This will allow small

several products to streamline the site permitting

businesses to participate in shellfish aquaculture,process. Due to many factors (for example

because the permitting process can be too expensive

regulatory complexities and social license), for aand uncertain for a small business to navigate alone.

number of years there have been difficulties with

The Northeast U.S. has been very successful inpermitting new sites for shellfish aquaculture in

streamlining the permitting process, and is seeingsome regions of the United States, particularly in theimpressive growth in the shellfish sector. For

Paciﬁc Northwest. Actions by the Washington State

example, over the last two years in Maryland, 1,100

25

2013 US Annual Report on Aquaculture for UJNR
acres have been leased for shellfish aquaculture,rock, and allowed for saltwater from the ocean toand 1,500 more acres are in the process. Over

enter and exit the ponds, cleaning them throughthe same period in Virginia, over 5,000 oyster

tidal flushing while preventing fish from escaping.

acres were permitted. The New England region

The most widely cultivated species were flathead(Maine, Vermont, New Hampshire, Massachusetts,mullet (Connecticut, and Rhode Island) is working on aand moi (general regional permit to streamline the permitting

hundreds of fishponds in use across the Hawaiian

process for new shellfish aquaculture operations.

Islands, but by the turn of the 20

In the state of Maine, state agencies and the ACOE

about a hundred remained. Today, most fishponds

are also pursuing a streamlined permitting system.

are in disrepair.

), milkfish ()). There were onceth

century only

Further, due to economic and fishery conditions,

The State of Hawaii, the NOAA Paciﬁc Regional

there are a growing number of fishermen andaquaculture coordinator, and various federal

fishing families in the Northeast who are starting

agencies and the environmental non-governmental

aquaculture in addition to or instead of ﬁshing.

organization Conservation International are

In Hawaii there is growing interest in raisingcollaborating on a general permit to make it possible

oysters. The Hawaii State Department of Healthto restore Hawaiian ﬁshponds for cultural heritage,

just developed a process to certify waters to raisesubsistence, and commercial production. The nextshellfish. The growth rate for oysters in Hawaii isstep is to develop a general permit from the ACOE

high, taking only 6‑8 months to reach market size.

to allow pond restoration.

In fact, there is at least one major land-based oyster

Part of the goal of this project is to introduce a

hatchery operating in Hawaii, which benefits frombroad segment of the population to aquaculture,these high growth rates.

through cultural and outreach activities, and foster

Native Hawaiian Aquaculture Site Permitting:grass-roots familiarity and support for aquaculture

Fishponds built by the original inhabitants of Hawaiiin Hawaii, a state with great marine aquaculture

were among the most sophisticated examples ofpotential.

aquaculture used by pre-historic native Pacificpeoples. Rock walls were built along the shoreline
to form ponds. The walls were made of porous lava

Fig. 1.

26

Marie L. Fujitani, Robert C. Jones, and Mike B. Rust
Production Trends

marine species make up a disproportionately largepart of the value of aquaculture in the U.S. Shellﬁsh

The main species for aquaculture production inin particular are produced in relatively smaller

the U.S. are trout, tilapia, catfish, crawfish, shrimp,

quantities but are high-value products.

salmon, oysters, mussels, and clams. The value of

Mariculture＊1: Major U.S. marine species are Atlantic

U.S. aquaculture showed 3% average year-over-year

salmon (), mussels (mainly blue mussels,), clams (largely hard clams

growth over the last 15 years, though total volume

and Manila clam

fell by an average of 0.5%. The last year data are
available is for 2011. From 2010 to 2011, total U.S.

and geoduck,), and oysters (mainly

andaquaculture went down ‑18.8% by volume (to 277,335). Saltwater

metric tons) but grew 4.2% by value (to 1.34billion

shrimp are excluded from mariculture because they

dollars).

are raised in inshore ponds.

By volume, most of U.S. aquaculture is from land-

From 2010 to 2011 mariculturewent down ‑12.4%

based ponds growing catfish, crawfish, and trout.

by volume (to 35,739 metric tons) while growing 0.3%

Marine species make up a very small proportionby value (to 314 million dollars).

of aquaculture production by volume. However,

2009‑2010, mariculture grew 21.6% by volume (toHowever, from40,823 metric tons) and 28.5% by value (to 312 million
dollars). The 15-year average growth for mariculture
is 4% by volume and 5% by value.
1. Finﬁsh
Atlantic salmon mariculture in the U.S. takes
place in Maine and Washington State. Salmon fromhatcheries are raised in tanks until they reach 40‑
120 grams, and then are transferred to marine
net-pens for grow-out. From 2010‑2011 salmon
production decreased ‑4.8% to 18,595 metric tons,
and grew 5.1% by value to 104 million dollars. The
average year-over-year growth over the last 15
Fig. 2.

Fig. 3.
＊1

years is 6% by volume and 11% by value.

Fig. 4.

We deﬁne Mariculture in this section as species cultured in the marine environment (e.g. oceans, bays, salt-water estuaries).

27

2013 US Annual Report on Aquaculture for UJNROther marine finfish species are cultured, but

clams are 7‑15 mm, they are placed into the final

due to issues such as the small number of farms,

grow-out system until they reach a harvest size of

consistent data are not available. To protect the

50 mm. For grow-out, clams are placed into diﬀerent

privacy of businesses, the USDA ARS cannot

structures (e.g. trays, pens, bags) and secured to thereport detailed statistics on farms when there aresubstrate of intertidal or subtidal areas. Harvesting

so few of them that it would be possible to identify

occurs by either collecting trays/bags or by raking

individual farms from the data. White sturgeon are

the substrate.

cultured (). Species in the

Geoduck seed are produced in hatcheries, andearly stages of commercializationor in research andjuveniles are raised in the hatchery until they reachdevelopment include sixﬁnger threadﬁn (about 5‑12mm in size. Geoduck are next raised), commonly known in Hawaii as moi, cobia((), yellowtail amberjack), Atlantic cod (yellowtail amberjack ((intertidal beach in PVC pipes. The pipe is placed in), red drum

the sediment, about 30cm deep, with 10 cm left at), sableﬁsh (),

the surface. Juvenile geoducks are very susceptible),

to predators and the pipe protects them until they), yellowfin

are large enough to evade predators on their own.

) and Florida pompano

On many farms, a net is placed over all of the tubes

summer flounder (tuna (

survival outdoors. Geoduck seed are planted on),

California flounder ((in a nursery where they grow large enough for).

to provide further protection from predators. Twoto three juveniles are placed into each pipe. While

2. Shellﬁsh

protected, the geoduck will grow and burrow itself

Clam culture in the U.S. is largely hard clam

into the sediment. After 1‑2 years, the netting is(() and Manila clam (removed since the geoducks are large and deep), with some native geoduck clams

enough to be safe from predators. The grow-out) grown in the Paciﬁc Northwest.

stage can take 4 to 7 years for a geoduck to reachHard clam culture in the U.S. involves seedproduction, nursery stage, and final grow-out.

harvest size of 2 pounds.
From 2010‑2011 clam production increased 12%

Producers rely on seed from hatcheries. At theto 4,683 metric tons, and grew 9.3% by value to 104

nursery stage, juvenile clams have a protected

million dollars.The average year-over-year growthenvironment where they can grow to a size thatover the last 15 years is 18% by volume and 13% by

provides optimum success for survival once placedvalue.

into a natural setting for grow-out. Once juvenile

Oyster culture in the U.S. mainly consists of two
species. The Paciﬁc oysteris thepredominant oyster grown on the west coast, andthe Virginia oyster () is the mostcommonly raised oyster on the east coast. However,on both coasts other species are raised in smaller
numbers, such as the native Olympia oyster () on the west coast. The majority ofoyster farms rely on hatchery production of seed,though wild seed collection also occurs. Oysters
are grown by on-bottom, off-bottom, or suspendedculture techniques. On-bottom culture is used whena site has a suitably ﬁrm substrate in an intertidal
or sub-tidal area. Off-bottom culture requires seed
to be put into mesh bags or trays and attached toFig. 5.

rope and wood frames in the intertidal zone to be28

Marie L. Fujitani, Robert C. Jones, and Mike B. RustFig. 6.

Fig. 7.

grown to market size. Suspended culture is where
oyster seed is hung from longlines and attached tohorizontal lines or rafts in deeper waters. Harvest
typically occurs at a size of around 75mm.
From 2010‑2011 oyster harvest dropped ‑27.8%
to 12,062 metric tons, and ‑11.9% by value to 98.4
million dollars. However, in the previous year,
compared to 2009, 2010 saw 15.0% growth byvolume to 16,721 metric tons, and 26.4% growth byvalue to 111.7 million dollars. The average year-overyear growth over the last 15 years is 6% by volume
and 6% by value.
Mussels are farmed either on- or off-bottom. On-

Fig. 8.

bottom mussel farming involves seeding sea bottom
areas, and dredging mussels for harvest. There areseveral methods of off-bottom farming, including

19.7% by volume (to 241,596 metric tons) while

longline culture and raft culture. From 2010‑2011

growing 7.2% by value (to 737 million dollars). The

mussel production decreased ‑0.7% to 402 metricaverage year-over-year growth over the last 15

tons, and grew 9.3% by value to 7.2 million dollars.

years is ‑0.3% by volume and 3% by value.

The average year-over-year growth over the last 15
1. Finﬁshyears is 8% by volume and 20% by value.

Channel catfish () are the＊2Inland Aquaculture : Major U.S. inland finfish

predominant aquacultured species in the U.S.,

aquaculture species are channel catfish (and only Channel catfish can be sold as ʻcatfishʼ), Rainbow trout (),

in U.S. markets. Catfish are grown primarily in),embankment or levee ponds, where the earth that is). Farmed shellfish include

removed to dig the ponds is used to build levees or

hybrid striped bass (and tilapia (the red swamp crawfish (whiteleg shrimp (

) and
).

From 2010 to 2011 inland aquaculture went down
＊2

embankments to encircle the ponds. Ponds range insize from 0.04 km2 and 2m deep to 0.1 km2 and 1m
deep.

In this section, inland aquaculture refers to the culture of freshwater species as well as marine species grown in land-based systems.

292013 US Annual Report on Aquaculture for UJNR

Fig. 9.

Fig. 11.

Fig. 10.

Fig. 12.

From 2010 to 2011 catﬁsh production went down-(to 51 million dollars). The average year-over-year

27.3% by volume (to 157,942 metric tons) while

growth over the last 15 years is ‑3% by volume andgrowing 4.2 % by value (to 391 million dollars). The

0% by value.

average year-over-year growth over the last 15

Hybrid striped bass are produced in the hatchery

years is ‑1% by volume and 1% by value.
Rainbow trout (by fertilizing female white bass (
) are

)

eggs with sperm from male striped bass (predominantly raised in raceways, though some). Hybrid striped bass have greater tolerance

farms use ponds or recirculating systems. Raceways,

to extremes in temperature and dissolved oxygen

also known as ﬂow-through systems, are essentiallythan either of its parents and is thus better suited

artiﬁcial streams. Gravity ﬂows water from naturalfor pond culture.The majority of hybrid striped bass

streams through the concrete raceways, and the

producers in the southern region raise their ﬁsh inwater is often filtered to remove waste products

freshwater ponds, but cages and tanks are also used

before being released back into the natural stream.

for hybrid striped bass production.

Steelhead trout () are sea-

From 2010 to 2011 hybrid striped bass production

run rainbow trout, and can be raised in net-pens indecreased 9.1% by volume (to 3,516 metric tons)

rivers and the marine environment. From 2010 towhile growing 1.4% by value (to 28 million dollars).

2011 trout production went down ‑1.8% by volume

The average year-over-year growth over the last 15(to 15,112 metric tons) while increasing 7.9% by value

years is 1% by volume and 3% by value.

30Marie L. Fujitani, Robert C. Jones, and Mike B. Rust

Tilapia are grown in ponds or tanks in the U.S., andmaterial to feed upon, they are often double-cropped

production did not change between 2010 and 2011,or rotated with field crops, most commonly rice

though there was an increase in value of 1.7%. In

but also soybeans and sorghum. From 2010 to 20112011, 9,979 metric tons of tilapia worth 54 million

crawﬁsh production grew 0.9% by volume (to 53,435

dollars were produced. The average year-over-year

metric tons) while growing 15.9% by value (to 206

growth over the last 15 years is 2% by volume andmillion dollars). The average year-over-year growth

7% by value.

over the last 15 years is 13% by volume and 17% byvalue.

2. Shellﬁsh

Shrimp in the U.S. are mostly wild-caught. However,

Crawfish are the predominant cultured crustaceanboth marine (penaeidae) and freshwater shrimp

in the U.S., and are cultivated and consumed forspecies are grown in ponds, tanks, and recirculating

food in several southern states. Louisiana dominates

systems. Whiteleg shrimp (the aquaculture and wild harvest crawﬁsh industry.

the most commonly cultured shrimp species, though

Crawfish are grown in shallow ponds 20 to 60 cm

culture of the Malaysian prawn () aredeep, and ponds are ﬂooded and drained each year.

) in land-based systems is increasingly

Crawfish in the U.S. are not fed formulated feeds,

popular. From 2010 to 2011 shrimp aquaculture grew

and instead primarily consume plant material as

19% by volume (to 1,612 metric tons) while growing

forage. In order to provide crawfish with plant

3.2% by value (to 6.1 million dollars). The average
year-over-year growth over the last 15 years is 5%
by volume and ‑0.6% by value.
3. Other land-based culture of marine species
There are several ongoing experimental andcommercial land-based marine finfish culture
operations in the U.S.: notably barramundi () has been commercialized in a large
land-based facility in Massachusetts. Several nonnative species of sturgeon () have been cultured in commercial
and demonstration projects in Florida (e.g.
Mote Marine Laboratory). Additionally, work to

Fig. 13.

commercialize cobia (black sea bass () and) continues inland based recirculating systems in Virginia.
Annotated Bibliography
Lowther A. (editor), 2013: Fisheries of The UnitedStates 2012. Current Fishery Statistics No. 2012.
National Marine Fisheries Service Office of
Science and Technology.
http://www.st.nmfs.noaa.gov/Assets/commerc
ial/fus/fus12/FUS2012.pdf
The annual Fisheries of the United States is theoﬃcial published source of ﬁsheries statistics for the
US. The 2012 report gives preliminary 2012 data
Fig. 14.

on US commercial ﬁsheries, and the ﬁnal report for2013 US Annual Report on Aquaculture for UJNR
2012 on US recreational fisheries. US aquaculture
statistics are reported for 2011. Summary statistics
are also provided on US production of processedfishery products, domestic supply and per capita
consumption, foreign trade, and world fisheries
production.
Kapetsky J. M., Aguilar Manjarrez J., and JennessJ., 2013: A global assessment of potential foroﬀshore mariculture development from a spatial
perspective. FAO Fisheries and Aquaculture
Technical Paper No. 549. Rome, FAO. 181 pp
The FAO produced this document to highlightthat mariculture, specifically offshore aquaculture,offers significant opportunities for sustainable foodproduction but is underutilized worldwide. The
report states that oﬀshore aquaculture could foster
development of many coastal communities, especiallyin regions with limited freshwater and arable land.
This report measures and compares for all maritime
nations the current status and potential for oﬀshore
mariculture development from a spatial perspective,
and identifies nations with high but unrealizedoﬀshore potential.

31

水研センター研報，第40号，33−38，平成27年
Bull. Fish. Res. Agen. No. 40，33−38，2015

33

The Challenge of Reconstructing Coho Salmon Aquaculture after theGreat East Japan Earthquake and Tsunami in 2011
Ikutaro SHIMIZU＊1, Tsuyoshi TANAKA＊2, Hideki MIURA＊3, and Kohichi SAOTOME＊4

Abstract: The wholesale price of farmed salmon in local ﬁsh markets showed higher prices before
the tsunami in 2011. Though farmed coho salmon started being landed again by the support ofnational funding in 2012, the price of the farmed salmon slumped. A higher price is necessary
to maintain the farming facilities for management. This paper aims to clarify the causes of theprice drop of Sanriku coho salmon and the issues in the reconstruction process of coho salmon
aquaculture. The most important cause of the price drop of Sanriku coho salmon was that
consumersʼ purchasing patterns changed from Sanriku coho salmon to imported salmon due tothe lack of Sanriku coho salmon in 2011. We hope to supply fresh Sanriku coho salmon at prices
between imported fresh Atlantic salmon and frozen rainbow trout in consumer markets. It isnecessary to improve the traditional aquaculture system and to develop brand value for Sanriku
coho salmon. Sales promotion is expected to drive the price of the fresh coho salmon up to a fairlevel.
Key words: coho salmon, aquaculture, Miyagi prefecture, tsunami, reconstructing, and priceanalysis

Aquaculture of coho salmon (farmed salmon slumped (less than 200 yen/kg)

in) is one of the most important fisheries

2012 (Fig. 2). Prices higher than 370 ‑ 380 yen/in Miyagi prefecture, Tohoku, where the annual

kg is necessary to maintain the farming facilities

production was more than 10,000 tons until 2010

and bread even. Understanding the causes of the(Fig. 1). However, the Great East Japan Earthquake

slump is necessary to reconstruct Sanriku salmon

hit the Paciﬁc coast area of eastern Japan on Marchaquaculture.

11 in 2011. The tsunami destroyed nearly all theThis paper clarifies the causes of the pricefarming facilities, local ﬁsh markets, and processing

drop of Sanriku coho salmon and the issues

firms in the Sanriku region, and suspended cohoin the reconstruction process of coho salmon

salmon farming. One year later, farmed coho salmon

aquaculture.

started being landed again with help from national

cooperatives, farming facilities, local fish markets,

funding. Fortunately, the tsunami did not aﬀect theseafood processing ﬁrms, and wholesale markets in

We conducted interviews at ﬁsheriescoho salmon juveniles which were located inland.

Miyagi prefecture in September and October 2012,

While the wholesale prices of the farmed salmon in

and general merchandising stores in the Tokyo

local ﬁsh markets showed higher prices (more thanarea in November 2012. In addition, the relational

400 yen/kg) before the tsunami, the price of theexpressions of salmon price and ﬁshmeal price were2015年 1 月30日受理（Received on January 30, 2015）＊1
National Research Institute of Fisheries Science, Fisheries Research Agency, 2‑12‑4 Fukuura, Kanazawa, Yokohama, Kanagawa, 236‑8648
Japan (Current aﬃliation: Hokkaido National Fisheries Research Institute, Fisheries Research Agency, 2‑4‑1 Nakanoshima 2jo, Toyohira,Sapporo, Hokkaido, 062‑0922 Japan)
E-mail: ikutaro@aﬀrc.go.jp＊2
Fishing Industry and Communities Promotion Organization, 1‑27 Kaisei, Ishinomaki, Miyagi, 986‑0032 Japan＊3
Japan Fisheries Cooperatives, 1‑1‑12 Uchikanda, Chiyoda, Tokyo, 101‑8503 Japan＊4
Tohoku National Fisheries Research Institute, Fisheries Research Agency, 3‑27‑5 Shinhamacho, Shiogama, Miyagi, 985‑0001 Japan (Current
aﬃliation: Fisheries Agency, Ministry of Agriculture, Forestry and Fisheries, 1‑2‑1 Kasumigaseki, Chiyoda, Tokyo, 100‑8950 Japan)

34

Ikutaro SHIMIZU，Tsuyoshi TANAKA，Hideki MIURA，and Kohichi SAOTOME

Fig. 1. Maps of Japan on the left and the Sanriku region in the Tohoku district on the right prepared by
the Google Maps.

25000

2000

20000

1600

Price (Yen/kg)

Production (t)
1400
1200

15000

1000
800

10000

600
400

5000

Sanriku coho salmon price (Yen/kg)

Sanriku coho salmon production (ton)

1800

200
0

0
1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012

Fig. 2. Variation of production and price of Sanriku farmed coho salmon from 1976 to 2012. The landing of 2011
was nothing by the damage of tsunami. These data were supplied by JF-Miyagi, 2012: Data of landing amounts
and values of farmed coho salmon in Miyagi prefecture.
measured with the software R (http://www.r-project.
org).

in Hokkaido and a part of eyed eggs are imported
from a company in the US (AquaSeed Corporationin Washington)

to inland ﬁsh farms in the Tohoku

Process of coho salmon aquaculture in Japan

district. Coho salmon fry are produced by inland

Domestic eyed eggs of coho salmon are supplied

December to the next October (Fig. 3).

Cohofrom inland hatcheries (Koshimizu and Sarabetsu)

salmon fry (total length: 140 ‑180 cm)

arefish farms in Iwate and Miyagi prefectures from

Challenge of Reconstructing Coho Salmon Aquaculture after Tsunami

35

Fig. 3. Farming schedule of Sanriku coho salmon. Inland farming is consisted from eyed egg productionin Hokkaido and fry farming in Miyagi and Iwate prefectures. Marine farming is operated along Sanriku
coast in Miyagi prefecture.
transported to marine cages in the Sanriku region.

plant decreased the demand for Sanriku coho

The fish farmers begin to land coho salmon after

salmon,they grow bigger than 1.5 kg body weight from

(５) Avoiding prolonged cold storage and increase

April until early August. Landed farmed cohoin fresh supply of Sanriku coho salmon reduced

salmon are transported to processing facilities andthe price of the farmed coho salmon, andthey are supplied to consumer markets around(６) The discontinuation of the supply of Sanriku

Japan.

coho salmon in 2011 changed consumersʼpurchasing pattern from Sanriku coho salmon

Causes of the price drop of Sanriku coho salmon

to imported salmon.

Based on the interviews, the causes of the price

Issues about coho salmon aquaculture

drop in domestic farmed coho salmon (Sanriku coho
salmon)can be as follows;

The price of Sanriku coho salmon is synchronized(１) The mass imports of Chilean farmed cohowith the import price of frozen farmed coho salmon

salmon starting autumn 2011 reduced theand frozen farmed rainbow trout from Chile, anddomestic wholesale price,

has decreased since the 1980s (Fig. 4). The import(２) Low seawater temperature in the winter 2012

price of frozen Chilean coho salmon (IPCCS) is

suppressed the growth of Sanriku coho salmon,

synchronized with the import price of frozen Chilean(３) Late landing of Sanriku coho salmon in therainbow trout (IPCRT). IPCCS (yen/kg/year) was

spring 2012 aﬀected supply in June,

explained by the imports (tons/year) of frozen(４) The spread of rumors about radiation caused

Chilean coho salmon (IQCCS) and IPCRT (yen/kg/

by the accident at Fukushima nuclear poweryear) from 1989 to 2010. The data was used by the

36

Ikutaro SHIMIZU，Tsuyoshi TANAKA，Hideki MIURA，and Kohichi SAOTOME
2000

Fresh Sanriku coho salmon price (Yen/kg)
Import price of frozen Chilean coho salmon (Yen/kg)

Salmon price (Yen/kg)

1600

Import price of frozen Chilean rainbow trout (Yen/kg)

1200

800

400

0
1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012

Fig. 4. Prices of fresh Sanriku coho salmon, frozen imports of coho salmon and rainbow trout from Chile between
1976 and 2012. The prices of fresh Sanriku coho salmon were supplied from JF-Miyagi and import prices were
provided by the trade statistics, Ministry of Finance of Japan.

trade statistics, Ministry of Finance of Japan Trade

Log (PSCS)＝2.494＋0.578Log (IPCRT)(4.114)＊＊＊(5.911)＊＊＊statistics (http://www.customs.go.jp/toukei/).
This study employed a conventional double-log
demand equation model with the estimation results

2

Adjusted R ＝0.618, n＝22
The result shows the import price of frozen
Chilean coho salmon increases by 0.91％ as theas follows:
Log (IPCCS)＝1.395−0.077Log (IQCCS)＋0.910Log (IPCRT)＊(1.308) (−2.104)(7.752)＊＊＊2

Adjusted R ＝0.928, DW＝2.54, n＝22
The statistical signiﬁcant code: ＊ ＊ ＊， ＊ ＊，and ＊suggests p-value under 0.005, under 0.01, and under0.1, respectively. DW is Durbin-Watson ratio.
The price (yen/kg/year) of fresh Sanriku coho

import price of frozen Chilean rainbow trout
increases 1％. In addition, the price of fresh Sanriku
coho salmon increases by 0.58％ as the import priceof frozen Chilean rainbow trout increases 1％ .
In the Japanese salmon market, the highest priced
salmon is fresh Atlantic salmon imported from
Norway (Fig. 5). The second highest priced salmon

salmon (PSCS)

was explained by the import priceare frozen sockeye salmon imported from Alaska,(yen/kg/year)of frozen Chilean rainbow trout

frozen rainbow trout or frozen coho salmon imported(IPCRT) from 1989 to 2010. The data was usedfrom Chile. The lowest priced salmon are domestic

by the local market price of JF (Japan Fisherieschum salmon and export chum salmon to China.

Cooperatives) −Miyagi and the trade statistics

One of the most important characteristics of Sanriku(http://www.customs.go.jp/toukei/)，Ministry ofcoho salmon aquaculture is market differentiation

Finance of Japan.

by being a fresh product. In addition, the supply

This regression model was estimated by step AIC
as follows:of fresh seafood in spring has been less than otherseasons in the Sanriku region. We hope to move the37

Challenge of Reconstructing Coho Salmon Aquaculture after Tsunami
1400

1200

Domestic Chum

Frozen Sockeye

Export Chum

Frozen Coho

Fresh Atlantic

Frozen Rainbow

Price (yen per kg)

1000

800

600

400

200

0
1992

1994

1996

1998

2000

2002

2004

2006

2008

2010

2012

Fig. 5. Prices of salmons in Japanese market. The highest price is fresh Atlantic salmon from Norway. The
secondary prices are frozen sockeye salmon imported from Alaska, and frozen rainbow trout and frozen coho
salmon imported from Chile. The third prices are domestic chum salmon in Japan and export chum salmon to
China. The prices were provided by the annual report (Hokkaido Stationary Net Fisheries Association, 2012:
Annual report of salmon distribution in Hokkaido. 96 pp.).

price of fresh Sanriku coho salmon to be between

data was used from 1984 to 2009 of the FAO

fresh Atlantic salmon and frozen rainbow trout instatistics, FishStatJ＊5. This model with estimation

consumer markets.

results by step AIC is as follows:
Log (JFIP)＝8.860−0.071Log (PAFQ)

Issue about ﬁshmeal in Japan(7.336)＊＊＊ (−1.465)−0.410Log (PFEQ)＋0.182Log (CFIQ)

One of the common issues about Sanriku coho
salmon aquaculture system (from eyed eggs(−3.226)＊＊(3.427)＊＊

Adjusted R2＝0.635, n＝26

production, and fry production, to seawater farming)

In addition, the import quantity (tons/year) ofis the impact on feed costs due to import price ofﬁshmeal in Japan (JFIQ) was explained by the ﬁshforeign produced fishmeal. The price increase of

aquaculture production (tons/year) in Japan (JAPQ)

imported fishmeal has impacted the economics ofand the export quantity (tons/year) of Peruvian

inland and seawater fed aquaculture. The importfishmeal (PFEQ). The data was used from 1979 toprice (yen/kg/year) of ﬁshmeal in Japan (JFIP) was2009 of the FAO statistics, FishStatJ. This demand

explained by the export quantity (tons/year) ofmodel is deﬁned below:

Peruvian fishmeal (PFEQ)，the fishery production

Log (JFIQ)＝27.001＋2.389Log (JAPQ)(tons/year) of Peruvian anchovy (PAFQ) and the(−2.057)＊ (2.089)＊

import quantity of fishmeal in China (CFIQ). The＊5(http://www.fao.org/ﬁshery/statistics/software/ﬁshstatj/en), Software for ﬁshery statistical time series of FAO

38

Ikutaro SHIMIZU，Tsuyoshi TANAKA，Hideki MIURA，and Kohichi SAOTOME

Fig. 6. Distribution image of Sanriku farmed coho salmon and import farmed salmon from Chile inJapanese market before and after the earthquake of 2011.

＋0.663Log (PFEQ)

the price of fresh coho salmon up to a fair level. We(3.930)＊＊＊believe that the development of Sanriku coho salmon

Adjusted R ＝0.614, DW＝1.86, n＝31

aquaculture contributes to the reconstruction of2

The import price of fishmeal in Japan decreases

regional industries in Miyagi prefecture. All of theby 0.41％ as the export quantity of Peruvian ﬁshmeal

Chilean farmed coho salmon and rainbow trout are

increases 1％. The import quantity of fishmeal inimported frozen. We believe that one of the mostJapan increases by 2.4％ as the fish aquaculture

important characteristics of Sanriku coho salmon for

production in Japan increases 1％. We hope ﬁshmeal

market diﬀerentiation is freshness (Fig. 6).

production would be stable in the near future. At
the same time, the development of feed with low
ﬁshmeal rate will be expected.
Conclusions
It is important to supply fresh salmon for
consumer markets by the Golden Week holidays(from late April to early May) in Japan. It isnecessary to improve the traditional aquaculture
system and to create a new brand value for Sanriku
coho salmon. Sales promotion is expected to drive水研センター研報，第40号，39−49，平成27年
Bull. Fish. Res. Agen. No. 40，39−49，2015

39

Development of a New Type of Fish Diet, Non-Fish Meal Extruded-Pellet
Noriko ISHIDA＊1, Tomohiko KOSHIISHI＊2, Tatsuo TSUZAKI＊3, Koji MAENO＊4,
Satoshi KATAYAMA＊5, Minoru SATOH＊5, and Shuichi SATOH＊6
Abstract: We developed a non-ﬁsh meal diet using plant and/or animal protein materials. Threekinds of non-ﬁsh meal diets and a control diet containing 50% ﬁsh meal were processed. In the nonﬁsh meal diets, the ﬁsh meal was replaced with commercially available plant or animal materials
and supplemented with taurine and materials for maintaining palatability. These diets were fed
to one year old yellowtail (BW 753±96g) in net cages. There was no diﬀerence in growth, daily
weight gain, daily feeding rate, feed conversion ratio and protein eﬃciency ratio among the diets.
Non-fish meal diets were processed in a factory and dietary properties were studied such as
uptake, stomach evacuation rate, and comparative disease resistance in ﬁsh fed the experimental
diets. In addition, palatability of each substitute protein material for fish was examined andmaterials to enhance palatability of the non-ﬁsh meal diets were clariﬁed. Non-ﬁsh meal diets havethe potential to support the growth of one year old yellowtail.
Key word: Fish diet, Non-fish meal, Extruded-pellet, Yellowtail, Palatability, Substitute proteinmaterial

In Japan, fish meal is the main material in fish

designed and fed to one year old yellowtail whichdiets used in aquaculture and is contained at a level

were at an immature and rapidly growing size. Weof over 50%. Over 90% of the ﬁsh meal used in ﬁsh

also studied dietary, uptake, stomach evacuate rate,

diets for aquaculture is imported. Globally, the pricepalatability, and comparative disease resistance ofof fish meal has increased dramatically in recent

fish. Then the meat quality fed the experimental

years due to reduced production caused by ﬁsherydiets was evaluated using a panel sensory test.

regulation in South America and the increased
worldwide demand for aquacultured fish. The

Materials and Methods

increasing ﬁsh meal price threatens of the viabilityof production for Japanese aquaculture farmers.

Experimental diets and processing: The control diet

Therefore, a new type of diet that is independentand three kinds of non-ﬁsh meal diets were preparedof fish meal is strongly required. Low or non-fishusing a variety of ingredients (Table 1) (Tsuzaki

meal extruded-pellets have been developed. In our., 2013 and 2014). The control diet (FM) contained

preliminary study, we prepared diets containing

50% ﬁsh meal to provide a suitable balance of amino

10% fish meal, and fed them to cultured yellowtailacids. Fish meal was replaced with commerciallyto compare the ﬁsh growth performances to a dietavailable plant (PP) and animal (AP) materials incontaining 50% fish meal. It was observed thatthe non-fish meal diets. Soy protein concentrate,

ﬁsh growth was similar in both diet groups. Threedefatted soy bean meal, and corn gluten meal wereexperimental non-fish meal extruded-pellets wereused for plant materials and pork meal, feather

2015年 1 月30日受理（Received on January 30, 2015）＊1
National Research Institute of Fisheries Science, FRA, Yokohama 236‑8648, Japan
E-mail: larry@aﬀrc.go.jp＊2
Marubeni Nisshin Feed Co., Ltd., Aichi 441‑3605, Japan＊3
National Research Institute of Fisheries and Environment of Inland Sea, FRA, Ehime 794‑2305, Japan＊4
Kagoshima Prefectural Fisheries Technology and Development Center, Kagoshima 891‑0315, Japan＊5
Tohoku University, Sendai 981‑8555, Japan＊6
Tokyo University of Marine Science and Technology, Tokyo 108‑8477, Japan

Noriko ISHIDA, Tomohiko KOSHIISHI, Tatsuo TSUZAKI, Koji MAENO, Satoshi KATAYAMA,
Minoru SATOH, and Shuichi SATOH

40

Table 1. Formulation of the experimental diets (%)

Diet group
IngredientFish meal

1

Soy bean concentrate2

Defatted soy bean meal
Corn gluten meal
Krill meal
Pork meal

4

5Feather meal
Blood meal
Fish oil
Wheat

3

5

6

7

Defatted rice brain

8

9

FM

PP-SP

PP-FS

AP-SP

50.0

0

0

0

0

30.0

30.0

0

3.5

11.0

11.0

8.0

0

12.0

12.0

9.0

0

3.0

3.0

3.0

0

0

0

30.0

0

0

0

3.0

0

0

0

3.0

15.4

17.0

17.0

14.0

13.0

10.8

10.8

12.0

8.5

0

0

1.8

6.6

8.0

8.0

8.0

Vitamin mixture

10

2.0

2.0

2.0

2.0

Mineral mixture
Taurine

10

1.0

1.0

1.0

1.0

Tapioca starch

0

1.0

1.0

1.0

Amino acid mixture

11

0

2.2

2.2

2.2

Calcium phosphate

12

0

2.0

2.0

2.0

Enzyme mixture

13(external addition)

0

(0.2)

(0.2)

(0.2)

Skipjack peptide

14(external addition)

0

(0.5)

0

(0.5)

0

0

(0.5)

0

Fish sauce

15(external addition)

1

Fish meal was made from anchovy meal as the main raw material, imported from Peru䠊

2

Soycomil; Archer Daniels Midland Japan Ltd䠊

3

Nisshin Oillio Co., Ltd䠊

4

Japan Corn Starch Co., Ltd䠊

5

Kagoshima Pure Foods Co., Ltd䠊6APC Japan Co., Ltd䠊

7

Tsuji Oil Mills Co., Ltd䠊

8

Suzuki Oil Mills Co., Ltd䠊

9

Sanguan Wongse Industries Co., Ltd䠊10

See Takagi et al 䠊 (2005)䠊

11

See Aoki et al 䠊(2000b)䠊

12

Onoda Chemical Industry Co., Ltd䠊

13

Alltech䠊

14

Marubeni Nisshin Feed Co., Ltd䠊

15

Sakaiovex Co., Ltd䠊

41

Non-Fish Meal Extruded-Pellet

among the four experimental diets. Small diﬀerences
Blend with materials

Spraywere seen in the fatty acid composition. AP-SP had
16% of n-3 PUFA while the other diets ranged from
22‑27%. AP-SP also had 41% of total monoenes,
higher than the 30‑35% found in the other diets

Cross-secƟon

Cross-secƟon

(Table 2).
Feeding experiment: One year old yellowtails which

Fig. 1. Method to add palatability materials andenzyme solution to ﬁsh pellets. Right is sprayed after
being shaped to reduce the material volume. Left isshaped after blending all the materials.

originated from seedling production in Goto Island,meal, and blood meal were used for animal materials

three times a week for six months from July toin this study. Taurine was added to the non-fish

January were reared in four 4 m x 4 m replicate

Nagasaki Prefecture were used in the experiments(Tsuzaki., 2013 and 2014). The average bodyweight of 220 fish was 753±96g. Four groups of
55 ﬁsh fed diﬀerent experimental diets to satiation

meal diets at a 1% composition. Experimental dietsnet cages on the sea surface. After January fish

were carefully shaped using an extruder to havecontinued to be fed with the leftover experimentsuitable physical properties in the sea and sink at andiet until March.

appropriate rate rather than ﬂoat.

The feeding experiment was divided into two

It is known that yellowtail become lessperiods, summer season (July to September) and

responsive to a diet as the amount of fish meal

autumn season (October to December). Sampling andincluded is reduced (Aoki

analysis were performed for each period.

., 2000a and 2000b).

Therefore, two commercially available materials,

Body weight and fork length were monthly

a skipjack peptide (SP) and a fish sauce (FS) were

measured for 20‑25 live ﬁsh at random from every

supplemented to maintain the palatability of the non-group.

ﬁsh meal diet. The skipjack peptide is concentrated

were sampled and weighed and the following datafrom the cooking water of skipjack. The ﬁsh sauce

were measured or calculated.

At the end of the experiment, all the fish3

3

is made from mackerel gut using a patent-pending

Condition factor＝(W / FL )×10

method for rapid production. An enzyme solution

Weight gain ratio (%) ＝ (and amino acid chelated trace metals were added to
the non-ﬁsh meal diets to improve digestibility andabsorption. These palatability materials and enzyme
solution were added by a spray method after the
extruder processing to minimize the volume (Fig. 1).
Therefore, the three kinds of non-ﬁsh meal diets
were as follows:
PP-SP was a plant protein based diet withthe skipjack peptide as a material to enhance

＋

−

)/

Daily weight gain ratio (%/day)＝(

＋

−

×100
)/[(

＋

＋

)/2× ]×100

Daily feeding rate (%/day)＝F/[(

＋

＋

＋

−

＋

−

)/2× ]×100
Feed conversion ratio (FCR)＝F/()
Protein eﬃciency ratio (PER) ＝ ()/

palatability.
PP-FS was a plant protein based diet with the

W: Body weight (g)ﬁsh sauce as a material to enhance palatability.

FL: Fork length (cm)

AP-SP was an animal protein based diet with

: Initial total body weight (g)the skipjack peptide as a material to enhance: Final total body weight (g)palatability.

: Total body weight of sampled fish andTwo sizes of experimental diets, 13mm and 15mm

dead ﬁsh (g)grain diameter, were prepared at the Kagoshima: Number of ﬁnal ﬁshplant of Marubeni Nisshin Feed Co., Ltd. There: Total feed intake (g)were minimal diﬀerences in the general composition: Rearing days

42

Noriko ISHIDA, Tomohiko KOSHIISHI, Tatsuo TSUZAKI, Koji MAENO, Satoshi KATAYAMA,
Minoru SATOH, and Shuichi SATOH
Table 2. Proximate composition, phosphorus content, and fatty acid composition of theexperimental diets

Analysis
Proximate composition (%)
Moisture
Crude ash
Crude protein
Crude lipid

FM

Diet group
PP-SP
PP-FS

7.6
9.1
39.8
18.9

7.7
5.6
38.1
16.1

8.8
5.6
37.4
17.4

4.9
7.4
42.0
17.6

Phosphorus content
P 䠄mg/g䠅

15.4

9.7

9.7

13.4

Fatty acid composition (%)
14: 0*1
16: 0
18: 0

4.8
18.3
3.8

4.2
16.5
3.5

4.3
18.3
4.1

5.4
15.8
4.2

16: 1n-7
18: 1n-9
20: 1n-9
20: 1n-11
22: 1n-11

5.8
16.0
2.6
2.2
4.0

5.2
16.8
2.7
2.3
4.1

5.5
19.0
4.5
1.5
4.5

5.5
16.4
7.5
1.5
9.8

18: 2n-6
20: 4n-6

4.5
1.0

7.0
0.9

7.1
0.9

5.6
0.4

18: 3n-3*2
18䠖4n-3
20: 4n-3
20: 5n-3 (EPA)
22: 5n-3 (DPA)
22: 6n-3 (DHA)
Othersn-3 HUFA

1.0
1.8
0.6
8.0
2.0
13.5
10.2
25.9

1.2
1.5
0.6
6.8
1.9
13.6
11.1
24.3

1.1
1.3
0.6
6.0
1.9
11.3
8.0
21.2

0.9
2.0
0.6
5.2
1.0
6.2
12.2
14.9

*1

AP-SP

Left figure represents the number of carbonatoms and right figure after colon
represents the number of carbon unsaturated bonds.
*2
䇾n-3”represents the omega-3 fatty acid.
n-3 HUFA䠙18: 4n-3+20: 4n-3+EPA+DPA+DHA.
Experimental diets are shown in Table 1.

43

Non-Fish Meal Extruded-Pellet
P: Total protein intake (g)at 2% of body weight directly into the stomach.

Blood, muscle, and liver samples were collected

Stomach residue was analyzed over time by thefrom 5 ﬁsh in every group at the start (initial), late

Kjeldahl nitrogen method and stomach evacuation

summer (intermediate), and late autumn (end). The

rate was estimated (Tohata., 2012).

ratio of liver weight per body weight was calculated.

Disease resistance was carried out using

Hematocrit value and plasma component levels were

, an infectious streptococcal

analyzed. General content, phosphorus content, fatty

bacterium. Fish were reared on each diet in indooracid composition, and amino acid composition oftanks for 3 months and fed the bacterium-containing

muscle were analyzed.

feed for 5 days. Fish mortality was observed over a

The statistical diﬀerences between trial diets were
determined by Tukeyʼs multiple range comparison

40 day period (Ishida., 2013).

Diet palatability: Fish behavior was divided into ﬁve

test ( <0.05).

responses after placing the diets into a fish tankExperimental diet uptake, stomach evacuate rate,

described as recognition, ingestion, orientation withinand comparative disease resistance of fish fed

the mouth, swallowing, or spitting out (Fig. 3). The

experimental diets: Nutritional absorption was

actions were analyzed by using a camera to ﬁlm red

analyzed from ﬁsh feces collected from ﬁsh reared insea bream, yellowtail, and coho salmon using single

a conical tank (Fig. 2) using special diets containing

dry pellets made from only one material (Fig. 4)

chrome oxide as an internal standard material (Satoh(Niinuma., 2013).

., 2012, Yamauchi., 2012).

Meat quality of raw sashimi: A panel test using the

Fish were anesthetized and administered the dietorder method, color-difference meter analysis, andelectronic nose system analysis were performed foreach experimental ﬁsh fed PP-SP, PP-FS, AP-SP, andcontrol diet (Tohata., 2013).
RESULTS

Processing: All the experimental diets were processedusing a double-screw extruder. At the beginning in
the autumn season, plant protein based diets did notsink at a suitable rate and flowed out from the net
cage compared to the other diets. Therefore thesediets were re-made. Palatability ingredients and an
enzyme solution were sprayed onto diets to minimize
materials used to 0.05% from the original 2% requiredin the blending method (Fig. 1).
Growth performances and biological indices inFig. 2. The conical tank used to collect feces of ﬁsh
fed the experimental diets to analyze nutritional
absorption.
Right: Feces at the bottom of the tankWaiƟngRecogniƟon

IngesƟon

yellowtail: The growth performances in both thesummer and autumn period are shown in Table 3.
In the summer period when the watertemperature was 22.8 ‑ 29.2 ℃ all diet groups

OrientaƟon
within themouthFig. 3. Fish behaviors for the diet.

SwallowingSpiƫng out

44

Noriko ISHIDA, Tomohiko KOSHIISHI, Tatsuo TSUZAKI, Koji MAENO, Satoshi KATAYAMA,
Minoru SATOH, and Shuichi SATOH

Diet

Filming
Red sea bream, yellowtail, coho salmon

Fig. 4. Behavioral analysis of ﬁsh after feeding the diets.
Table 3. Growth performance and feed utilization by yellowtail fed the experimental diets
Rearing season (days)

InitialSummer (90 days)

Autumn (91 days)

FM

PP-SP

PP-FS

AP-SP

FM

PP-SP

PP-FS

AP-SP

Dead

0

0

1

2

1

0

1

1

Sampled

3

4

3

4

7

7

7

7

Final

52

51

51

49

44

44

43

41

113.0

113.9

113.6

118.0

98.5

102.9

101.0

90.5

＊1

Number of ﬁsh

220

Total food intake (kg)−＊2

Body weight (g)753±96a＊2

Fork length (cm)

a

a

a

1663±204 1700±221 1698±156 1688±204

Final

a

a

a

a

2242±333 2178±285 2234±244 2205±222

36.9±1.5
Final＊2

Condition factor

44.8±2.3aa44.8±1.8a45.0±1.2

44.1±1.2a48.4±2.2aa48.6±1.8a49.0±1.748.5±1.5a15.0±0.8b18.3±1.1

18.8±0.9＊3Weight gain ratio (%)＊3

Daily weight gain (%/day)

−

116.4

−

0.82

＊3

Daily feeding rate (%/day)−＊3

Feed conversion ratio＊3

Protein eﬃciency ratio

b

b

a

19.6±1.2a18.9±1.2b19.0±1.1

ab18.6±0.7

19.7±1.4

119.9

119.1

113.6

34.2

30.4

31.0

32.3

0.83

0.83

0.80

0.32

0.29

0.30

0.31

1.92

1.91

2.02

1.91

1.08

1.15

1.05

1.12

−

2.35

2.29

2.30

2.51

3.33

3.91

3.76

3.39

−

1.07

1.14

1.16

0.94

0.75

0.67

0.71

0.70

Final

19.3±1.0ab＊1Fishes were divided into 4 diet groups, each group contains 55 ﬁshes.
Data are shown as mean ± standard deviation ( =20‑44)．Values having diﬀerent superscript letters are signiﬁcantly diﬀerent ( ＜0.05).
＊3
Shown in the text.
Experimental diets are shown in Table 1.
＊2

showed a similar total feed intake ranging from 113

Final fork length and body weight were 48.4 cm

to 118 kg and a similar body weight gain (Fig. 5).

and 2,242 g in the control (FM), 48.6 cm and 2,178

In the autumn period when the water temperature

g in the PP-SP, 49.0 cm and 2,234 g in the PP-SF,

was 11.5 ℃ lower than during the summer period

and 48.5 cm and 2,205 g in the AP-SP. Differences

total food intake of all groups was less than in the

between control and non-fish meal diets were notsummer ranging from 90 to 103 kg. No seasonalobserved and the growth was almost the same.

difference in weight gain was seen among diet

There was no seasonal difference between dailygroups.

weight gain (%/day), daily feeding rate (%/day), and45

Non-Fish Meal Extruded-Pellet
weight gain ratio (%). For example, feed conversion

not seen in any of the dead ﬁsh.

ratio in the autumn season was 3.33 in the control

Plasma components measured included glucose,(FM) and 3.91 in PP-SP, 3.76 in PP-FS, and 3.39 intotal protein, total cholesterol, glutamate pyruvate

AP-SP.

transaminase (GPL), and alkaline phosphatase (ALP).

Table 4 shows the results of the biological indicesNo significant difference was observed in plasmaof cultured ﬁsh ( =5). The liver to body weight ratio

components between the treatments in the summer

in every group was 1.2 to 1.3 and no significant

period. Total cholesterol exhibited a significant

diﬀerence was seen. The average hematocrit value

seasonal variability between the treatments withof 5 fish in every group was 46.2 to 50.6 with nolower values of 246 and 266 mg/dl in PP-SP and PP-

significant difference observed. There were three,

FS respectively ( <0.05) (Table 4).

two, and one mortalities of ﬁsh in AP-SP, PP-FS, andCharacteristics such as uptake, stomach evacuate

FM respectively. Green liver, which is thought to be

rate, and disease resistance of fish fed thea sign of taurine deﬁciency (Takagi

experimental diets: The uptake of protein from non-., 2005), wasﬁsh meal diets was reduced by 4‑9 % compared to

Average body weight (g)2,500

the FM control (Satoh., 2013).

Evacuation of feed from fish stomachs was
2,000

observed for all three diets. The order of the rate ofevacuation was PP-SP > AP-SP 㲔FM (Fig. 6).

1,500

Survival rate at 42 days after the challenge test
was 80% in FM group, and 95% in PP-SP and AP-SP

1,000

group (Ishida., 2013).

Diet palatability: Fish spitting out feed was almost

500

zero for single dry fish meal pellet. The spitting
0

Jul

Aug

Sep

Oct

Nov

Dec

rate was higher in single dry pellet made fromJan

plant protein than those made from animal protein.

Month

To improve palatability of PP and AP pellets we

Fig. 5. Growth curves of yellowtail fed theexperimental ﬁsh meal diet (FM： ● ) and non-ﬁshmeal diet (PP-SP： ○ , PP-FS： ○ , and AP‒SP：△ ) in net cages for 6 months．Experimental diets
are detailed in Table 1.

washed the original materials with 80% ethanol toremove the extractive component and made eachsingle dry pellet from those pre-treated materials.
The spitting rate in single dry pellet made from pre-Table 4. Changes in hepatosomatic index, heamatocrit value, and hematochemical characteristics in yellowtail( =5) fed the experimental deits
Rearing season (days)

Hepatosomatic index (%)＊1

Hematocrit value (%)

Initial1.1±0.053.6±3.4

Summer (90 days)FM
1.4±0.1

PP-SP
a

a

61.5±7.450.8±0.7AP-SPa

1.2±0.1aAutumn (91 days)

PP-FS
1.2±0.2a

1.3±0.3

a

FM
a

a

a

57.0±4.9

PP-SP55.3±7.3

PP-FS
a

AP-SP
a

1.4±0.2

1.3±0.1

1.2±0.0aaa46.2±2.145.7±4.5a

1.2±0.1a50.6±6.5

48.0±6.2

Heamatochemical characteristicsaaa

Glucose（mg/ ）144±25.2 237.8±51.8 262.6±30.8 267.0±41.3 280.8±146.1

Total protein (g/ ）4.0±0.5

Total cholesterol（mg/ )＊2

GPT (IU/ )＊3

ALP (IU/ )a

4.3±0.7

3.7±0.2aaa

4.3±1.5a

4.8±0.7aaa32.4±4.2

a

a

a

a

a

59.2±44.1

34.6±14.544.4±19.9

aaa148.6±33.8 156.4±34.3 134.8±14.4 181.0±64.0 200.6±22.9

a

a

a

a

138.6±10.5 127.6±21.3 143.0±12.1 130.0±28.5
4.1±0.5a349.4±63.0 282.0±30.8 203.4±21.7 267.0±79.5 276.4±101.0
25.4±2.6ab

3.7±0.2abb

3.9±0.5aba

4.4±0.4bab

396.8±72.3 245.6±29.0 266.4±61.0 346.0±82.1aa32.0±13.4

30.8±14.8

a

a

a

a

23.4±8.6

33.8±11.4
aa143.4±43.4 125.2±16.9 126.5±23.7 147.8±42.3

Mean ± standard deviation ( =5)．Values having diﬀerent superscript letters are signiﬁcantly diﬀerent ( ＜0.05）.
＊1
The ratio of liver weight to body weight.
＊2
Glutamate-pyruvate transaminase.
＊3
Alkaline phosphates.
Experimental diets are shown in Table 1.

Noriko ISHIDA, Tomohiko KOSHIISHI, Tatsuo TSUZAKI, Koji MAENO, Satoshi KATAYAMA,
Minoru SATOH, and Shuichi SATOH

46

Discussion

㸦%㸧

It has long been thought that fish meal was a100

necessary ingredient in fish feed. Generally, theformulated feeds for yellowtail contain about 50%
80

fish meal. Recently, formulated feeds containing
lower amounts of fish meal such as 30% have60FMbeen produced as a commercial product. However

PP-SP

a fish meal content of less than 30% would haveAP-SP

considerable financial benefits for aquaculture
enabling continued economically viable production.

40

Therefore, we developed non-fish meal feeds andevaluated those in a ﬁeld experiment with a similar

20

aquaculture environment. We used one year old
㸦h㸧

0
0

5

10

15

20

Fig. 6. Evacuation of the diet from the ﬁsh stomach
after a single oral administration of diet at 2% bodyweight. Value are shown the percentage of nitrogenin the stomach residue per administered nitrogen.
Experimental diets are shown in Table 1.

yellowtail which is an intermediate size during
culturing and shows rapid growth requiring highfeed costs. Experimental periods included the higher
feeding activity season of summer and the lower
feeding activity season of winter.
In the present study (Tsuzaki., 2014), theplant protein based diets of PP-SP and PP-SP showeda similar daily feeding rate and daily weight gain,
better feed conversion ratio and protein efficiency
ratio than the control FM in summer season. On

treated plant protein decreased. On the other hand,the other hand, the animal protein based diet ofthe opposite result was shown in pre-treated animal

AP-SP showed a higher daily feeding rate andproteins that the spitting rate increased (Niinuma

feed conversion ratio, lower protein eﬃciency ratio., 2012).

demonstrating lower protein uptake than others.

Fish meat analysis: Table 5 shows the results of the., 2012, Yamauchi

We also studied uptake of nutrient in these non-ﬁsh

ﬁsh meat analysis of cultured ﬁsh. In the proximate

meal diets and FM by analysis of yellowtail feces. It

composition of all the diet groups, moisture content

is clear that the uptake of protein in these non-ﬁshdeclined and fat content increased until the end

meal diets were reduced by 4 ‑ 9 % compared toof experiment. No difference was seen in thethe control FM.

phosphorus content among the diet groups. The

Then, in the autumn season, the plant protein

EPA, DHA and n3HUFA content of ﬁsh fed AP-SP

based diets of PP-SP and PP-FS showed a low

pellets was always lower than in ﬁsh fed the otherperformance because of the higher daily feeding rate

diets, at times significantly less ( <0.05) in bothand feed conversion ratio, lower daily weight gain.

summer and autumn. These results were similar to

These results are thought to be due to the physicalthe analysis of experimental diets.

properties of the diets. Because these plant protein
based diets did not sink appropriately and flowed
out from the net cage compared to the other diets.
From these results, more studies are needed for the
improvement of uptake for animal protein based
diets and adjustment of the physical characteristics
for plant protein based diets. However, theseresults demonstrate that non-fish meal diets havethe potential to support the growth of one year old

47

Non-Fish Meal Extruded-Pellet

Table 5. Proximate composition, phoshorus conntent, and fatty acid composition of muscle in yellowtail ( =5) fed
the experimental diets
Rearing season (days)
FM

Summer (90 days)
PP-SP
PP-FS

InitialProximate composition (%)
Moisture
71.9 ± 0.8
Crude ash
2.3 ± 0.5
Crude protein
22.1 ± 0.2
Crude lipid
3.7 ± 0.964.7 ± 1.7
1.3 ± 0.3
21.4 ± 0.6
11.5 ± 2.1

64.5 ± 1.4
1.3 ± 0.0
20.3 ± 1.1
12.8 ± 2.6

Phosphorus content
P (mg/g)15.5 ± 4.4

4.2 ± 1.7

Fatty acid composition (%)
14: 0
4.2 ± 0.4
16: 0
19.6 ± 0.9
18: 05.6 ± 0.5

Autumn (91 days)
PP-SP
PP-FS

AP-SP

FM

AP-SP

64.8 ± 1.7
1.4 ± 0.1
20.5 ± 0.8
10.6 ± 1.2

65.6 ± 4.1
1.4 ± 0.1
20.7 ± 1.3
10.1 ± 3.3

60.6 ± 4.6
1.4 ± 0.1
20.1 ± 2.3
16.0 ± 6.2

60.0 ± 3.0
1.3 ± 0.1
20.5 ± 1.6
17.9 ± 4.7

60.3 ± 1.3
1.4 ± 0.2
22.0 ± 1.0
16.0 ± 1.2

62.5 ± 0.9
1.4 ± 0.1
22.0 ± 0.5
14.3 ± 1.5

5.5 ± 2.8

5.0 ± 1.5

4.3 ± 2.5

4.6 ± 1.1

4.5 ± 1.6

4.4 ± 0.7

4.7 ± 1.4

4.0 ± 0.3
18.0 ±0.8
4.2 ± 0.3

3.1 ± 0.6
16.2 ± 1.3
4.0 ± 0.1

3.4 ± 0.7
16.8 ± 1.1
4.2 ± 0.5

3.9 ± 0.2
16.1 ± 0.3
4.5 ± 0.4

4.3 ± 0.2
16.4 ± 0.4
3.4 ± 0.1

3.9 ± 0.4
16.5 ± 0.7
3.4 ± 0.1

3.8 ± 0.3
15.6 ± 0.6
3.3 ± 0.1

4.2 ± 0.2
16.0 ± 0.5
3.9 ± 0.0

16: 1n-7
18: 1n-9
20: 1n-9
20: 1n-11
22: 1n-11

5.9 ± 0.8
21.4 ± 1.4
3.1 ± 0.8
0.6 ± 0.9
2.7 ± 0.3

6.6 ± 0.3
23.2 ± 2.2
5.1 ± 0.3
0.2 ± 0.0
3.4 ± 0.4

5.8 ± 0.5
25.0 ± 0.8
5.4 ± 0.3
0.2 ± 0.0
3.7 ± 0.5

5.9 ± 0.4
25.6 ± 1.3
5.7 ± 0.5
0.2 ± 0.0
4.0 ± 0.6

5.9 ± 0.3
25.0 ± 1.9
9.4 ± 1.4
0.3 ± 0.0
8.7 ± 0.7

7.0 ± 0.3
20.5 ± 1.9
5.6 ± 1.2
0.6 ± 0.9
3.6 ± 0.6

6.3 ± 0.2
23.5 ± 1.6
5.3 ± 0.1
0.2 ± 0.0
3.1 ± 0.1

6.2 ± 0.2
22.6 ± 2.0
5.6 ± 0.3
0.2 ± 0.0
3.7 ± 0.4

6.6 ± 0.3
22.1 ± 0.3
6.5 ± 0.2
2.9 ± 0.1
6.3 ± 0.4

18: 2n-6
20: 4n-6

5.6 ± 0.2
1.2 ± 0.2

5.8 ± 0.2
0.9 ± 0.0

8.6 ± 0.3
0.9 ± 0.1

8.6 ± 0.1
0.8 ± 0.1

7.2 ± 0.2
0.5 ± 0.1

5.3 ± 0.1
0.8 ± 0.0

8.4 ± 0.1
0.8 ± 0.1

8.4 ± 0.1
0.8 ± 0.0

7.4 ± 0.2
0.4 ± 0.0

18: 3n-3
18: 4n-3
20: 4n-3
20: 5n-3 (EPA䠅
22: 5n-3 (DPA䠅
22: 6n-3 (DHA䠅Othersn-3 HUFA

0.6 ± 0.1
0.9 ± 0.1
0.5 ± 0.0
5.3 ± 0.2
2.2 ± 0.2
11.4 ± 1.6
9.2 ± 2.8
20.3 ± 1.4

1.1 ± 0.0
1.3 ± 0.1
1.2 ± 0.1
0.9 ± 0.1
1.6 ± 0.1
1.3 ± 0.0
1.3 ± 0.1
1.6 ± 0.3
0.9 ± 0.0
0.7 ± 0.0
0.7 ± 0.0
0.6 ± 0.1
6.2 ± 0.5a 5.3 ± 0.4ab 4.8 ± 0.9ab 4.6 ± 1.1baabbc
2.2 ± 0.2
2.1 ± 0.2
1.5 ± 0.1
2.4 ± 0.1aaab
11.3 ± 1.0
9.4 ± 3.1
4.9 ± 1.2
10.9
5.9 ± 1.5
5.0 ± 0.3
5.2 ± 1.0
4.5 ± 2.4
aaabb
21.7 ±2.5 20.9 ± 1.6 18.3 ± 4.3 13.2 ± 2.9

1.0 ± 0.0
1.2 ± 0.0
1.2 ± 0.0
0.8 ± 0.0
1.5 ± 0.1
1.3 ± 0.1
1.4 ± 0.1
1.5 ± 0.0
0.7 ± 0.0
0.6 ± 0.0
0.7 ± 0.0
0.6 ± 0.0abcbc
6.6 ± 0.2
5.0 ± 0.3
5.1 ± 0.3
4.7 ± 0.1abbc
2.4 ± 0.2
2.0 ± 0.1
2.1 ± 0.1
1.4 ± 0.1aaab
11.4 ± 0.6 10.2 ± 0.8 10.5 ± 1.2
5.1 ± 0.5
8.9 ± 1.9
7.9 ± 2.0
8.5 ± 2.0
9.8 ± 0.2
22.7 ± 0.6a 19.2 ± 1.2b 19.8 ± 1.5b 13.2 ± 0.6c

Mean ± standard deviation (n =5)䠊Values having different superscript letters are significantly different (P 䠘0.05).
Experimental diets are shown in Table 1.
Fatty acid compositions are shown in Table 2.

yellowtail compared to the results of Takagi.

digestion using only one protein material

For substitute materials, plant protein material2012). More studies are needed in future using low

such as soy bean concentrate has been studied

cost materials such as defatted soy bean meal, corn

(Aoki

gluten meal and canola oil.

(2013).

diet, and found minimal difference (Tohata., 2000a, 2000b). We also used soy bean.,

concentrate and confirmed its performance as a

As for substitute animal protein materials, there

substitute protein for ﬁsh meal in the present study.

are meat meal, chicken meal, feather meal, and pork

In the field, it is sometimes believed that plant

meal. Feather meal and meat meal were reported toprotein materials stay longer in the stomach thanbe useful for yellowtail (Shimeno., 1993a, 1993b,

fish meal. However, in our experiment, the plant

2000). However, the animal protein studies areprotein based diets did not stay longer among the

fewer than plant protein studies. There is potential

diet groups. Fish meal based diet was the first toto use chicken meal and pork meal. As there are

be evacuated from the stomach. In addition, it isfew studies about pork meal, we tried to use pork

sometime believed that plant protein fed yellowtail

meal in the present study. Pork meal has less DHA,

is more susceptible to epidemic disease than fish

DPA and n3HUFA of fatty acid than FM. A similar

meal based diet. However, in our experiment, thosetendency was seen in the whole ﬁsh body and thephenomena were not seen (Ishida., 2013). We

meat of yellowtail fed by pork meal based diet.

also compared the stomach evacuation rate and the

However, there is no signiﬁcant eﬀect on growth and48

Noriko ISHIDA, Tomohiko KOSHIISHI, Tatsuo TSUZAKI, Koji MAENO, Satoshi KATAYAMA,
Minoru SATOH, and Shuichi SATOH

survival. Only slightly less protein digestion in pork

and enzyme mixtures (Table 1). As we usedmeal based diet was seen than the plant protein

both plant and animal materials, we suggest thebased diet. And feeding activity on pork meal based

possibility of many materials for non-ﬁsh meal diets.

diets was active. From these results, pork meal was

More study is needed to improve low cost diets andthought to be available as a substitute material.

produce higher quality meat.

The biggest problem has been shown to be theThis work was supported by Research andreduction of feeding activity when ﬁsh are fed low

Development Projects for application in promoting

fish meal content diets (Aoki., 2000a, 2000b,

new policy of agriculture, forestry and ﬁsheries from2000c). However, it is reported that an added

the Ministry of Agriculture, Forestry and Fisheries

palatability ingredients improved the feedingof Japan.

activity in eel (Takii., 1984), yellowtail (Takii., 1994), and tiger puﬀer (Takii., 1998). In the

References

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prefer plant protein to animal protein and ﬁsh meal.

Aoki H., Sanada Y., Furuichi M., Kimoto R., Maita

However, after washing the feed ingredients toM., Akimoto A., Yamagata Y., and Watanabe

remove the extractive component, the spitting ratioT., 2000a: Partial or complete replacement ofdecreased in plant ingredient feeds and increased

ﬁsh meal by alternate protein source in diets forin animal protein feeds, which suggested that theyellowtail and red sea bream.

extractive components might aﬀect feeding activity

48, 53‑63.

(Niinuma., 2012, Yamauchi.,., 2012). Then,

Aoki H., Watanabe K., and Satoh S., 2000b: Use

we added some palatability ingredients to the plantof low non-fish meal diets for red sea bream.

based diet, ﬁsh swallowing ratio was increased and., 48, 73‑79.

spitting ratio was decreased. From these results, we

Aoki H., Sanada Y., Furuichi M., Watanabe K., Satohused the skipjack peptide (SP) and fish sauce (FS)S., Yamagata Y., and Watanabe T., 2000c: Useas palatability ingredients in order to improve theof non-ﬁsh meal diets for yellowtail.

feeding activity. In the results, higher palatability
was seen when using both ingredients. The biggest., 48, 65‑72.
Ishida N., Maeno K., Yanagi S., Koshiishi T., andproblem of reduction of feeding activity in non-ﬁsh

Satoh S., 2013: Feeding experiment in room

meal diets was thought to be almost resolved by thetanks of the yellowtail

addition of palatability ingredients.

with non-ﬁsh meal diet.

The meat quality of yellowtail fed with non-fishmeal diets were compared to control yellowtail by. pp131 (in Japanese).

a panel test, which showed a diﬀerence in color andNiinuma T., Yamauchi A., Nakano T., Yamaguchi T.,

smell. Then we used colorimeter and Electronic

Satoh M., Katayama S., Aoyama T., Udagawa T.,

Nose System of Alpha MOS (http://www.alpha-mos.

and Ishida N., 2012: Palatability improvementco.jp/products/sensory.html). The fish fed by non-eﬀect of vegetable protein feed by the addition

fish meal diets had whiter muscle and a less fishyof material attractive to the target species.

smell (Tohata., 2013). Now, the effect of non-ﬁsh meal diets on ﬁsh meat quality and as a methodof enhancing the meat quality control is being
determined.

. pp45 (inJapanese).
Satoh S., Abe Y., Haga Y., Matsukura K., Koshiishi T.,

As described above, we alternated fish meal toand Ishida N., 2013: Digestibility and absorption

plant or animal protein materials to make non-fishof nutrient in low/non-ﬁsh meal feed for marine

meal diets, and fed them to one year old yellowtail,fish.

and got similar growth, body and blood compositions.
The main reason for the similar growth rates wasthought to be the addition of palatability ingredients. pp34 (in Japanese).
Shimeno S., Matsumoto T., Hujita T., Mima T., and49

Non-Fish Meal Extruded-Pellet
Ueno S., 1993a: Alternative protein sources for

growth and digestive enzyme secretion in Tiger

fish meal in diets of young yellowtail.

puﬀer.

., 6, 159‑166.

Tohata K., Koshiishi T., Satoh S., and Ishida N., 2012:, 59, 137‑143.

andShimeno S., Mima T., Imanaga T., and Tomaru
K., 1993b: Inclusion of combination of defatted

digestibleness of extruded

diets of non-ﬁsh meal in red sea bream.

soybean meal, meat meal, and corn gluten meal
to yellowtail diets.

. pp112 (in, 59,Japanese).

1889‑1895.
Shimeno S., Matsumoto M., Hosokawa H., Matsumoto

Tohata K., Ishida N., Koshiishi T., Satoh S., HottaT., and Ukawa M., 2000: Inclusion of poultryT., Nakagawa M., Yoshida K., Tsusaki T., andfeather meal in diet for fingerling yellowtail.

Miyazaki T., 2013: Evaluation of meat qualityof yellow tail fed by extruded diets of non-ﬁsh., 48, 99‑104.
Takagi S., Murata H., Goto T., Ichiki T., Munasinghe

meal.

D. MS, Endo M., Matsumoto T., Sakurai A.,
Hatae H., Yoshida T., Sakai T., Yamashita H.,. pp 43 (in Japanese).

Ukawa M., and Kuramoto T., 2005: The Green

Tsuzaki T., Hotta T., Yoshida K., Nakagawa M.,

Liver syndrome is caused by taurine deﬁciency

Koshiishi T., Yamada M., Satoh S., and Ishidain yellowtail,fed diets

N., 2013: Feeding experiment at sea of the., 53, 279‑290.

yellowtail

Takagi S., Murata H., Goto T., Hatae H., Yamashita

meal diet.

without ﬁshmeal.

with non-fish.

H., Takano A., and the late Ukawa M., 2013:
Long-term feeding of the yellowtail

pp131 (in Japanese).

with soy protein concentrate-

Tsuzaki T., Yoshida K., Hotta T., Nakagawa M.,

based non-fishmeal diet supplemented with

Yamada M., Satoh S., Koshiishi T., Maeno K.,

taurine.

Akiyama T., and Ishida N., 2015: Long-term., 61, 349‑358.

Takii K., Takeda M., and Nakao Y., 1984: Eﬀects ofexperimental feeding of yellowtail
with complete non-fish meal

supplement of feeding stimulants to formulated
feeds on feeding activity and growth of juvenile

extruded-pellet (EP) diets.

eel.

29‑38.

., 50, 1039‑1043.

Takii K., Shimeno S., Akutsu M., and Takeda M.,., 63,

Yamauchi A., Niinuma A., Nakano T., Yamaguchi

1994: Dietary supplement of feeding stimulants

T., Satoh M., Katayama S., and Ishida N., 2012:

on performance and digestive function of

Palatability evaluation of alternative fish feedyellowtail,ingredients using coho salmon.
., 4, 127‑137.
. pp45 (inTakii K., Shimeno S., Akutsu M., and Takeda M.,
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水研センター研報，第40号，51−59，平成27年
Bull. Fish. Res. Agen. No. 40，51−59，2015

51

The Importance of Taurine and n-3 Fatty Acids in Cobia,, Nutrition
Aaron M. WATSON＊1, Frederic T. BARROWS＊2, and Allen R. PLACE＊3

Abstract: In the wild, fish can adapt to their diet by down regulating or eliminating biosynthetic
pathways for nutrients found in abundance in their prey. Upon domestication for aquaculture theseﬁsh may now have unique dietary requirements which must be met through supplementation. To
examine this hypothesis and aid in determining potential minimum requirement levels for aquaculture
production, we have examined the synthetic capacity for taurine and polyunsatured fatty acids incobia,, through molecular methods and growth trials, respectively.

Taurine, a non-protein amino acid, is found in high concentrations in the natural diet of cobia andplays a variety of physiological roles. The taurine biosynthesis pathway in vertebrates is well known;
however it appears to be nonfunctional in some terrestrial (Felidae) and marine (Rachycentridae)
carnivores, thereby necessitating dietary supplementation. Taurine is not found in plant protein
sources used for replacing ﬁshmeal in aquaculture feeds, an increasingly important priority.
We examined the effects of graded levels of taurine addition (0%, 0.5%, 1.5%, 5.0%) toplant protein and fishmeal based diets formulated and manufactured by the USDA-ARS. At
the conclusion of a feeding trial, RNA was extracted from liver, muscle, and brain tissue forquantitative-RT-PCR analysis of the genes involved in taurine synthesis. Cysteine dioxygenase (CDO),
cysteamine dioxygenase (ADO), and taurine transporter (TauT) activity and expression levels were
examined and no diﬀerences in transcript abundance was detected within the tissues between the
dietary taurine levels. Increasing dietary taurine resulted in increased tissue taurine concentrations.
To examine the eﬀects of alternative sources completely replacing ﬁsh oil, two replacements
were examined as the lipid sources in a ﬁshmeal free, plant based feed (USDA-ARS) and compared
to a ﬁsh oil version of the diet. A thraustochytrid meal plus soybean oil (TM+SOY) and a canola
oil with exogenous docosahexaenoic (DHA) and arachidonic (ARA) acids (CO+EFA) were utilized.
At the conclusion of an eight week growth trial, whole body and fillet fatty acid profiles were
examined.
The TM+SOY diet worked equivalently to the USDA-ARS control diet, however the CO+EFA
diet resulted in signiﬁcantly lower growth and survival as well as an increased feed conversion
ratio. Whole body fatty acid proﬁles revealed a signiﬁcant reduction in total essential fatty acid
(EFA) concentration in the CO+EFA fed ﬁsh, indicating the supplemented levels of DHA and ARA
were insuﬃcient to meet requirements. Although all three diets were suﬃcient in the precursors
for EFA synthesis, this reduction in growth, survival, and whole body EFA concentration suggests
cobia have limited synthetic capacity for DHA and ARA. Both alternative lipid diets were devoidof eicosapentanoic acid (EPA) supplementation, which does not appear to be essential for cobia as
the TM+SOY diet performed equivalently to the ﬁsh oil control.
Key words: Cobia, Taurine, Docosahexaenoic Acid, Fishmeal and Fish Oil Replacement

2015年 1 月30日受理（Received on January 30, 2015）＊1
Assistant Marine Scientist, Marine Resources Research Institute, South Carolina Department of Natural Resources E-mail: WatsonA@dnr.
sc.gov＊2
U.S. Department of Agriculture, Agricultural Research Service＊3
Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science

52

Aaron M. WATSON, Frederic T. BARROWS, and Allen R. PLACEWorld fish supply has grown on average 3.2
percent per year over the last five decades,with much of the growth occurring in various

health are lacking in terrestrial oil sources whencompared to ﬁsh oil.
Taurine, a non-protein amino acid, is found in highaquaculture sectors (FAO, 2012). With worldwide

concentrations in ﬁshmeal, wild prey items, and meat

capture fisheries holding relatively static over thebased meals but is devoid from plant protein sources.

last few decades, and expected to remain at current

Taurine plays multiple important physiologicallevels, aquaculture must continue to expand rapidly

roles as an osmolyte, antioxidant, photoreceptor

to meet the protein demands of the ever increasingprotectant, and as a bile salt conjugate (Schuller-

world population. Traditionally, ﬁshmeal and ﬁsh oil

Levis and Park, 2003). Multiple studies have shownacquired through reduction of wild capture ﬁsheries

advantages to supplementing taurine when reducing

have served as major protein and lipid sources,

fishmeal in a variety of species (Chatzifotis

respectively, for compounded ﬁsh feeds utilized for

2008; Kim

many species in culture. In order for aquaculture., 2005, 2003; Lunger.,., 2007; Pinto., 2010).

to continue to grow at current and desired rates,

Long chain polyunsaturated fatty acids, such as

the inclusion levels of these ingredients must

docosahexaenoic (DHA) and arachidonic (ARA) acids

be continually reduced or eliminated, as theirare also not typically observed in terrestrial oils, andabundance is finite. The reduction of fishmeal andthe ability of teleost species to convert precursors

fish oil use as well as alternate protein and lipid

into these important fatty acids is typically low andsources have been studied extensively in manyhighly variable, rendering them essential for many

species and has been established as a priority formarine species. As with taurine and several otherresearch efforts and the aquaculture industry as acompounds, these fatty acids in particular must bewhole (Rustsupplemented to feeds utilizing alternative protein., 2011).

Reductions in productivity including decreased
growth rates, increased feed conversion ratios,or lipid sources.
Cobia,, is a fast growing

low feed intake, and negative health impacts havemarine carnivore whose natural diet is high in ﬁshbeen observed in many species when reducing

and crabs (Arendt

fishmeal below species specific threshold levels

and is therefore likely suﬃcient in both taurine and(Glencross., 2012; Luo., 1996).,

the essential fatty acids DHA and ARA. Cobia is a., 2012; Xie

promising species for intensive aquaculture as they., 1998). This is often related to deficiencies incan be spawned and maintained in captivity, are2006; Thiessen., 2011; Kader., 2001; Franks., 2004; Wacyk

amino acids, fatty acids, vitamins, or minerals thathighly fecund serial spawners, and have high quality

are not present in sufficient quantities in sources

ﬂesh suitable for multiple consumer types (Holtutilized as ﬁshmeal replacements. This is especially2007). A great deal of research has focused on cobia.,

true when utilizing plant proteins (wheat flour,

nutrition and reducing and replacing both ﬁshmeal

corn gluten, soybean meal, barley, etc) as plants

and fish oil with alternative sources to develop

have substantially different amino acid profiles

sustainable feeds (Lunger

when compared to fishmeals or other meat based

2010; Trushenski., 2006; Salze., 2012; Watson.,., 2012).

protein meals. Many of these deﬁciencies have been

Here, several studies were carried out withidentified (lysine, methionine, threonine) and canjuvenile cobia to determine the potential synthetic

be corrected for by simple crystalline amino acidcapacity for taurine present in various tissues and to

addition to feed formulations. One challenge brought

evaluate two ﬁshmeal and ﬁsh oil free diets.

about by reducing ﬁshmeal in favor of plant proteinsis in identifying deficiencies on a species specificMaterials and Methods

basis and then determining minimal and optimal
supplementation levels. Similarly, when reducing or

To examine the effects of dietary taurine on

replacing ﬁsh oil in favor of alternative lipid sources,

growth and performance, as well as the taurine

some specific fatty acids critical to growth andsynthetic capacity of juvenile cobia, four diets

Taurine and n-3 Fatty Acid Eﬀects on Juvenile Cobia
Table 1. Dietary formulations for the diets withgraded levels of taurine. From Watson. 2014.

53−1
canola oil plus exogenous DHA (~2.85 g kg ) andARA (~0.32 g kg−1) (CO+EFA diet) while the otherFM4experimental diet utilized a thraustochytrid meal

Menhaden Fish Meal
345
345
345
345
Corn Protein concentrate
44.3 44.3 44.3 44.3
Poultry by-product meal
118
118
118
118
Wheat Flour
242.7 237.7 227.7 192.7
Soybean meal, solvent
90
90
90
90
extracted
Blood meal, spray
39
39
39
39
dehydrated
Menhaden Fish Oil
90
90
90
90
20
20
20
20
Vitamin Pre-mix1
Choline CL66
6
6
Stay-C
2
2
2
2
1
1
1
1
Trace mineral pre-mix2
Mycozorb
2
2
2
2
Taurine0
515
50
1Contributed per kg diet; vitamin A, 9650 IU; vitamin D,
6.6 IU; vitamin E, 132 IU; menadione sodium bisulfite,
4.7 mg; thiamine mononitrate, 9.1 mg; riboflavin, 9.6
mg; pyridoxine hydrochloride, 13.7 mg; pantothenate,
DL-calcium, 101.1 mg; cyanocobalamine, 0.03 mg;
nictonic acid, 21.8 mg; biotin, 0.33 mg; folic acid, 2.5
mg.
2
Contributed in mg kg-¹ of diet; zinc 37; manganese, 10;
iodine, 5; copper, 1.

plus soybean oil (TM+SOY) as the lipid source. The

Ingredient (g kg-1)

FM1

FM2

FM3

control diet consisted of the same plant proteinbase as the experimental diets, but utilized ﬁsh oilas the lipid source. An eight week growth trial was
conducted beginning with ~130g juvenile cobia andat the conclusion growth and feed conversion ratio
were calculated, and fatty acid proﬁles of ﬁllets were
determined through gas chromatography (GC). Fullmaterials and methods are available in Watson.,

2013a.
Results
Full results of both studies are available in thecited references, only a brief synopsis is presented
here. Taurine supplementation to a ﬁshmeal (~35%)
diet did not have signiﬁcant eﬀects on growth rate,
protein eﬃciency ratio, total bile salt concentration,or feed conversion ratio (Table 3). Transcript
abundances of CDO, ADO, and TauT were also notaﬀected by dietary taurine input level in any of thewere formulated to contain 0.0%, 0.5%, 1.5%, andtissues examined (Fig. 1), however the rate limiting

5.0% supplemental taurine to a ﬁshmeal based dietstep of the reaction, cysteinesulﬁnate decarboxylase(Table 1). Juvenile cobia (~125g) were housed in a(CSD) was not detected through the q-PCR methods

recirculating aquaculture system at the Institute ofutilized. Tissue taurine levels for plasma, liver, andMarine and Environmental Technology in Baltimore,

muscle did increase with increasing dietary taurine.

Maryland, USA and fed for 8 weeks. At the

In contrast, taurine supplementation to theconclusion of the eight week feeding trial, growthplant protein based diet did have significant

and feed conversion ratios were calculated, andeffects on growth rate and feed conversion ratio

tissues (muscle, liver, and brain) were preserved inalthough overall growth was significantly lower

RNA later for quantitative-PCR analysis of several

in all treatments of the plant protein formulation

genes involved in taurine synthesis. Plasma, liver,when compared to the fishmeal based series

and muscle tissues were also taken for taurine(Table 4). Similarly to the ﬁshmeal series, transcript

content analysis. Full materials and methods areabundances for CDO, ADO, and TauT wereavailable in Watson

unaﬀected by dietary taurine input (Fig. 1).

. (2014). A parallel study

was conducted with a fishmeal free, plant protein

In the fish oil replacement study, the TM+SOY

based formulation also containing 0.0%, 0.5%, 1.5%,

diet performed similarly to the ARS control fish

and 5.0% supplemental taurine and similar analyses

oil diet while the CO+EFA diet significantly

were conducted at the conclusion of the trial.

underperformed in regards to growth rate, feed

To examine the effects of fish oil replacement

conversion ratio, and survival (Table 5; Watson

in a fishmeal free, plant protein based diet similar., 2013a). The diﬀerent lipid sources utilized in themethods were employed. Two experimental diets

diets did affect whole body and fillet lipid profiles,

were formulated and manufactured by the USDA-

with direct similarity to the profiles of the diets

ARS (Table 2). One experimental diet utilized(Fig. 2; Watson., 2013a). The alternate lipid

54

Aaron M. WATSON, Frederic T. BARROWS, and Allen R. PLACE
Table 2. Diet formulations and fatty acid compositions of the diets used for ﬁsh
oil replacement. FromWatson., 2013a.

Ingredient (g kg-1)
Soy Protein Concentrate
Corn Gluten
Wheat Flour
Soybean Meal
Solvent Extracted
Menhaden Oil
Soybean Oil
Algamac 3050
Canola Oil + DHA + ARA
Dicalcium Phosphate
Vitamin Pre-mix1
Lysine-HCL
Choline CL
Trace Mineral Pre-mix2
Magnesium Oxide
Stay-CDL-Methionine
Threonine
Potassium Chloride
TaurineFatty Acid (g 100g-1)1
12:0
14:0
16:0
17:0
18:0
20:0
22:0
SFA3
16:1Q-7
18:1Q-7
18:1Q-9+6
20:1Q-15+cis-8
20:1Q-9
24:1Q-9
MUFA4
16:3Q-4
16:4Q-1
18:2Q-6
18:3Q-3
20:2Q-6
20:4Q-6
20:5Q-3
22:5Q-6
22:5Q-3
22:6Q-3
PUFA5
Q-36
Q-67
Q-3:Q-6
1

TM+SOY
269.3
211
226.5

Diet
CAN+EFA
269.3
211
226.5

ARS Control
269.3
211
226.5

121
0
5
79
0
23.7
10
15.5
6
1
0.5
3
5.8
2.1
5.6
15

121
0
0
0
84
23.7
10
15.5
6
1
0.5
3
5.8
2.1
5.6
15

121
84
0
0
0
23.7
10
15.5
6
1
0.5
3
5.8
2.1
5.6
15

0.15
4.33
17.17
0.16
2.77
0.28
0.24
25.10
0.23
0.87
13.86
0.06
0.14
0.0
15.15
0.05
0.04
33.27
3.94
0.04
0.70
0.42
6.09
0.16
14.93
59.75
19.45
40.10
0.49

0.34
1.21
7.73
0.15
3.49
0.82
0.38
14.12
0.46
2.58
48.28
0.03
0.76
0.13
52.24
0.06
0.06
22.86
6.25
0.13
0.32
0.27
0.66
0.16
2.85
33.64
9.53
23.97
0.40

0.10
4.88
22.89
0.47
5.31
0.30
0.14
34.08
5.44
2.46
19.53
0.22
1.21
0.32
29.18
0.57
0.79
12.74
1.19
0.18
0.84
8.90
0.31
1.51
8.57
36.75
20.17
14.07
1.43

Contributed per kg diet; vitamin A, 13510 IU; vitamin D, 9.2 IU; vitamin E, 184.4 IU;
menadione sodium bisulfite, 6.6 mg; thiamine mononitrate, 12.7 mg; riboflavin, 13.4 mg;
pyridoxine hydrochloride, 19.2 mg; pantothenate, DL-calcium, 141.5 mg;
cyanocobalamine, 0.04 mg; nictonic acid, 30.5 mg; biotin, 0.46 mg; folic acid, 3.5 mg.
2Contributed in mg kg-¹ of diet; zinc 37; manganese, 10; iodine, 5; copper, 1.
3
Saturated fatty acids = sum of all fatty acids without double bonds.
4
Monounsaturated fatty acids = sum of all fatty acids with a single double bond.
5
Polyunsaturated fatty acids = sum of all fatty acids with two or more double bonds.
6
Sum of all n-3 fatty acids.
7
Sum of all n-6 fatty acids

55

Taurine and n-3 Fatty Acid Eﬀects on Juvenile Cobia
Table 3. Performance characteristics from the graded taurinein a ﬁshmeal diet growth trial. Within arow, diﬀerent letters indicate signiﬁcant diﬀerences ( <0.05). From Watson. 2014.

Diet
Initial Weight (mean ± SD)
Survival (%)
Weight Gain (g fish-1)1
FCR2
SGR3
PER4
HSI 5
Total Bile Salts (mM)

FM1
127.44 ± 13.31
100
227.83 ± 18.15
1.52 ± 0.10
2.1 ± 0.12
1.35 ± 0.15
3.14 ± 0.14
28.48 ± 4.79

FM2
122.70 ± 14.97
100
297.62 ± 24.20
1.42 ± 0.02
2.2 ± 0.04
1.44 ± 0.04
2.99 ± 0.18
32.69 ± 6.04

FM3
130.64 ± 12.37
100
313.47 ± 31.36
1.43 ± 0.11
2.2 ± 0.14
1.26 ± 0.43
2.78 ± 0.45
30.02 ± 4.34

FM4127.36 ± 14.52
100
290.08 ± 33.11
1.51 ± 0.05
2.1 ± 0.09
1.29 ± 0.07
2.72 ± 0.19
29.59 ± 2.32

1 ሺሻ ൌ  ሺሺሻ െ ሺሻ
୤୭୭ୢ୤ୣୢሺ୥ሻ ሺ ሻ ൌ ሺሻ23୵ୣ୧୥୦୲୥ୟ୧୬ୣୢሺ୥ሻ୪୬ ୤୧୬ୟ୪୵ୣ୧୥୦୲ሺ୥ሻି୪୬ ୧୬୧୲୧ୟ୪୵ୣ୧୥୦୲ሺ୥ሻ

4ୢୟ୷ୱ୭୤୥୰୭୵୲୦୲୰୧ୟ୪
୵ୣ୧୥୦୲୥ୟ୧୬ୣୢሺ୥ሻ   ሺ ሻ ൌ ͳͲͲ ‫ כ‬ሺ  ሺሻ ൌ ሺ

5

ሻ

ሻ

୮୰୭୲ୣ୧୬୤ୣୢሺ୥ሻ
୪୧୴ୣ୰୵ୣ୧୥୦୲ሺ୥ሻ

  ሺ  ሻ ൌ ͳͲͲ ‫ כ‬ሺሻୠ୭ୢ୷୵ୣ୧୥୦୲ሺ୥ሻ

Table 4. Performance characteristics from the graded taurine in a plant based diet growth trial (128.37
g initial weight). Within a row, values that share common superscripts are not signiﬁcantly diﬀerent
from one another ( >0.05).

Diet (Taurine %)Weight Gain (%)
FCR1
SGR2
Total Bile Salts (mM)

PP1 (0.02)
PP2 (0.39)
a23.35 ± 22.82 80.57 ± 60.24a
6.38 ± 1.49a
2.97 ± 1.17ba0.57 ± 0.12
1.31 ± 0.26b
38.76 ± 7.40
28.37 ± 1.94

PP3 (1.35)
130.87 ± 25.20b
1.98 ± 0.19b
1.47 ± 0.19b
36.30 ± 5.69

PP4 (4.08)
133.82 ± 10.83b
2.12 ± 0.23b
1.51 ± 0.09b
28.75 ± 3.22

୤୭୭ୢ୤ୣୢሺ୥ሻ

1 ሺ ሻ ൌ ሺ୵ୣ୧୥୦୲୥ୟ୧୬ୣୢሺ୥ሻሻ୪୬ ୤୧୬ୟ୪୵ୣ୧୥୦୲ሺ୥ሻି୪୬ ୧୬୧୲୧ୟ୪୵ୣ୧୥୦୲ሺ୥ሻ

2

   ሺ ሻ ൌ ͳͲͲ ‫ כ‬ሺୢୟ୷ୱ୭୤୥୰୭୵୲୦୲୰୧ୟ୪

ሻ

Table 5. Production characteristics from the eight week grow out trial with ﬁsh oil replacement sources.
Data from a previous grow out with juvenile cobia on a ﬁsh meal and ﬁsh oil based, commercially produced
diet are included to show overall eﬀects of ﬁsh meal and ﬁsh oil replacement. From Watson., 2013a.

p

Weight Gain (%)1
Fillet Yield (%)2
FCR3
Plasma Osmolality
PCB Content (ng g-1)
Mercury Content(ng g-1)
Hepatosomatic index4
Specific Growth Rate5
Survival
1

TM+SOYCAN+EFAARS Control

Commercial Diet(2009 grow out data)6288a
25.93 ± 3.34
1.42a358.25 ± 30.59a
8.40 ± 1.54
30.20 ± 3.53
1.83 ± 0.19a
2.48a
100%b

117b
20.88 ± 4.34
2.98b
311.75 ± 36.82b9.30 ± 3.41
48.17 ± 16.24
2.93 ± 0.79b
1.42b
58%a

275a
25.71 ± 1.90
1.46a327.58 ± 13.83a,b13.2 ± 4.2
20.14 ± 2.96
2.66 ± 0.55a,b
2.36a
98%b

218
20.12 ± 0.01
1.85
na
45.5 ± 3.8
71.37 ± 3.56
3.15 ± 0.002
1.93
100%

Weight gain=(final tank weight - initial tank weight)/ initial tank weight*100.
Fillet yield=(fillet weight/body weight)*100.
3
FCR=Feed conversion ratio= grams fed/grams gained.
4
Hepatomsomatic index=liver weight/ body weight.
5
SGR=specific growth rate= ((lnBW2-lnBW1)*(days of growth trial-1))*100.
6
Initital Weight 120g, eight week growth trial (Watson HWDO 2012).
Values in the same row with different superscripts are significantly different (p<0.05), no superscript indicates no significant difference within acategory.
2

56

Aaron M. WATSON, Frederic T. BARROWS, and Allen R. PLACE

CDO

Liver
ADO

B-1
TauT

15

20

Percent expression compared
to beta actin

Percent expression compared
to beta actin

A-1 2000PP1(0.02%)

B-2 10
Brain
CDO

ADO

TauT

6

4

2

Percent expression compared tobeta actin

Percent expression compared tobeta actin

A-2 10

8

TauT

4

2

PP1 (0.02%) PP2 (0.39%) PP3 (1.35%) PP4 (4.08%)

B-3 0.40
MuscleCDO ADO

TauT

Percent expression compared tobeta actin

Percent expression compared tobeta actin

A-3 0.30

0.05

Brain
ADO

FM1
(0.24%)

6

FM1 (0.24%) FM2 (0.44%) FM3 (1.41%) FM4 (4.55%)

0.10

CDO

PP4
(4.08%)

0

0

0.15

PP3
(1.35%)

5

FM1 (0.24%) FM2 (0.44%) FM3 (1.41%) FM4 (4.55%)

0.20

PP2
(0.39%)

TauT

10

5

0.25

Liver
ADO

15

10

8

CDO

0.35
0.30

CDO

Muscle
ADO

TauT

0.25
0.20
0.15
0.10
0.05
0.00

0.00
FM1 (0.24%) FM2 (0.44%) FM3 (1.41%) FM4 (4.55%)

PP1 (0.02%) PP2 (0.39%) PP3 (1.35%) PP4 (4.08%)

Fig. 1. Relative transcript expression (mean ±SE) of genes involved in taurine synthesis and transport injuvenile cobia,, as a percent expression compared to reference gene beta-actin for eachdiet (% taurine) in liver (1), brain (2), and muscle (3). Equivalent cDNA input (10 ng) for triplicate samples ofeach tissue and six ﬁsh sampled per treatment group and tissue ( = 6 per data point). Numbers in parenthesis
indicate measured taurine in each diet. Panels A-1, A-2, and A-3 depict results from Watson. (2014). withﬁshmeal based diet series and panels B-1, B-2, and B-3 depict results from ﬁshmeal free, plant protein based dietseries.

Taurine and n-3 Fatty Acid Eﬀects on Juvenile Cobia

Fig. 2. Fatty acid compositions of fillet and whole body tissues from juvenile
cobia fed experimental ﬁsh oil replacement diets expressed as a fraction of dietary
total lipid profile. Values were calculated from relative fatty acid methyl ester(FAME) composition (Fillet or whole body fatty acid concentration/Diet fatty acidconcentration). Based on this calculation, a value of 1 represents equality between
ﬁllet and dietary fatty acid composition. From Watson., 2013a.

57

58

Aaron M. WATSON, Frederic T. BARROWS, and Allen R. PLACE

source diets also resulted in lower total lipid content

type of work will allow for the aquaculture industryof the fillets with the TM+SOY diet resulting into continue to expand to meet growing global

5.81 ± 0.77% lipid, the CO+EFA diet resulting inneeds. Species specific work also has the potential

5.53 ± 3.15% lipid, and the control ARS ﬁsh oil dietto produce optimal diets which not only maximize

resulting in 7.90 ± 1.75% lipid.

production but reduce losses due to waste. These
two characteristics, high production and low waste,

Discussion

will be critical in expanding aquaculture operationsof various scales and intensity while maintaining

These studies illustrate the importance ofprofitability around the world as global priorities

determining species specific requirement levels

shift to food and water supply safety and thefor compounds which need to be supplemented

environmental impacts of aquaculture.

separately to feeds. Results from the taurine
supplementation to a fishmeal based diet indicate

Referencesthat diets containing at least 35% fishmeal containsufficient taurine and do not require additional
supplementation. However, the trial with a ﬁshmeal

Arendt M. D., Olney J. E., and Lucy J. A., 2001:
Stomach content analysis of cobia,

free, plant protein based formulation reveal negativefrom lower Chesapeake Bay.

eﬀects of low taurine such as low growth and high., 99, 665 670.

feed conversion, which are alleviated by increasing

Chatzifotis S., Polemitou I., Divanach P., anddietary taurine, up to ~5.0% supplementation. These

Antonopoulou E., 2008: Eﬀect of dietary taurine

effects, combined with the lack of modulation at

supplementation on growth performance andthe transcript level of genes involved in potential

bile salt activated lipase activity of common

taurine synthesis and transport appear to indicate

dentex,that juvenile cobia are unable to synthesize taurine

protein concentrate-based diet.

in sufficient quantities, if at all. Taurine should

201 208.

therefore be considered essential, especially whenreducing ﬁshmeal in favor of plant based proteins.
DHA and ARA levels were most likely insuﬃcient, fed a fish meal/soy., 275,

FAO, 2012: The State of World Fisheries andAquaculture. food Agric. Organ. United Nations
Rome, Italy.

in the CO+EFA diet to meet the nutritionalFranks J. S., Garber N. M., and Warren J. R.,

requirements of juvenile cobia. This also potentially

1996: Stomach contents of juvenile cobia,

indicates limited or no synthetic capacity forthese specific fatty acids in this species. Giltheadsea bream,, in contrast performed, from the northern Gulf
of Mexico.

., 94, 374 380.

Glencross B., Rutherford N., and Jones B., 2011:equivalently on all three of the exact same diets

Evaluating options for ﬁshmeal replacement infrom this study (Watson

diets for juvenile barramundi (., 2013b). The diﬀerencein performance of these two species on the same
diets could highlight the difference in requirementlevels for these fatty acids between species, or., 17, 722 732.
Holt G. J., Faulk C. K., and Schwarz M. H., 2007: A
review of the larviculture of cobia

different synthetic or conversion capabilities
between these two carnivores.
Taken together, these studies demonstrate the).

, a warm water marine ﬁsh.

.,268, 181 187.
Kader M. A., Bulbul M., Koshio S., Ishikawa M.,

ability to produce fishmeal and fish oil free diets

Yokoyama S., Nguyen B. T., and Komilus C.

for a fast growing, high value marine carnivore

F., 2012: Effect of complete replacement of

without signiﬁcant loss of production characteristics.

ﬁshmeal by dehulled soybean meal with crude

Targeted supplementation of specific compounds

attractants supplementation in diets for red sea

found to be lacking in fishmeal and fish oil

bream,

replacement sources can alleviate deﬁciencies. This116.

., 350‑353, 109

59

Taurine and n-3 Fatty Acid Eﬀects on Juvenile Cobia
Kim S., Takeuchi T., Akimoto A., Furuita H.,

new implications for an old amino acid.

Yamamoto T., Yokoyama M., and Murata Y.,., 226, 195 202.

2005: Effect of taurine supplemented practicalThiessen D. L., Maenz D. D., Newkirk R. W., Classen

diet on growth performance and taurine

H. L., and Drew M. D., 2004: Replacement ofcontents in whole body and tissues of juvenile

ﬁshmeal by canola protein concentrate in diets

Japanese ﬂounder

fed to rainbow trout (., 71, 627 632.

).

., 10, 379 388.

Kim S., Takeuchi T., Yokoyama M., and Murata Y.,

Trushenski J., Schwarz M., Bergman A., Rombenso

2003: Effect of dietary supplementation with

A., and Delbos B., 2012: DHA is essential,

taurine, b-alanine and GABA on the growth

EPA appears largely expendable, in meetingof juvenile and fingerling Japanese flounder

the n‑3 long-chain polyunsaturated fatty acid., 69, 242 248.

requirements of juvenile cobia

Lunger A. N., Craig S. R., and McLean E., 2006:
Replacement of ﬁsh meal in cobia (Wacyk J., Powell M., Rodnick K., Overturf K., Hill

) diets using an organically certified
protein.

., 326‑329, 81 89.

., 257, 393 399.

R. A., and Hardy R., 2012: Dietary protein
source signiﬁcantly alters growth performance,

Lunger A. N., McLean E., Gaylord T. G., Kuhn D.,

plasma variables and hepatic gene expression

and Craig S. R., 2007: Taurine supplementationin rainbow trout (to alternative dietary proteins used in ﬁsh mealamino acid balanced diets.

replacement enhances growth of juvenile cobia().

., 271, 401) fed., 356‑357,

223 234.
Watson A. M., Barrows F. T., and Place A. R., 2014:410.

Effects of graded taurine levels on juvenile

Luo L., Xue M., Wu X., Cai X., Cao H., and Liang Y.,

cobia,

2006: Partial or total replacement of ﬁshmeal bysolvent-extracted cottonseed meal in diets forjuvenile rainbow trout ().

., 12, 418 424.

., 76, 190‑200.
Watson A. M., Barrows F. T., and Place A. R., 2013a:
Taurine supplementation of plant derived
protein and n-3 fatty acids are critical for

Pinto W., Figueira L., Ribeiro L., Yúfera M., Dinis

optimal growth and development of cobia,

M. T., and Aragão C., 2010: Dietary taurine
supplementation enhances metamorphosis andgrowth potential oflarvae.

., 309, 159 164.

, 48:9, 899‑913.
Watson A. M., Barrows F. T., and Place A. R., 2013b:
Taurine supplemented plant protein based
diets with alternative lipid sources for juvenile

Rust M. B., Barrows F. T., Hardy R. W., Lazur A. L.,

gilthead sea bream

Naughten K., and Silverstein J., 2011: NOAA/., 4, 59 66.

USDA Alternative Feeds Initiative. The Future

Watson A. M., Kissil G. W., Barrows F. T., and Placeof Aquafeeds. NOAA Tech. Memo. NMFS‒F/

A. R., 2012: Developing a plant-based diet for

SPO‒124 103 p.

cobia

Salze G., McLean E., Battle P. R., Schwarz M. H., and, 15,

34 38.

Craig S. R., 2010: Use of soy protein concentrate

Xie S., He X., and Yang Y., 1998: Eﬀects on growthand novel ingredients in the total elimination ofand feed utilization of Chinese longsnout catﬁsh

ﬁsh meal and ﬁsh oil in diets for juvenile cobia,

Günther of replacement of., 298, 294 299.
Schuller Levis G. B., and Park E., 2003: Taurine:

dietary ﬁshmeal by soybean cake.
4, 187 192.

.,

水研センター研報，第40号，61−65，平成27年
Bull. Fish. Res. Agen. No. 40，61−65，2015

61

Availability of Fisheries By-Product Materials with Cadmium Removal
Treatment as a Feed Ingredient for Fingerling Black Rockﬁsh

Nobukazu SATOH＊1, Motoomi WAKASUGI＊2, and Shigeharu NOBUTA＊3

Abstract: Squid liver and scallop-mid gut glands, which are generated and discarded as waste fromthe Japanese common squid and Japanese scallop, are rich in amino acids and lipids. However,the squid liver and scallop mid-gut glands contain cadmium (Cd) which is generally known as aharmful heavy metal for human health. Hirama.＊4 recently studied a technique for removal ofCd from squid viscera and scallop mid-gut glands and conﬁrmed the eﬃcacy of the acid leachingand electrolysis method to produce the ﬁsheries by-product materials (meal and extract) with Cd
removal treatment as a feed ingredient for cultured ﬁsh.
So far we have evaluated the nutritional value of squid viscera meal with Cd removal treatment(dCSVM), which contained 1.5‑2.0 mg/kg Cd, as an alternative protein source to sardine meal indiets for fingerling black rockfish. In our study, it was clearly demonstrated

that dCSVM with good protein digestibility could be substituted for 60% of sardine meal in diets
for ﬁngerling black rockﬁsh without growth retardation, poor palatability and the problem of Cd
accumulation. We suggested that dCSVM was superior to commercial squid viscera meal without
Cd removal treatment (CSVM) as a high-quality feed ingredient for black rockﬁsh based on theheavy metal accumulation in ﬁsh tissues.
Recently, we are investigating availability of mid-gut glands extract with Cd removal treatment(dCSMGE) as a feed ingredient for ﬁngerling black rockﬁsh. In the recent study, we found thatweight gain, speciﬁc growth rate, and feed eﬃciency of ﬁsh fed the diet containing dCSMGE at
2% were signiﬁcantly higher than those of the control. Furthermore, we conﬁrmed that test diets
containing dCSMGE was safe in terms of accumulation of Cd.
These results demonstrate that proper inclusion of dCSVM and dCSMGE is eﬀective for the
improvement of feed quality in practical diets for ﬁngerling black rockﬁsh.
Key words: squid viscera, scallop mid-gut glands extract, cadmium, alternative ingredients for ﬁshmeal with Cd removal treatment, black rockﬁsh

The increasing feed cost in aquaculture

feed, in diets has been increasingly realized (Gatlin

production due to the increasing price of ﬁsh meal., 2007). However, it is widely recognized thathas been a serious problem recently. Therefore, thehigh inclusion of alternative protein source in diets

importance of studies on alternative protein sources

may result in poor palatability, particularly at low

for fish meal, a major ingredient for aquaculture

temperatures.

2015年 1 月30日受理（Received on January 30, 2015）＊1
Fisheries Research Department Mariculture Fisheries Research Institute, Hokkaido Research Organization, Muroran, Hokkaido 051‑0013, Japan
E-mail: sato-nobukazu@hro.or.jp＊2
IndustrialResearch Institute, Hokkaido Research Organization, Sapporo, Hokkaido 060‑0819, Japan＊3
Fisheries Research Department Kushiro Fisheries Research Institute, Hokkaido Research Organization, Kushiro, Hokkaido 085‑0027, Japan＊4
Hirama M., WakasugiM., TomitaK., KamadaT., TakahashiT., NobutaS., AsoS., SatohN., and IshidaR., 2013: Studies on the technique for removalof Cadmium from scallop mid-gut glands to produce ﬁsheries by-product materials with Cadmium removal treatment as a feed ingredient forﬁsh farming (in Japanese). Abstracts of the Annual Meeting of the Japanese Society of Fisheries Science. Japanese Society of Fisheries Science,
Mie, Japan, September, p.77.

62

Nobukazu SATOH, Motoomi WAKASUGI, and Shigeharu NOBUTA

Fig. 1. Original production method of scallop mid-gut glands exract with Cd removal treatment (dCSMGE).
Squid liver, squid viscera and scallop mid-gut

species in Japan (Nakagawa., 2007) and Korea

glands, which are generated and discarded as(Bai, 2009) that is cultured for stock enhancement

waste from the Japanese common squidand aquaculture. However, information on theand Japanese scallop

alternative protein sources of ﬁsh meal in diets for, are rich in amino acids and lipids (Sakuta

aquaculture is still limited. Aso. (1999) reported

and Shimakage, 2004). Particularly, by-products ofthat scallop viscera meal with Cd removal treatment

squid processing are important feed ingredients incould substitute 10 % of brown fish meal in theaquaculture shrimps diets, since the diets contain

diets for fingerling black rockfish.Lim

squid by-products are eﬀective for the improvement

showed that dehulled soybean meal could replaceof growth performances and feed utilization infish meal up to 20% withoutsupplementation of

prawn species (Hertramp and Piedad-Pascual, 2000).

methionine and lysine and 30% with these aminol. (2004)

In Hokkaido, Japan, these fisheries wastes areacids being supplemented in ﬁngerling and growing

generated and wasted every year, and the volumes

black rockfish. Therefore, we studied availabilityof squid viscera and scallop mid-gut glands wereof dCSVM and dCSMGE as a feed ingredient for

9,324 and 30,533 metric ton in the 2010 ﬁscal year.

ﬁngerling black rockﬁsh.The present article reviews

However, the squid liver and scallop mid-gut
glands contain cadmium (Cd) at 19 and 39 mg per

information on the feed values of dCSVM anddCSMGE for black rockﬁsh.

kg wet weight on average, respectively (Wakasugi., 2005; Kruzynski, 2004). Cd is generally known

Availability of squid viscera as a feed ingredientfor cultured-ﬁsh

as a harmful heavy metal for human health (Mai., 2006). Wakasugi. (2009) recentlystudied

a technique (Fig. 1) for removal of Cd from squid＊5

Availability of various by-products in ﬁsheries as

and conﬁrmed

a feed ingredient for aquaculture fish species was

the efficacy of acid leaching and electrolysis tostudied recentlyby many researchers, because by-produce fisheries by product materials (mealproducts fromfisheries hold promise as potentialand extract) with Cd removal.This material was

protein substitutes for ﬁsh meal (Liviscera and scallop mid-gut glands

evaluated as a feed ingredient for cultured fish., 2004).

It has been reported that extrudeddiets containing(Wakasugi, 2009). Consequently, they have developed

20‑30 % raw squid liver could be used for rainbow

squid viscera mealand scallop mid-gut glands exract

troutwith Cd removal treatment, dCSVM and dCSMGE,
respectively.
Black rockfish,＊5, and red seabream(Mastuda

an important, yellowtail., 2001a, b). Feeding studies haveshown that the commercial squid viscera meal

Hirama M., WakasugiM., TomitaK., KamadaT., TakahashiT., NobutaS., AsoS., SatohN., and IshidaR., 2013: Studies on the technique for removalof Cadmium from scallop mid-gut glands to produce ﬁsheries by-product materials with Cadmium removal treatment as a feed ingredient forﬁsh farming (in Japanese). Abstracts of the Annual Meeting of the Japanese Society of Fisheries Science. Japanese Society of Fisheries Science,
Mie, Japan, September, p.77.

63

Fisheries By-Product Materials as a Feed Ingredient for Fingerlingwithout Cd removal treatment (CSVM) can be usedof dCSVM inclusion. In that paper, we suggested

as an alternative protein source in diets for variousthat dCSVM was superior to CSVM as a high-fish species; In fingerling black rockfish, Satohquality feed ingredient for black rockfish based. (2006) showed that CSVM could be substitutedon the heavy metal accumulation in fish tissues.

for 30% of sardine meal in a diet when growth

Furthermore, we found that appropriate replacement

performance was used as the dependent variable,(30‑60%) of sardine meal by dCSVM was effective

while substitution of only 10% of sardine meal

for stimulation of feeding in ﬁngerling black rockﬁsh,

could be recommendedin terms of the heavy metal

and that supplementation of methionine and lysine

accumulation in the liver. Mai

was not necessary with the inclusion of dCSVM.

. (2006) evaluatedthe effects of dietary CSVM on the growth andFrom these results, we concluded that dCSVMwas

Cd accumulation in tissues of Japanese seabass,safe and useful as an alternative protein source in. They demonstrated thatﬁngerling black rockﬁsh diets.

growth rates in ﬁsh fed diets contained 50 and 100g
CSVM per kg diet were significantly higher than

Availability of dCSMGE as a feed ingredient forthat in control fish, and that Japanese seabass did

ﬁngerling black rockﬁsh

not accumulate Cd in the muscle (edible portion),
although Cd concentrations in fish kidneys, livers

We also investigated availability of dCSMGE,

and gills were at high levels (0.4‑5.9 mg per kg drywhich contains about 0.5 mg/kg Cd, as a feed

weight). In the paper, they also pronounced that a＊4
ingredient for ﬁngerling black rockﬁsh . The total

long-term feeding trial was necessary to investigate

free amino acids content (mg/kg dry matter) of

Cd accumulation in cultured Japanese seabass. MoredCSMGE is about 30,000, while that of ﬁsh meal isrecently, Wang

about 800. dCSMGE contain amino acids effective. (2012) reviewed that several

ﬁsh diets supplemented with CSVM contained highfor feeding stimulants and growth enhancement

Cd levels, 51‑116 mg/kg diet.

in various fish species (Mackie and Mitchell, 1985,
Shimizu., 1999, Ikeda., 2012). In the recent

Availability of dCSVM as a feed ingredient for

feeding trial for 65 days, we found that weight gain,

ﬁngerling black rockﬁsh

speciﬁc growth rate, and feed eﬃciency of ﬁsh fed
the diet containing dCSMGE at 2% of the diet were

We have evaluated the nutritional value ofsignificantly higher than those of the control ＊6.

dCSVM, which contained 1.5‑2.0 mg/kg Cd, as an

Moreover, we confirmed that test diets containing

alternative protein source to sardine meal in diets

dCSMGE was safe in terms of accumulation of Cd＊6.

for fingerling black rockfish (Satoh., 2013a).

The Cd level of dCSVM was less than 3.0 mg/

Kumai. (1989) found that commercial

scallop extract as a flavor in the diet for ocelatekg, which is the allowable level of Cd in livestock

puﬀer

feed ingredients according to the Law Concerning

digestive and absorptive functions in the ocelate

promoted feed intake and

Safety Assurance and Quality Improvement of

puﬀer. Kikuchi and Furuta (2009) reported that blue

Feeds in Japan. In this study, it was demonstrated

mussel extract in diets based on fish and soybeanthat dCSVMwith good protein digestibility could

meals for tiger puﬀer would be an eﬀective feeding

be substituted for 60% of sardine meal in diets forstimulant. We observed that dCSMGE improved

juvenile black rockﬁsh without growth retardation,

feeding activity of barfin flounderpoor palatability and the Cd accumulation, although

in the short-term feeding experiment at low

feed eﬃciency gradually decreased with the increase

temperatures2 ＊ 7. dCSMGE may be also an eﬀective＊6＊7

Satoh N., IshidaR., NobutaS., AsoS., WakasugiM., TomitaK., HiramaM., and TakahashiT., 2013: Availability of scallop mid-guts extract withcadmium removal treatment as a feed ingredient for ﬁngerling black rockﬁsh (in Japanese). Abstracts of the Annual Meeting of the JapaneseSociety of Fisheries Science.Japanese Society of Fisheries Science, Mie, Japan, September, p.42.
Satoh N., IshidaR., NobutaS., AsoS., WakasugiM., TomitaK., HiramaM., and TakahashiT., 2013: Availability of scallop mid-guts extract withcadmium removal treatment as a feed ingredient for juvenile barﬁn ﬂounder (in Japanese). Abstracts of the Annual Meeting of the JapaneseSociety of Fisheries Science. Japanese Society of Fisheries Science, Tokyo, Japan, March, p.135.

64

Nobukazu SATOH, Motoomi WAKASUGI, and Shigeharu NOBUTA

feed ingredient for the improvement of growth

Utilization of scallop visceral meal in the diets

performance, feed utilization, and feeding activity infor fingerling rockfish,ﬁsh.

Japnanese with English abstract).

Krill extract and meal are often added toaquaculture feed to improve of platability (Shimizu,
1999) and digestibility (Satoh, 2003), but the(in., 56, 143‑147.
Bai S. C., and Okorie O. E., 2009: Korean rockfish
production in Korea.

supplementation in the diets is mostly limited for, Jan./Feb, 41‑42.

larval and juvenile ﬁsh because of the high price. To

GatlinⅢ D. M., Barrows F. T., Brown P., Dabrowski

date, we estimate the cost of producing dCSMGE

K., Gaylord G., Hardy R. W., Herman E., Hu G.,(semi-liquid form) will be below half of cost of krill

Krogdahl A., Nelson R., Overturf K., Rust M.,

ingredients. On the other hand, the producing cost of

Sealey W., Skonberg D., Souza E. J., Stone D.,

dCSMGE in powder form is higher than that in semi-

Wilson R., and Wurtele E., 2007: Expanding

liquid form, since producing of dCSMGE powder

the utilization of sustainable plant products

requires energy to dry. Interestingly, Kaderinaquafeeds: a review.

.

(2012) found that fermented soybean meal and squid., 38, 551‑

579.

by-product blend (1:1) could be substituted for about

Hertramph J. W., and Piedad Pacual F., 2000:

40% of fish meal protein in the diet of Japanese

Handbook on ingredients for aquaculture feeds.

flounder. Likewise, fermented soybean meal and1st ed., Kluwer Academic, Dordrecht, p.403‑410.

scallop by-product blend (1:1) was able to replace

Ikeda I., Okamoto Y., and Oda K., 2012: Identiﬁcation

at least 30% ﬁsh meal protein in red seabream dietof feeding stimulants for Japanese flounder in(Kadermuscle extract of Jack mackerel.

., 2011). These blends are effective for

producing low-cost and healthy aquaculture feed,
and the fermentation technique may be an eﬀective
measure of producing dCSMGE powder.

.,60, 195‑198.
Kader M. A., Koshio S., Ishikawa M., Yokoyma
S., Bulbul M., Honda Y., Mamauag R. E., andLaining A., 2010: Growth, nutrient utilization,

Conclusionoxidative condition, and element composition ofjuvenile red sea bream

Our results demonstrate that proper inclusion

fed withfermented soybean meal and scallop by-productof dCSVM and dCSMGE is effective for the

blend as fish meal replacement.

improvement of feed quality in practical diets for

119‑128.

., 77,

ﬁngerling black rockﬁsh. Future research is needed

Kader M. A., Koshio S., Ishikawa M., Yokoyma S.,

to clarify the nutritional value of dCSVM and

Bulbul M., Nguyen B. T., GaoJ., and Laining A.,

dCSMGE as a feed ingredient for aquaculture fish

2012: Can fermented soybean meal and squid by-

species other than black rockﬁsh.

product blend be used as ﬁshmeal replacements
for Japanese flounder (Acknowledgements)?

., 43, 1427‑1438.
Kikuchi K., and Furuta T., 2009: Inclusion of blue

The research was financially supported bymussel extract in diets based on fish anda Research Project for Utilizing Advanced

soybean meals for tiger puﬀer

Technologies in Agriculture, Forestry, and Fisheries., 75, 183‑189.

(grant no. 18031), Ministry of Agriculture, Forestry,

Kumai H., Kimura I., Nakamura M., Takii K., andand Fisheries, Government of Japan and partly by

Ishida H., Studies on digestive system andHokkaido Government.

assimilation of a ﬂavored diet in Ocellate puﬀer.
, 55, 1035‑1043.

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Fisheries By-Product Materials as a Feed Ingredient for Fingerling
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Min T. S., and Bai S. C., 2004: Eﬀects of dehulledsoybean meal as a ﬁsh meal replacer in diets forﬁngerling and growing Korean rockﬁshhydrometallurgical process (in Japanese withEnglish abstract).

, 120, 71‑77.

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extract and krill meal to diet on the growth, 231, 457‑468.

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Feeding in Fish, Academic Press, London, p.177‑

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diets of fingerling rockfish (in Japanese withMai K., Li H., Ai Q., Duan Q., Xu W., Zhang C., Zang
L., Tan B., and Liufu Z., 2006: Eﬀects of dietary

English abstract).

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squid viscera meal on growth and cadmium

Satoh N., Nobuta S., Wakasugi M., Satoh S., andaccumulation in tissues of Japanese seabass,

Takeuchi T., 2013: Availability of squid viscera(Cuiver 1828).

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meal with cadmium removal treatment as a feed
ingredient for ﬁngerling black rockﬁshMatsuda Y., Hayashi A., Yoshitomi B., TakeuchiT., 2001: Preliminary studies on effect of the
supplementation of raw squid liver to extruded
pellet on the digestibility of dietary nutrientsand the feed performance for raibow trout (inJapanese with English abstract).

.,49, 377‑381.

., 79, 259‑267.
Shimizu C., Allahpichay I., Tokoro T., Shirakawa Y.,
1990: Feeding stimulation in sea bream,, fed diets supplementedwith Antarctic
krill meals.

, 89, 43 53.

Wang W. X., Onsaint S., and Dang F., 2012: Dietarybioavailability of cadmium, inorganic mercury,Matsuda Y., Hayashi A., Yoshitomi B., Kanazawa A.,
and Takeuchi T., 2001: Eﬀect of the combinationof raw squid liver to extruded pellet on growthand zinc to a marine fish: Effects of foodcomposition and type.

, 356‑357,

98‑104.

performance and digestibility of dietary

Wakasugi M., Tomita K., Takahashi T., Nagano N.,

nutrients for Yelowtail and Red seabream (inand Sakuta Y., 2005: Development of processing

Japanese with English abstract).

.,49, 483‑488.

and utilizing technology of squid viscera (Part Ⅳ)(in Japnanese with English abstract). Sci. Rep.

Nakagawa M., Okouchi H., AdachiJ., Hattori K.,., 306, 41‑51.

and Yamashita Y., 2007: Eﬀectiveness of stock

Wakasugi M., 2009. Development of high quality

enhancement of hatchery-reared black rockﬁshdiet using squid viscera for fish farming (inSebastesschlegeli in Yamada Bay-Evaluation by

Japanese).

., 574, 28‑31.

水研センター研報，第40号，67−68，平成27年
Bull. Fish. Res. Agen. No. 40，67−68，2015

67(

Cholecystokinin and Trypsin Responses of Larval Red Drum) in Response to Algae, Live Prey, and Inert Particles
Ken WEBB＊1

Abstract: In an attempt to better understand the problems in weaning larval ﬁsh to artiﬁcial diets,
our lab has begun to investigate the role of the digestive hormone cholecystokinin (CCK). While
there are a number of other labs also investigating CCK and other digestive hormones such asbombesin, PPY, and gastrin; research into the roles of these hormones in ﬁsh is still in its infancy.
Previous research with red drum larvae suggests that some component of rotifers and algae
enable red drum larvae to more eﬃciently utilize microparticulate diets than when these are notincluded in the culture system. The current work investigated the impact of soluble componentsof rotifers and algae on the CCK and trypsin responses of larval red drum at 6 and 10 days post
hatch (DPH) as well as the response of red drum larvae to ingestion of inert polystyrene particles
at 10 DPH. Introduction of homogenized rotifers was shown to signiﬁcantly increase whole body
CCK levels, CCK mRNA, and trypsin activity in 6 DPH red drum larvae, but not in 10 DPH
larvae. Homogenates of

did not significantly affect CCK or trypsin at either

age. Ingestion of the polystyrene particles was increased in response to the presence of rotifer
homogenate and both CCK mRNA and trypsin activity was increased as well. This researchsuggests that there is a soluble component of rotifers that can upregulate digestive function inlarval red drum, at least in 6 DPH larvae, as well as inﬂuence consumption.

Annotated Bibliography

Liou A. P., Chavez D. I., Espero E., Hao S., Wank S. A.,
and Raybould H. E., 2010: Protein hydrolysate-

Dockray G. J., 2012: Cholecystokinin. Current

induced cholecystokinin secretion from

Opinion in Endocrinology, Diabetes & Obesity,

enteroendocrine cells is indirectly mediated by

19(1), pp 8 12.

the intestinal oligopeptide transporter PepT1.
.,

The author provides an excellent review of thecurrent understanding of the role of Cholecystokinin

300: G, G895-G902.

(CCK) in satiety and to a lesser extent, digestion.

The authors investigate the role of the PepT 1

The author focuses on the role of CCK in thetransporter as the direct mediator of Cholecystokinin

cephalic phase and reviews current knowledge of(CCK) secretion in response to protein hydrolysate.

both CCK activators and targets. The author also

Before this work, PepT 1 was considered a likely

discussed the role of Leptin in potentiating the eﬀect

mediator of direct mediation of CCK secretion due

on CCK on vagal aﬀerent neurons. Of particular noteto previous work that showed a synthetic dipeptide,in this manuscript, the author mentions the role of

Gly-Sar, used in PepT1 kinetic studies caused a dose-

GPR40 and long-chain fatty acids in the secretion

dependent inhibition of gastric motility consonantof fatty acids while much of the other literature

with CCK secretion. The work demonstrated that

focuses on protein hydrolysate / amino acid roles inwhile Gly-Sar did inhibit gastric motility it had no

promoting CCK secretion.

effect on eliciting CCK secretion from CCK-eGFP

2015年 1 月30日受理（Received on January 30, 2015）＊1
Environmental and Fisheries Sciences, National Marine Fisheries Service, Northwest Fisheries Science Center, 2725 Montlake Boulevard East,
Seattle, WA 98112
E-mail: Ken.Webb@NOAA.gov

68

Ken WEBB

cells. Based on this work, the authors concluded
that protein detection by intestinal I cells likely
includes both direct and indirect detection but that
PepT1 does not have a direct role in CCK secretion.
The authors do however suggest that PepT1 may
function indirectly by stimulating a diazepambinding inhibitor.
Cudenneca B., Fouchereau-Peron M., Ferry F.,
Duclos E., and Ravallec R., 2012:

and

evidence for a satiating effect of fishprotein hydrolysate obtained from blue whiting() muscle.
., 4, 271‑277.

The authors in this work examine the role ofprotein hydrolysate from blue whiting on CCK
secretion in STC-1 cells as well as on long and shortterm food intake of rats fed blue whiting muscle
hydrolysate (BWMH). The authors found that inthestudies on STC-1 cells, peptides fromthe BWMH stimulated CCK secretion in a dosedependent response similar to that seen from othertypes of protein hydrolysate. In the

study,

the authors found that BWMH produced short-term
reductions in food intake but that this reduction was
not reflected in the long term. The

study

did show that blood CCK and GLP-1 levels weremore than doubled in rats fed 100 and 250mg of
BWMH following a 24h fast lending credence thatthese may be involved in the short-term decrease in
consumption.

水研センター研報，第40号，69−74，平成27年
Bull. Fish. Res. Agen. No. 40，69−74，2015

69

Eﬀect of Feed Ingredients on Digestive Enzyme Secretion in Fish
Koji MURASHITA＊1, Haruhisa FUKADA＊2, Noriyuki TAKAHASHI＊2, Noriko HOSOMI＊2,
Hiroyuki MATSUNARI＊1, Hirofumi FURUITA＊1, Hiromi OKU＊1, and Takeshi YAMAMOTO＊1
Abstract: In response to the limitation in the global supply of ﬁsh meal, the traditional protein
source used in aquaculture feed, eﬀorts have increasingly been focused on the use of alternative
protein sources of plant origin. However, plant ingredients may cause growth retardation inaquaculture ﬁsh species. Feed nutrients must be digested for their utilization, and so pancreatic
digestive enzymes have essential roles for the digestion. Also, cholecystokinin (Cck) is known to
be a hormone that stimulates the secretion of digestive enzymes in vertebrates. To improve theutilization of plant based diets in ﬁsh, we investigated the eﬀects of various feed ingredients on
secretion of digestive enzymes in red seabreamand yellowtail,

which are commercially important aquaculture species in Japan.
We ﬁrst investigated the eﬀect of soybean meal on digestive enzyme secretion in red seabream.
Activities of pancreatic digestive enzymes (trypsin, chymotrypsin, lipase, amylase) in theintestinal content of a soybean meal based diet (SBM) fed fish were lower than those of a fishmeal based diet (FM) fed ﬁsh. Also, lower gene expression levels of the digestive enzymes in thehepatopancreas were observed in the SBM fed ﬁsh compared with the FM fed ﬁsh. These data
indicate the FM diet stimulated the secretion/synthesis of pancreatic digestive enzymes to agreater degree than the SBM diet. Then, we tried to identify the stimulation factor in ﬁsh meal.
Administration of a FM water-soluble fraction increased the gene expression of() in yellowtail, suggesting the enzyme stimulation factor may exist in

the water-soluble fraction of FM. Supplementation of the enzyme stimulation factor, although not
identiﬁed yet, may improve the utilization of plant based diets in aquaculture ﬁsh species.
Key words: red seabream (), yellowtail (), pancreatic digestive

enzymes, ﬁsh meal, soybean meal

Fish meal (FM), which is primarily produced

most common ﬁsh meal-replacement due to its price,from small pelagic fish such as jack mackerel

high availability in the market and relatively well-and anchovy, has long been used as the majorbalanced amino acid proﬁle. However, ﬁsh fed withprotein ingredient for fish feeds in aquaculture.

diets containing high concentrations of plant protein

However, as the wild stocks of these pelagic fish

often show low growth performance and digestibility

species become increasingly limited, developmentof feed (Gaylord., 2008; Glencross., 2007).

of alternative feeds is considered essential to theFeed nutrients must be digested for their

expansion of sustainable aquaculture worldwide. In

utilization, and pancreatic digestive enzymes

response to such a limitation in the global supply of

have essential roles for the digestion; trypsin andFM, efforts have increasingly been focused on the

chymotrypsin are the main pancreatic proteases,

use of alternative protein sources of plant origin.

lipase is the major pancreatic lipolytic enzyme, andEspecially, soybean meal (SBM) is known to be theamylase is known as the major pancreatic digestive

2015年 1 月30日受理（Received on January 30, 2015）＊1
National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie 519‑0423, Japan
E-mail: kojim at aﬀrc.go.jp＊2
Faculty of Agriculture, Kochi University, 200 Monobe, Nankoku, Kochi 783‑8502, Japan

Koji MURASHITA, Haruhisa FUKADA, Noriyuki TAKAHASHI, Noriko HOSOMI, Hiroyuki MATSUNARI,
Hirofumi FURUITA, Hiromi OKU, and Takeshi YAMAMOTO

70

enzyme for carbohydrates. The exocrine pancreatic

Materials and Methods

enzymes secretion is controlled by both neuronaland hormonal factors (Konturek., 2003). In mostEffect of soybean meal on digestive enzymes

vertebrates, a peptide hormone cholecystokinin

secretion in red seabream: Juvenile red seabream(Cck) is known to be one of the key physiologicalwere used for this experiment. Isonitrogenous andregulators of pancreatic secretion (Einarsson

isolipidic FM based and SBM based diets weregene

prepared (diet FM and diet SBM). The fish were

has been identified in several fish species such as., 1997; Kofuji., 2007). Recently, thefed the experimental diets for 6 weeks, and theAtlantic herring(Kamisaka.,hepatopancreas and intestinal content of the fish2005), Atlantic salmon(Murashita.,

were sampled (2009), yellowtail., 2006) and red drum

= 6). Activities of pancreatic(Murashita

digestive enzymes (trypsin, chymotrypsin, lipase(Webband amylase) in the intestinal content ( = 7) were., 2010), and is mainly expressed in the anterior

analyzed according to Murashita. (2007).

part of the intestine (including the pyloric caeca)

Gene expression levels of the enzymes in the(Murashita

hepatopancreas were evaluated by real-time(., 2006). Further, the) gene has also been identified in yellowtail

quantitative PCR (qPCR) using the speciﬁc primers

and is principally expressed in the pyloric caeca

for red seabream digestive enzymes.

(Furutani

Effect of fish meal on gene expression levels of., 2013).

To improve the utilization of plant based diets

,

,

and

in yellowtail: Water-in fish, we investigated the effects of various feedsoluble and water-insoluble fractions of FM wereingredients on secretion of digestive enzymes in

prepared. The same amount of the two FM fractions

red seabreamon crude protein basis were orally administrated

and yellowtail, which

are commercially important aquaculture species into juvenile yellowtail according to Murashita

Japan.

(2008). The pyloric caeca was sampled from the ﬁsh,Fig. 1. Eﬀects of ﬁsh meal (FM) and soybean meal (SBM) based diets on activities of thepancreatic digestive enzymes in intestinal content of red seabream. Values are mean ± SE
( = 7 ﬁsh).

.

71

Eﬀect of feed ingredients on digestive enzymes
and the gene expression levels ofdigestive enzymes secretion/synthesis.

andwere analyzed by qPCR according to

Effect of fish meal on gene expression levels

Furutani. (2013).

of

,

,

and

in yellowtail:Administration of a FM water-soluble fraction
Results and Discussion

increased the gene expressions ofandin yellowtail, whereas those of fish

Effect of soybean meal on digestive enzymes

administrated a FM water-insoluble fraction did

secretion in red seabream: Activities of all fournot (Fig. 3, brief results), suggesting the enzyme

pancreatic digestive enzymes examined in thestimulation factor may exist in the water-soluble

intestinal content of SBM fed ﬁsh were lower thanfraction of FM. We are trying to identify the enzyme

those of FM fed ﬁsh (Fig. 1), indicating the amounts

stimulation factor from the FM water-solubleof enzymes secreted into the intestine in the SBM

fraction.

fed ﬁsh were lower than those of the FM fed ﬁsh.

The main component of the FM water-solubleAlso, lower gene expression levels of the digestive

fraction may be very small peptides and/or free

enzymes in the hepatopancreas were observed inamino acids (data not shown). It is known that somethe SBM fed fish compared with the FM fed fish

amino acids strongly stimulate CCK release in(Fig. 2), which is in line with the report in yellowtail;rodents (Dalyorally administrated FM increased thealso exist in fish. Supplementation of the enzyme

andstimulation factor may improve the utilization ofgene expressions in the pyloric caeca,

but not in fish administrated SBM (Furutani.,., 2013); such a mechanism mightplant based diets in aquaculture ﬁsh species.

2012). These data indicate that SBM does not fully
stimulate the secretion/synthesis of the pancreatic
digestive enzymes, or, FM strongly stimulates theFig. 2. Eﬀects of ﬁsh meal (FM) and soybean meal (SBM) based diets on gene expressionof pancreatic digestive enzymes in the hepatopancreas of red seabream. Data for eachgene is represented as mean calculated copy number normalized against red seabream
copy numbers. Values are mean ± SE ( = 6 ﬁsh).

72

Koji MURASHITA, Haruhisa FUKADA, Noriyuki TAKAHASHI, Noriko HOSOMI, Hiroyuki MATSUNARI,
Hirofumi FURUITA, Hiromi OKU, and Takeshi YAMAMOTO
Furutani T., Masumoto T., and Fukada H., 2013:
Molecular cloning and tissue distribution ofcholecystokinin-1 receptor (CCK-1R) in yellowtail
and its response to
feeding and in vitro CCK treatment.
., 186, 1 8.
Gaylord T. G., Barrows F. T., and Rawles S. D., 2008:
Apparent digestibility of gross nutrients fromfeedstuffs in extruded feeds for rainbow trout,.,39, 827 834.
Glencross B. D., Booth M., and Allan G. L., 2007:
A feed is only as good as its ingredients ‑ areview of ingredient evaluation strategies foraquaculture feeds.

., 13, 17 34.

Kamisaka Y., Drivenes Ø., Kurokawa T., Tagawa
Fig. 3. Eﬀect of ﬁsh meal on gene expression levels
of
,
,
and
in yellowtail. The
schematic diagram of the experiment is presented
with brief results.

M., Rønnestad I., Tanaka M., and Helvik J.
V., 2005: Cholecystokinin mRNA in Atlantic
herring,̶molecular cloning,

characterization, and distribution in thedigestive tract during the early life stages.
, 26, 385 393.

Acknowledgments

Kofuji P. Y. M., Murashita K., Hosokawa H., andMasumoto T., 2007: Effects of exogenous

This work was partly supported by JSPS Grant-cholecystokinin and gastrin on the secretion

in-Aid for Scientific Research (B) (Grant Number:of trypsin and chymotrypsin from yellowtail

24380117) to H. Fukada and K. Murashtia, and by() isolated pyloric caeca.

the Fisheries Agency, Government of Japan.

, 146, 124 130.
Konturek S. J., Pepera J., Zabielski K., Konturek

References

P. C., Pawlik T., Szlachcic A., and Hahn E. G.,
2003: Brain-gut axis in pancreatic secretion and

Daly K., Al-Rammahi M., Moran A., Marcello M.,
Ninomiya Y., and Shirazi-Beechey S. P., 2013:

appetite control.

., 54,

293 317.

Sensing of amino acids by the gut-expressed

Murashita K., Fukada H., Hosokawa H., andtaste receptor T1R1-T1R3 stimulates CCK

Masumoto T., 2006: Cholecystokinin andsecretion.

peptide Y in yellowtail (., 304, G271‒G282.

Einarsson S., Davies P. S., and Talbot C., 1997: Eﬀect
of exogenous cholecystokinin on the discharge

Molecular cloning, real-time quantitative RTPCR, and response to feeding and fasting.

of the gallbladder and the secretion of trypsin
and chymotrypsin from the pancreas of theAtlantic salmon,L.

, 117, 63 67.
Furutani T., Masumoto T., and Fukada H., 2012:
Response of cholecystokinin and digestive
enzyme mRNA levels to various feed):., 145, 287 297.
Murashita K., Fukada H., Hosokawa H., andMasumoto T., 2007: Changes in cholecystokinin
and peptide Y gene expression with feeding inyellowtail (): Relation topancreatic exocrine regulation.
, 146, 318 325.

ingredients in yellowtail

Murashita K., Fukada H., Rønnestad I., Kurokawa., 78, 1075 1082.

T., and Masumoto T., 2008: Nutrient control

73

Eﬀect of feed ingredients on digestive enzymes
of release of pancreatic enzymes in yellowtail

while starch and PBS did not aﬀect the activity of(

any of these enzymes.

): Involvement of CCK

and PY in the regulatory loop.

mRNA levels rose, while

mRNA levels were reduced in ﬁsh administered, 150, 438 443.

casein, oleic acid and tri-olein. These results suggest

Murashita K., Kurokawa T., Nilsen T. O., andthat in yellowtail, Cck and Py maintain antagonistic

Rønnestad I., 2009: Ghrelin, cholecystokinin, andcontrol of pancreatic enzyme secretion after intake

peptide YY in Atlantic salmon (of protein and/or fat.

):molecular cloning and tissue expression.
., 160, 223 235.

Furutani T., Masumoto T., and Fukada H., 2012:

Webb K. A., Khan I. A., Nunez B. S., Rønnestad I.,

Response of cholecystokinin and digestiveand Holt G. J., 2010: Cholecystokinin: Molecularenzyme mRNA levels to various feedcloning and immunohistochemical localization

ingredients in yellowtailin the gastrointestinal tract of larval red drum,. 78, 1075‑1082.

(L.).

.,

166, 152 159.

Cholecystokinin (Cck) is the key regulator hormonethat stimulates the secretion of digestive pancreatic
enzymes in vertebrates. In ﬁsh, little is known about

Annotated Bibliography

the mechanism of induction of Cck in the digestive
tract by different feed ingredients. To investigate

Murashita K., Fukada H., Rønnestad I., Kurokawa

the response ofand digestive enzymes to fishT., and Masumoto T., 2008: Nutrient control

feed ingredients in yellowtailof release of pancreatic enzymes in yellowtail

we performed a series of experiments in which(

we measured the mRNA levels of): Involvement of CCK

and PY in the regulatory loop.

,
,

, and

after oral administration of a single bolus of, 150, 438‑443.

various ingredients. We administered ﬁsh meal andCholecystokinin (Cck) and neuropeptide Y (Npy)-ﬁsh oil in experiment 1; high and low concentrations

related peptides are the key regulators of pancreaticof fish meal in experiment 2; and five different

enzyme secretion in vertebrates. Cck stimulates

dietary protein sources (ﬁsh meal, soybean meal, soy

enzyme secretion whereas peptide Y (Py or Pyyb),

protein concentrate, corn gluten meal, and glutamica Npy-related peptide, plays an antagonistic role toacid fermentation by-products) in experiment 3. Inthat of Cck. In fish, very little is known about theexperiments 1 and 3, only fish meal significantly

eﬀects of diﬀerent nutrients on the synthesis of Cck

increased the mRNA level ofand Py in the digestive tract, and the mechanism

enzyme. In experiment 2, a high concentration of ﬁshby which Cck and Py actually regulate digestive

meal [20 % (w/v)] signiﬁcantly increased theand digestive
andenzyme secretion is not well understood. In order tomRNA levels, but a low concentration of ﬁshdetermine stimulating effects of different nutrients

meal [1 % (w/v)] did not. These results suggest thaton the synthesis of Cck and Py in yellowtail

high concentrations of ﬁsh meal (the protein source(in ﬁsh feed) has the most potent eﬀect on stimulation

),

and

mRNA levelsin the digestive tract were measured after oralofadministration of a single bolus of either phosphate-in yellowtail.

synthesis and secretion of digestive enzymes

buﬀered saline (PBS: control), starch (carbohydrate),casein (protein), oleic acid (fatty acid) or tri-olein

Furutani T., Masumoto T., and Fukada H., 2013:(triglyceride). In addition, in order to confirm the

Molecular cloning and tissue distribution ofsynthesis and secretion of digestive enzymes, thecholecystokinin-1 receptor (CCK-1R) in yellowtail

mRNA levels and enzymatic activities of three

and its response todigestive enzymes (and)

were also analyzed. Casein, oleic acid and tri-oleinincreased the synthesis of

,

and

,

feeding and

CCK treatment.

. 186, 1‑8.
In vertebrates, the peptide cholecystokinin (Cck) is

74

Koji MURASHITA, Haruhisa FUKADA, Noriyuki TAKAHASHI, Noriko HOSOMI, Hiroyuki MATSUNARI,
Hirofumi FURUITA, Hiromi OKU, and Takeshi YAMAMOTO

one of the most important neuroregulatory digestive
hormones. Cck acts via Cck receptors that areclassified into two subtypes, Cck-1 receptor (Cck1r; formally Cck-a) and Cck-2 receptor (formally
Cck-b). In particular, the Cck-1r is involved indigestion and is regulated by Cck. However, very
little information is known about Cck-1r in fish.
Therefore, we performed molecular cloning of
cDNA from the digestive tract of yellowtail. Phylogenetic tree analysis
showed a high sequence identity between thecloned yellowtail

cDNA and

which belongs to the,cluster. Furthermore,

the expression of yellowtail

mRNA wasobserved in gallbladder, pyloric caeca, and intestines,
similarly tomRNA expression in mammals,suggesting that the cloned cDNA is the yellowtail. In

experiments, themRNAlevels increased in the gallbladder and pyloric caeca
after feeding, whereas, mRNA levels ofand digestive enzymes in cultured pyloric caeca
increased by the addition of Cck. These results
suggest that Cck-1r plays an important role indigestion stimulated by Cck in yellowtail.

水研センター研報，第40号，75−77，平成27年
Bull. Fish. Res. Agen. No. 40，75−77，2015

75

Development and Characterization of Several Open Formula Reference
Diets for Marine Fish Larvae
Michael B. RUST＊1, Frederic T. BARROWS＊2, Mark DRAWBRIDGE＊3, Emily R. HART＊1,
Kevin STUART＊3, Ken WEBB＊1, Harold J. BARNETT＊1, Peter M. NICKLASON＊1,
and Ronald B. JOHNSON＊1

Abstract: A limiting constraint in the development and growth of marine aquaculture is successful
hatchery production. Presently, larval finfish are raised using live feeds such as copepods,
rotifers and. Live feeds are expensive, time-consuming, labor intensive, unreliable andnutritionally imperfect. An alternative to live feeds is microparticulate feeds, however, highperformance microparticulate diets for larval finfish are typically closed formula commercial
diets that make scientiﬁc inference diﬃcult or impossible. We describe a series of open formula
microparticulate reference diets that have been formulated and produced to facilitate comparisonsacross species and systems for use by the scientiﬁc community. Open formula diets can produce
consistent results among trials and species, they can be simply formulated with well-defined
ingredients and provide a basic platform for improvement. The reference diets were processedby three methods: flaking (F), microextrusion followed by marumerization (MEM) and particleassisted rotational agglomeration (PARA). An additional two diets were made by using the ﬂake
diet and further processing it using the MEM (F-MEM) or PARA (F-PARA) methods. The F,
F-PARA and F-MEM diets had the same formulation and only differed by processing method.
PARA and MEM had unique formulations. Each diet was screened to the 400‑700 µm range. All
microparticulate diets were compared to enriched

and rotifers () forchemical analysis. Scanning electron microscopy (SEM) was used to visualize the microparticles
between 22x and 4000x magniﬁcation. It was clear that micropellet structure, sinking rate andleaching was inﬂuenced by both formulation and manufacture method. Basic structure remainedlargely intact for all but the F-PARA particles even after being immersed in fresh water for 15
minutes. Compositional data, leaching half-life and sinking rates are given in the table below (values
in a column with diﬀerences at P<0.05 are denoted by superscripts). Data on feeding trials withlarval ﬁsh indicate that two of the open formula diets performed as well as, or almost as well as,
a popular commercial diet (Otohime, Marubeni Nisshin Feed, Tokyo, Japan) with larval yellowtail(
drum (), white sea bass () Pompano () and red). This performance illustrates that these diets provides a good base on

which to make improvements.

Annotated Bibliography

Aquaculture. John Wiley & Sons, New York.
525‑528.

Barrows F. T., and Lellis W. A., 2000: MicroboundIn addition to a review of methods to make older

feeds. In R. R. Stickney (ed) Encyclopedia of

micro bound feeds for aquaculture, methods to2015年 1 月30日受理（Received on January 30, 2015）＊1
NOAA, Northwest Fisheries Science Center, 2725 Montlake Blvd. E. Seattle, WA 98121
E-mail: Mike.rust@noaa.gov＊2
USDA/ARS-Fish Technology Center, 4050 Bridger Canyon Rd, Bozeman, Montana 59715＊3
Hubbs-SeaWorld Research Institute, 2595 Ingraham Street, San Diego, CA 92109

76

Michael B. RUST, Frederic T. BARROWS, Mark DRAWBRIDGE, Emily R. HART, Kevin STUART,
Ken WEBB, Harold J. BARNETT, Peter M. NICKLASON, and Ronald B. JOHNSON

produce microparticulate feeds using micro extrusion

behaviour that determines the kind and quantityand particle assisted rotational agglomeration areof food ingested by fish larvae as well as how

explained.

live prey and food particles are detected,

Cho C. Y., Cowey C. B., and Watanabe T., 1985.

on the progressive development of anatomical

Finfish Nutrition in Asia: Methodologicalcharacteristics and physiological functions and on thecaptured and ingested. Feeding success depends

approaches to research and development. IDRC,

availability of suitable food items throughout larval

Ottawa, Ont. Canada. 154 p.

development. The fish larval stages present eco-

Step by step instructions to produce zein andmorpho-physiological features very different fromcarrageenan bound diets developed in Japan are

adults and differ from one species to another. The

explained in English.

organoleptic properties, dimensions, detectability,
movements characteristics and buoyancy ofHolt G. J. (editor) 2010: Larval Fish Nutrition. John
Wiley & Sons, New York. 435 p.

food items are all crucial features that should beconsidered, but is often ignored, in feeding regimes.

Nutrition is particularly important in the healthy

Ontogenetic changes in digestive function lead todevelopment of fish during their early-life stages.

limitations in the ability to process certain feedstuﬀs.

Understanding the unique nutritional needs ofThere is still a lack of knowledge about the digestion

larval fish can improve the efficiency and quality

and absorption of various nutrients and about theof fish reared in a culture setting.

ontogeny of basic physiological mechanisms in ﬁshcomprehensively explores the nutritional

larvae, including how they are aﬀected by genetic,

requirements, developmental physiology, anddietary and environmental factors. The neural andfeeding and weaning strategies that will allow

hormonal regulation of the digestive process and of

aquaculture researchers and professionals to develop

appetite is critical for optimizing digestion. Theseand implement improved culture practices.

processes are still poorly described in fish larvae

is divided into three sections. The

and attempts to develop optimal feeding regimes

first section looks at the role of specific nutrient

are often still on a ʻtrial and errorʼ basis. A holistic

requirements in the healthy digestive development

understanding of feeding ecology and digestiveof fish. The second section looks at the impacts

functions is important for designing diets for fish

if nutritional physiology on fish through several

larvae and the adaptation of rearing conditions toearly-life stages. The final section looks at feeding

meet requirements for the best presentation of prey

behaviors and the benefits and drawbacks to bothand microdiets, and their optimal ingestion, digestion

live feed and microparticulate diets in developing

and absorption. More research that targets gaps inﬁsh.

our knowledge should advance larval rearing.

Langdon C. L., 2003: Microparticle types for deliveryof nutrients to marine ﬁsh larvae.

,

227, 259‑275.
Good review and discussion of several typesof microparticulate diets including complex
microparticles.
Ronnestad I., Yufera M., Ueberscchar B., Ribeiro
L., Saele O., and Boglione C., 2013: Feeding
behaviour and digestive physiology in larval ﬁsh:Current knowledge and gaps and bottlenecks inresearch.

, S59-S98.

Food uptake follows rules defined by feeding

77

Open Formula Reference Diets for Marine Fish Larvae

Feed

Moisture

Proximate Composition

Fatty Acid Concentrations(%as fed)(% dry weight)(mg fatty acid/100mg total fatty acids)

ProteinProtein
Leaching
t1/2 /% lost

Sinking Rate

Lipid

Ash

ARA (n-6)

EPA (n-3)

DHA (n-3)(sec)/(%)(cm/sec)
a

MEM

5.7

53.8

30.5

8.4

0.4

7.5

5.7

98/23.0

0.84±0.010

PARA

5.3

54.2

29.7

8.9

0.4

7.4

5.6

94/30.2

0.34±0.001

F

9.0

75.9

10.9

7.3

1.0

6.0

15.2

20/44.3

0.33±0.001

F-MEM

13.3

75.6

13.4

7.5

1.0

5.3

13.0

50/41.8

0.78±0.060

F-PARA

11.0

75.6

12.6

7.3

1.0

6.0

15.2

33/47.2

0.61±0.001

Artemia

91.3

NA

15.3

13.7

5.6

12.1

7.1

NA/0.1

NA

Rotifers

90.1

49.6

15.5

17.7

1.5

5.7

23.3

NA

NA

c

c

a

b

水研センター研報，第40号，79−82，平成27年
Bull. Fish. Res. Agen. No. 40，79−82，2015

79

An Analysis of the Causality between the Market Price of Imported
Fishmeal and the Market Price of Marine Farmed Fish
Yoshifumi TAKAHASHI＊1

Abstract: The purposes of this paper are to introduce the historical background and price trendsof the imported fishmeal market, and to reveal the pattern of causality between prices in theimported fishmeal market and the market prices of yellowtail and sea bream inlanding areas.
Granger causality test was used in this analysis.
The results were as follows: 1) there is causality between the price of imported ﬁsh meal andthe market price of sea bream in landing areas, 2) but there is no causality among the market
prices of all combinations without imported ﬁshmeal and sea bream.
Key words: Granger causality test, market price of imported ﬁshmeal, market prices of pisciculture

In Japan, many piscicultural farmers are facing

The domestic production of ﬁshmeal anddiﬃculties due to the impact of falling ﬁsh prices andthe market price of major farmed ﬁshes

the rising cost of feed. These are serious problems
because they can result in a great reduction in

The domestic production of ﬁshmeal came mainly

profits for many farmers. According to statistics

from inshore fisheries in northern Japan between

produced by the Japanese Corporation of Feed

1959 and 1991 (Fig. 1), and the main ingredient,for Pisciculture (Nihon Yougyo Shiryou Kyoukai),

Pollack, was used on the grounds that it was freshthe production of feed reached 360,000 tons inand suitable for processing. However, the catch of

2011, the majority of which was for yellowtail andPollack has decreased year by year from then as

sea bream. These feedstock were made from raw

a result of increasing demand for fish pastes andingredients such as fishmeal, starch, and fish oil.

the impact of ﬁshery treaties such as the 200 mile

Fishmeal was especially important, making up 50%

exclusive fishing zone. As a result, the amount ofor more of the feed content. Therefore, piscicultural

imported ﬁshmeal has increased gradually to makefarming yellowtail and sea bream always worry

up for insufficient supplies of local ingredients inabout soaring imported fishmeal prices. This

Japan. More than half current fishmeal stocks areresearch investigates the relationship of priceimported from three countries in South Americachanges in imported ﬁshmeal on the market price ofpiscicultural products.
The purposes of this paper are to introduce

1,200
1,000
800

Fishmeal:1000t

Feed production:1000t

Fishmeal from fishing in northern Japan
Fishmeal from inshore fisheries
Imported fishmeal
Feed for pisciculture

600
500
400

Latest trends
Slightly
decreasing

imported ﬁshmeal market, and to reveal the pattern

400

200

Decreasingof causality between prices in the imported ﬁshmeal

200

100

0

0

market and the market prices of yellowtail and sea
bream in landing areas. Granger causality test wasused in this analysis.

2015年 1 月30日受理（Received on January 30, 2015）＊1
Tohoku National Fisheries Research Institute, FRA
E-mail: gibun@aﬀrc.go.jp

1955
1959
1963
1967
1971
1975
1979
1983
1987
1991
1995
1999
2003
2007

300

+

600

the historical background and price trends of the

Increasing
Increasing

Fig. 1. Domestic production of ﬁshmeal and feed for
pisciculture in Japan

80

Yoshifumi TAKAHASHI
Imported fishmeal(yen/kg)

1,200 Farmed fish(yen/kg)

180

Yellowtail priceSea bream priceSep-12

May-12

Jan-12

Sep-11

May-11

Jan-11

Sep-100May-100

Jan-1030

Sep-09200

May-0960

Jan-09400

Sep-0890

May-08600

Jan-08120

Sep-07800

May-07150

Jan-071,000

Imported fishmeal price㻺㼛㼠㼑㻦㻌㼀㼔㼛㼟㼑 㼜㼞㼕㼏㼑㼟㻌㼣㼑㼞㼑㻌㼐㼑㼒㼘㼍㼠㼑㼐㻌㼡㼟㼕㼚㼓㻌㻯㻼㻵㻌㼍㼚㼐㻌㻵㻼㻵㻌㻔㻞㻜㻝㻜㻩㻝㻜㻜㻕
Fig. 2. Market prices for yellowtail, sea bream and imported ﬁshmeal(Peru, Ecuador and Chile).

Table 1. Result of the DF-GLS test

Fig. 2 shows the trend of market prices inimported ﬁshmeal, farmed yellowtail and farmed sea
bream during the six years from 2007. The market
price changes of yellowtail and sea bream have been
subject to violent ﬂuctuations within certain limits.
As for only yellowtail, there is an exception thatthe price of yellowtail soared upwards in June 2008
because of little production. Imported fishmeal has
also ranged in price from 90 to 175 yen/kg.

㻰㻲㻙
㻳㻸㻿
㼀㼑㼟㼠

㻲㼕㼞㼟㼠㻌㼐㼕㼒㼒㼑㼞㼑㼚㼏㼑㻌㼐㼍㼠㼍㼅㼑㼘㼘㼛㼣㼠㼍㼕㼘㻌㼜㼞㼕㼏㼑㻿㼑㼍㻌㼎㼞㼑㼍㼙㻌㼜㼞㼕㼏㼑㻵㼙㼜㼛㼞㼠㼑㼐㻌㼒㼕㼟㼔㼙㼑㼍㼘㻌㼜㼞㼕㼏㼑㼘㼍㼓 㼟㼠㼍㼠㼕㼟㼠㼕㼏 㼟㼕㼓㼚㼕㼒㼕㼏㼍㼚㼠 㼘㼍㼓 㼟㼠㼍㼠㼕㼟㼠㼕㼏 㼟㼕㼓㼚㼕㼒㼕㼏㼍㼚㼠 㼘㼍㼓 㼟㼠㼍㼠㼕㼟㼠㼕㼏 㼟㼕㼓㼚㼕㼒㼕㼏㼍㼚㼠㻜 㻙㻢㻚㻟㻞㻢 㻖㻖㻜 㻙㻜㻚㻣㻜㻞 㻖㻜 㻙㻝㻚㻠㻣㻢 㻖㻝 㻠㻚㻠㻥㻠 㻖㻝 㻙㻜㻚㻠㻣㻠㻝 㻜㻚㻣㻣㻟㻞 㻟㻚㻤㻜㻟 㻖㻞 㻙㻜㻚㻢㻝㻞 㻖㻞 㻜㻚㻣㻥㻥㻟 㻟㻚㻞㻡㻤㻟 㻙㻜㻚㻢㻝㻝 㻖㻟 㻜㻚㻠㻥㻥㻠 㻞㻚㻤㻝㻣㻠 㻙㻜㻚㻠㻥㻠㻠 㻜㻚㻡㻣㻜㻡 㻞㻚㻠㻜㻝㻡 㻙㻜㻚㻠㻥㻢㻡 㻜㻚㻢㻠㻢㻢 㻞㻚㻜㻜㻠㻢 㻙㻜㻚㻟㻥㻢㻢 㻜㻚㻠㻟㻤㻣 㻝㻚㻢㻞㻜㻣 㻙㻜㻚㻟㻞㻡㻣 㻜㻚㻜㻢㻥㻤 㻝㻚㻞㻠㻥㻤 㻙㻜㻚㻠㻠㻤 㻖㻤 㻜㻚㻞㻞㻥㻥 㻜㻚㻥㻡㻣㻥 㻙㻜㻚㻟㻡㻢㻥 㻜㻚㻟㻜㻡㻝㻜 㻜㻚㻢㻟㻢㻝㻜 㻙㻜㻚㻞㻝㻢㻝㻜 㻙㻜㻚㻜㻝㻡㻝㻝 㻜㻚㻟㻞㻟㻝㻝 㻙㻜㻚㻝㻟㻡㻝㻝 㻜㻚㻝㻝㻠㻝㻞 㻜㻚㻝㻝㻥㻝㻞 㻙㻜㻚㻞㻠㻟㻝㻞 㻜㻚㻜㻥㻠

㻺㼛㼠㼑㻦㻌㻎㻖㻖㻎㻌㼕㼚㼐㼕㼏㼍㼠㼑㼟㻌㼟㼠㼍㼠㼕㼟㼠㼕㼏㼍㼘㻌㼟㼕㼓㼚㼕㼒㼕㼏㼍㼚㼏㼑㻌㼍㼠㻌㼠㼔㼑㻌㻝㻑㼘㼑㼢㼑㼘㻘㻌㻎㻖㻎㻌㼍㼠㻌㻡㻑㻌㼘㼑㼢㼑㼘

Generally, the market price of a commodity
tends to rise when the price of ingredients rises;

Finance), and have also been deﬂated with “ﬁshmeal”

this phenomenon is called “cost push inflation”. As

in the IPI (Import Price Index).

seen in Fig. 2, there have been price changes overThe data ﬁrst had to be tested for “stationarity” ortime, but we cannot conclude that there has been

“not stationarity” before processing in the Granger

any relationship between changes in the price of

causality test, because time series data in economics

one commodity and that of another. Therefore we

has an almost unit root. Regression among data sets

decided to analyze the relationships among prices

having unit roots has no meaning because of theusing statistical approaches.

potential for high correlations among those data. The
DF-GLS (Dicky-Fuller is Based on GLS Detrending)

Data and Method

test is reliable statistical method to check for unit
root, so this was applied. The first test with raw

Data: Time series data about piscicultural products

data was not suitable for null hypothesis testing. As

such as yellowtail and sea bream are available froma countermeasure for the unit root problem, first

the website of JAFIC (Japan Fisheries Informationdifference data was used and then retested in theService Center). These original data are nominal, sosame way. The result of this retest made it possible

for this paper these were deﬂated with “yellowtail”

for null hypothesis testing to function correctly

and “sea bream” in the CPI (Consumer Price Index).

(Table 1). From this, ﬁrst diﬀerence data whose lag

Also time series data of imported fishmeal are

is 0 was adopted.

available from the website of the MOF (Ministry ofAnalysis method: Analysis method used in this paper

81

Price Causality between Imported Fishmeal and Marine Farmed Fish
was the Granger causality test in a VAR (Vector
Autoregression) model. An AR (Autoregression)
single variable model was used, and current value
was determined by past own value and white
noise (for more information on the VAR model andGranger Causality Test, see chapter 12 and chapter
13 in Maddla (1992)). The VAR model is an expanded
AR model, from single to multivariable cases,
although the current values of the multivariables arealso determined by past own values and white noise.
In this section, the two variables VAR(1) model that

F-statistic=0.6805

F-statistic=5.1805*
F-statistic=0.0161

Importedfishmeal price F-statistic=0.5635
F-statistic=0.014

Sea bream
price

F-statistic=0.5635

Yellowtail
price㻺㼛㼠㼑㻦㻌㻎㻖㻎㻌㼕㼟㻌㼟㼕㼓㼚㼕㼒㼕㼏㼍㼚㼏㼑㻌㼘㼑㼢㼑㼘㻌㻨㻌㻜㻚㻜㻡
Fig. 3. Result of market linkages in Japan

was actually used is described. The values given inare thecarried out with four model types which were “none”,

price data of commodity at time t, ε denotes error“const”, “trend” and “both”. “None” has no drift andterms, and αij is an individual parameter.

trend, “const” has only drift, “trend” has only trend,

parentheses are lag numbers. Here,

t

＝α11

t−1

＝α21

t−1

t

+α12

t−1

+ε t

+α22

t−1

+ε t

t

and

t

and “both” has both of these. The estimation of these……………………(1)Information Criteria). Consequently, “none” and “lag

The Granger causality test is examined byequation (1). The basic idea of the Granger causality
test is that the past value of variablevariablefour types were judged by using S.C. (Schwarz

can affect(or not). It can determine that variable

length = 1” are selected in all combinations of two
variable VAR models.
Fig. 3 shows the results of the Granger causality
test. There is no causality of price between

if the null hypothesis of “α12=0”

yellowtail and sea bream, and this is natural because

is rejected by F test. The following equation (3) isyellowtail cannot be substituted for sea bream. Also,

the form of equation (2) where “α12=0”．

changes in the price of farmed ﬁsh have no inﬂuencecan aﬀect variable

t

＝α11

t−1

＋α12

t−1

t

＝α11

t−1

＋ε t ……………………(2)＋ε t………………………………(3)The residual sum of squares of equation (2)replaces, the residual sum of squares ofequation (3) replaces, and we can deﬁne the Fvalue using the following equation:
＝

(

Where

of imported fishmeal can affect the market priceof sea bream in Japan. We do not have a scientiﬁc
explanation for the fact that this relationshiponly affects the price of sea bream. Nevertheless,according to Demura (2010), feed stocks for farmed
sea bream are dependent on fishmeal even if theprice of imports rises. Conversely, feed stocks for−)/,/( − −1)0

is the number of samples,on the price of imported fishmeal. Why the priceyellowtail can be changed to other ingredients suchas raw ﬁsh if the price of imported ﬁshmeal rises toois thehigh. This situation is probably the explanation.

number of constraint conditions such as “α12＝0”,
anddenotes the number of variables without a

References

constant term in equation (2).
Demura M., 2010: A trend of fishmeal price andResults and Discussion

impact on the aquacultural industry,, 45, 601‑605 (inThe VAR model has a problem with respect
to lag length selection, so it is necessary to selectoptimal lag length to obtain an accurate result

Japanese).
ndMaddla G. S., 1992: Introduction to Econometrics, 2
edition, Prentice-Hall, USA.

before applying the Granger causality test. In thisJapanese Corporation of Feed for Pisciculture,

paper, the estimation of lag length selection was

2007: Pisciculture and formula feed in Japan: A82

Yoshifumi TAKAHASHI
historical record of 40th anniversary of JapaneseCorporation of Feed for Pisciculture, JapaneseCorporation of Feed for Pisciculture, Tokyo,
Japan (in Japanese).

Yano I., 2010: Heading towards construction ofrecycling-oriented society associated withfood production and consumption: food wastes
problem and recycle problem. in Yamao,
M. (Ed.), Our “food safety and security” andenvironmental problem, Hirodai Seibutsuken
Shuppankai, Hiroshima, Japan: 83‑92 (inJapanese).

水研センター研報，第40号，83−88，平成27年
Bull. Fish. Res. Agen. No. 40，83−88，2015

83

Good Aquaculture Practices to Reduce the Use of Chemotherapeutic
Agents, Minimize Bacterial Resistance, and Control Product Quality
Stanley SERFLING＊

Abstract: A significant challenge to the expansion of aquaculture production is controlling theoutbreak of disease. Many farmers who experience the potential loss of stock from disease may
choose to use chemotherapeutic agents to minimize their loss. It is generally understood thata disease in aquaculture is a combination of the health of the animal, the condition of the environment
and the presence of a pathogen. From this concept there are a number of precautionary measuresthat farmers may practice to minimize disease outbreaks. The principles of Hazard Analysis
Critical Control Point (HACCP) may be useful risk management tools to reduce pathogens, animal
stress and the need for chemotherapeutic agents to control disease outbreaks on the farm.
Key Words: good aquaculture practices, chemotherapeutic agents, bacterial resistance, food safety

Introduction
As the global demand for seafood increases,
the trend towards high density aquaculture
systems grows. These systems referred to as“intensive aquaculture” increasingly rely on inputsof oxygen, formulated feed, and the applicationof agrochemicals, and antibiotics. This method of
culture may also create stressful conditions for the
animals and increase the risk of bacterial infections
and therefore the use of chemotherapeutic agents(Sapkota

Fig. 1.

., 2008).

Shrimp is the single most popular type of seafoodin the United States and over 90 percent of shrimp

for fecal coliforms,

products are farm raised and imported primarily

A signiﬁcant relationship was found between the log

from Southeast Asian countries. The primary

number of fecal bacteria and the probability that any

hazards associated with aquaculture shrimp are

given sample would contain. The study

contamination from pathogens and residues fromconcluded the occurrence ofbacteria inunapproved drugs. The origins for these hazards are

shrimp from aquaculture farms is directly related to

, and

.

from production farms and can remain in or on thethe concentration of fecal bacteria in the source andproduct throughout the normal cleaning, rinsing andproduction pond water (Koonsepackaging process.

., 2005).

The importance of chemical and antimicrobial

Between 2001 and 2003 the U.S. Food and Drugcompounds in the protection of animals has been

Administration analyzed 1,234 samples from 103

acknowledged, but the negative impact and use of

shrimp aquaculture farms representing six countries

these agents in animals raised for food is a concern.

2015年 1 月30日受理（Received on January 30, 2015）＊
U.S. Food and Drug Administration, Oﬃce of Food Safety, College Park, MD 20740
E-mail: stanley.serﬂing@fda.hhs.gov

84

Stanley SERFLINGThe detection of certain banned chemotherapeutic

are the seven basic principles with an example:

agents in ﬁsh and crustaceans in international tradeduring 2001‑2002 led to greater attention about

1.

Hazard and Risk Analysis: Collect andthe potential health risks from farm raised seafood

evaluate information on hazards associatedproducts (Iddya, 2012).

with the product under consideration todecide the likelihood of the occurrence andthe severity of the occurrence. To help

Good Aquaculture Practices

identify a potential hazard a farmer should
Good aquaculture practices (GAqP) can be deﬁned

construct a ﬂow diagram with all of the inputs

in many ways. In general it is based on preventive

to the farm and then evaluate a control forpractices that are developed and implemented toeach potential hazard.

meet the needs of the species, culture methods and− See Fig. 3 for a shrimp farm ﬂow diagram.

local environmental conditions. When aquaculture
producers take time to identify potential risks to2.

Identify Critical Control Point (CCP): This step

their operation and implement controls to manage

is essential to prevent or eliminate a hazard

the risk, then the potential for disease and the use ofor reduce it to an acceptable level.

chemotherapeutic agents is minimized.

− For example a shrimp farmer may choose

The Hazard Analysis Critical Control Point

critical water quality parameters such

(HACCP) principles can be used as a preventive

as minimum and maximum oxygen andrisk management system to control the introduction

temperature levels.

of pathogens at aquaculture facilities. The HACCP
approach is based on prevention and requires a3.

Establish Critical Limits: A maximum and/or

hazard analysis that identifies a potential hazard

minimum value to which a physical, chemicalin the system and then a Critical Limit, with aor biological hazard must be controlled tomaximum and or minimum point, is set for each

prevent, eliminate or reduce to an acceptablecomponent of the system. When monitoring thelevel the occurrence of a hazard.

aquaculture system and a critical limit has been in− For example a critical limit could beviolation, then a corrective action is taken to bringstocking density at a hatchery tank andthe system back into compliance (Jahncke

the ratio of live feed to animal.

.,

2002).
These HACCP principles provide a step by step4.

Establish Monitoring Procedures: Monitoring

approach to identify and control hazards found in

is a process that the aquaculture operation

the environment and production process for bothrelies on to maintain control of a Criticalfreshwater and marine aquaculture. The following

Control Point. Accurate monitoring isimportant to help determine when a CCP has
deviated from the desired range. Failure toproperly use and monitor chemotherapeutic
agents may result in detectable residues inthe tissue of aquacultured products.
− For example, monitoring a speciﬁc water
quality parameter every day or more
frequently depending on the production
system.
5.

Establish Corrective Actions: The farmer
should identify procedures to follow when aFig. 2.

deviation from the acceptable range occurs.

85Good Aquaculture Practices

Broodstock Maturation Reproduction

Water quality (CCP)
Fresh/prepared feed (CCP)
Vector control (SOP)
Equipment (SOP)

Live food Production

Water quality (CCP)
Live food source (CCP)
Vector control (SOP)
Equipment (SOP)

Larval Culture

Water quality (CCP)
Fresh/frozen food (CCP)
Vector control (SOP)
Equipment (SOP)

Grow-out ponds

Stocking Density (CCP)
Prepared feed (CCP)
Vector control (SOP)
Equipment (SOP)

Pond Side Harvest

Water quality/ice (CCP)
Equipment (SOP)
Trained Staff (SOP)
Farm conditions (SOP)

CCP – Critical Control Point
SOP – Standard Operating Procedure
Fig. 3. Process ﬂow diagram of an integrated shrimp farm with hatchery.
The farmer will implement a corrective action

records to determine if the production

plan after the deviation has been identified

system is meeting expected output.

and prepare a long term solution as soon as
possible.
− For example if a farmer has identified

7.

Establish Record Keeping Procedures: The
final step is to document all previous steps,

the feed supply for the hatchery does notfrom the Evaluation of Hazards and flow

conform to required standard, the productdiagram, the identiﬁcation of Critical Control

is rejected and the farmer contacts an

Points and Critical Limits, and any Corrective

alternate feed supplier for new feed.

Actions taken, and all Veriﬁcation Procedures.
− Records should be made for all identiﬁed

6.

Establish Verification Procedures: This

Critical Control Points and be accessible toprocedure helps to determine the validity

staﬀ in paper or electronic format.

of the operation and verify that the farm is
operating according to plan.
− For example the farmer may review dailywater quality parameter sand feeding

Fig. 3 is an example of an operational flow
diagram for an integrated shrimp farm with broodstock, hatchery, nursery, and growout production.

86

Stanley SERFLING
Preventive Measuresof pathogens by isolating and testing incoming
seed stock. In addition, providing clean water andA standard operating procedure (SOP) manual issanitary facilities for staﬀ and proper farm security

an important document the farmer should assemble.

help to control the spread of pathogens from humans

This should include facility design and ﬂow diagram,and animals.

general husbandry procedures for each step in theChemical: Includes measures that are used to

production process, restricted access areas, waste

prevent the introduction of pathogens or vectors

management, pest control and staff rules. Goodby treating materials before they enter the facility.

Aquaculture Practices may also include welfare

For example, chlorination or ozone can be used toof the animals, environmental integrity and social

treat incoming water, and iodine and chlorine can be

responsibility.

used to treat other potential vectors such as tools,

There are a number of biological, chemical

footwear and clothing.

and physical precautionary measures that can be

Physical: Includes barriers that may be useful toundertaken. The following are examples of how toprevent vectors from contaminating a farm site.

apply these practices on the farm.

Physical barriers can be stress reducing practices

Biological: Includes measures to prevent orlike proper stocking densities, optimal levels fortreat infections such as the proper use of

dissolved oxygen and temperatures, and proper

chemotherapeutic agents or the use of vaccinesas

feeding practices.

well as management practices to prevent the spread
Table 1. The following hazard analysis for an intensive marine shrimp farm.

,GHQWLI\3RWHQWLDO
+D]DUG
Seed stock,VWKH+D]DUG
6LJQLILFDQWYes

Water Source

Yes

Potential
pathogens

Feed Source

Yes

Stocking Density

Yes

Effluent or
Recirculated Water

Yes

Animal and Human
Vectors

Yes

Farm Equipment
and SanitationYes

May result inand poor
production
Important to
optimize
production
Effluent may
containpathogens
May transfer
contaminants
and pathogens
Equipment
may becomecontaminatedwith pathogens

SPF – Specific Pathogen Free
CCP – Critical Control Point
SOP – Standard Operating Procedure-XVWLILFDWLRQ

3UHYHQWLYH0HDVXUHV

&&3

Shrimp larvae
may containdetectable
pathogen

SPF certification foreach receipt.
Quarantine
procedures and test
periodically (CCP)
Provide pre-treatment
to ensure watersupply is safe (CCP)
Certificate from feed
supplier to ensure
standard (CCP)
Controlled byStandard Operating
Procedure (SOP)
Controlled byStandard Operating
Procedure (SOP)
Controlled byStandard Operating
Procedure (SOP)
Controlled byStandard Operating
Procedure (SOP)

Yes

Yes
Yes
Yes
and NoYesand NoNo
No87

Good Aquaculture Practices
Conclusion

risks: Current knowledge and future priorities.
., 34, 1215‑1226.

Good Aquaculture Practices may be applied tomany different kinds of aquaculture systems in a

Annotated Bibliographyvariety of management strategies. The key elements
can be summarized as reliable sources of stock,

Jahncke M. L., Browdy C. L., Schwarz M. H., Segars

good diagnostic and detection methods for disease,A., Silva J. L., Smith D. C., Stokes A. D., 2002:

disinfection and eradication of pathogens and best

Risk management, disease prevention, andmanagement practices. HACCP principles provide

HACCP principles: Application of hazard

a logical step by step approach to help prevent

analysis critical control point (HACCP)

pathogen contamination and the outbreak of diseaseprinciples as a risk management tool to control

on the farm site. This management approach allows

viral pathogens at shrimp aquaculture facilities.

the farmer to choose the most important process

NOAA Office of Sea Grant, Virginia Graduate

control steps and then choose appropriate critical

Marine Science Consortium and the Virginia

control points and control measures to minimize risk

Sea Grant College Program, Publication VSG-02‑

to the animals and the farm operation and insure

10, Pages 1‑33.

that chemotherapeutic agents if needed will be usedwisely.

The authors identify how the principles of HACCP(Hazard Analysis Critical Control Point) can be used
as a preventive risk management system to control

References

the introduction of pathogens and the outbreak of
disease in shrimp aquaculture facilities. The HACCP

Jahncke M. L., Browdy C. L., Schwarz M. H., Silva

approach requires a hazard analysis that identifies

J. L., Smith D. C., and Stokes A. D., 2002: Risk

a potential hazard in the system and then a Criticalmanagement, disease prevention, and HACCP

Limit with a maximum and or minimum point is set

principles: Application of hazard analysis criticalfor each component of the system. When monitoring

control point (HACCP) principles as a risk

the aquaculture system and a critical limit has been

management tool to control viral pathogens atin violation, then a corrective action is taken to bring

shrimp aquaculture facilities. NOAA Office ofthe system back into compliance.

Sea Grant, Virginia Graduate Marine Science

To develop an eﬀective biosecurity program ﬁshConsortium and the Virginia Sea Grant College

and shrimp farmers should follow these principles

Program, Publication VSG-02‑10, Pages 1‑33.

based on seafood HACCP: 1) Perform Systematic

Karunasagar I., 2012: Public health and trade

Hazard Analysis, 2) Determine Critical Control

impact of antimicrobial use in aquaculture,

Points, 3) Establish Critical Limits, 4) Determine

in “Improving biosecurity through prudent

Appropriate Corrective Actions, 5) Establish

and responsible use of veterinary medicines

Monitoring Procedures, 6) Establish Verification

in aquatic food production” (ed by Bondad-

Procedures, 7) Establish Record Keeping Systems.

Reantaso M. G., Arthur J. R. and Subasinghe
R.P.) FAO Fisheries and Aquaculture TechnicalPaper No. 547. Rome, FAO, pp. 1‑9.

Karunasagar I., 2012: Public health and trade
impact of antimicrobial use in aquaculture,

Koonse B., Burkhardt W., Chirtel S., and Hoskins G.,

in “Improving biosecurity through prudent

2005: Food safety and aquaculture, controlling

and responsible use of veterinary medicinesrisk, coliform bacteria: Salmonella and Sanitary

in aquatic food production” (ed by Bondad-

Quality of Aquaculture Shrimp.

Reantaso, M.G., Arthur J. R. and Subasinghe.,

68, No 12, 2527‑2532.
Sapkota A., Sapkota A., Kucharski M., Burke J.,

R.P.) FAO Fisheries and Aquaculture TechnicalPaper No. 547. Rome, FAO,pp. 1‑9.

McKenszie S., Walker P., Lawrence R., 2008:

The Food and Agriculture Organization of

Aquaculture practices and potential healththe United Nations (FAO), the World Health

88

Stanley SERFLING

Organization (WHO) and the World Organization

search with 145 peer reviewed papers to identify

for Animal Health (OIE) organized consultations andcurrent global aquaculture practices and thetechnical meetings to review the global seafood tradepotential health risks from aquacultured seafood.

and the use of antimicrobial agents in aquaculture

The authors focused on aquaculture production inand develop recommendations. The author outlines

Asia as having 11 of the top 15 producing countries

how detection of certain banned antibiotics in fishin the world. The paper identifies chemical andand crustaceans in international trade during 2001‑

biological contaminants used by the aquaculture

2002 lead to greater attention on the public healthindustry and the potential impacts on food safety

risks owing to the use of antimicrobial agents inand human health. Tables identify the top 15

aquaculture. Most fish importing countries adopt

countries that use antibiotics and the prevalencea zero tolerance approach regarding residues ofof antibiotic resistance in the environment as a

antimicrobials that are banned for use in foodpotential health concern. As global aquaculture

animals. In such cases, residue levels that attract

production continues to increase worldwide with an

regulatory action are based on analytical capability

emphasis on intensive systems, exposure to various

rather than toxicology of the residues.

hazardous chemical and biological compounds is aconcern. The authors conclude the increased use ofKoonse B., Burkhardt W., Chirtel S., and Hoskins G.,

chemotherapeutic agents is contributing to elevated2005: Food safety and aquaculture, controllinglevels of antibiotic residues and antibiotic resistantrisk, coliform bacteria: Salmonella and Sanitarybacteria.

Quality of Aquaculture Shrimp.

.,

68, No 12, 2527‑2532.
The authors examined the prevalence ofand coliform bacteria on shrimp
aquaculture farms in several Asian countries todevelop guidelines or preventative measures for
reducing

and fecal contamination on

products harvested from these farms. A signiﬁcantrelationship was found between the log number of
fecal bacteria and the probability that any given
sample would containof finding. The likelihoodin grow-out pond water

increased 2.7 times with each log unit increase infecal coliform concentration and 3.0 times with eachlog unit increase inconcentration.

is not part of the natural ﬂora of the shrimp culture
environment nor is it inherently present in shrimp
grow-out ponds. The occurrence ofbacteria in shrimp from aquaculture operations isrelated to the concentration of fecal bacteria in thesource and grow-out pond water.
Sapkota A., Sapkota A., Kucharski M., Burke J.,
McKenszie S., Walker P., Lawrence R., 2008:
Aquaculture practices and potential healthrisks: Current knowledge and future priorities.
., 34, 1215‑1226.
The authors conducted an extensive literature水研センター研報，第40号，89−96，平成27年
Bull. Fish. Res. Agen. No. 40，89−96，2015

89

Modeling Intraspeciﬁc Genetic Eﬀects for Managementof Aquaculture Programs
Jason D. VOLK＊1, Michael B. RUST＊2, Gregory R. BLAIR＊1, Lars E. MOBRAND＊1,
Conrad V. W. MAHNKEN＊3, and Walton W. DICKHOFF＊2

Abstract: Rapid worldwide development of marine finfish cage farming has raised awarenessover the possible genetic and ecological eﬀects of escaped ﬁsh on wild populations. With increased
interest in implementation of marine aquaculture in the United States, NOAA Fisheries andother regulators charged with stewardship of marine ecosystems need tools to understand andmitigate risks presented by aquaculture escapees. To develop an understanding of genetic andecological effects of escapes and design management strategies to address potential risks tomarine resources, NOAA Fisheries has developed a numerical decision-support tool: the Oﬀ-shore
Mariculture Escapes Genetics/Ecological Assessment (OMEGA) model. The OMEGA model is anextension of concepts from another model, the All-H Analyzer (AHA) that is used successfully in
the U.S. Paciﬁc Northwest to evaluate genetic and ecological interactions between hatchery andwild salmon and trout.
OMEGA model input parameters include size and growth characteristics of cultured fish,
frequency and magnitude of escape events, survival of escapees in the wild, probability of escapees
encountering a conspeciﬁc natural population and interbreeding, and population dynamics of thenatural population. Model results describe the influence of aquaculture escapees on spawning
biomass, juvenile production, and genetic ﬁtness of the composite population. Eﬀects of interactions
on ﬁtness and abundance are based on the frequency and relative abundance of cultured ﬁsh thatescape and survive to encounter a natural population, the difference in survival characteristics
between the artiﬁcial and the natural environments, and the genetic legacy of the cultured andnatural populations.
NOAA Fisheries is using the OMEGA model to identify and evaluate risks of marine
aquaculture operations, design sustainable aquaculture programs, explore the eﬀects of regulation,and identify research priorities for areas of uncertainty.
This talk will describe the model and present results for a hypothetical sableﬁsh () culture program along the U.S. West Coast. We are interested in speaking withany and all individuals interested in collaborating on the further development of the model,
applying the model to other species of interest such as rockﬁsh (), salmon (spp.), yellowtail (.), or any other aquaculture candidate species, and to

identify opportunities to validate model results.
Key words: escapes, survival, introgression, simulation, ﬁtness, selection, OMEGA.

2015年 1 月30日受理（Received on January 30, 2015）＊1
ICF International, 710 Second Avenue Suite 550, Seattle, WA 98104, U.S.A.
＊2
NOAA Fisheries, Northwest Fisheries Science Center, 2725 Montlake Blvd. East, Seattle WA, 98112, U.S.A.
＊3
Aquatic Resource Consultants, 9933 NE Lafayette, Bainbridge Island WA 98110, U.S.A.
Corresponding author: Jason D. Volk E-mail jason.volk@icﬁ.com

Jason D. VOLK, Michael B. RUST, Gregory R. BLAIR, Lars E. MOBRAND,
Conrad V. W. MAHNKEN, and Walton W. DICKHOFF

90

Modeling intraspeciﬁc genetic eﬀects for

to: 1) provide insights about factors aﬀecting risks

Management of Aquaculture Programs

associated with escapes from aquaculture operations,
2) help identify research priorities, 3) explore

The development of world cage farming ofoptions for the design of sustainable aquaculture

marine ﬁshes has raised concerns over the possible

programs, and 4) inform policy and management

genetic and ecological impact of escaped ﬁsh on wilddecisions related to the genetic and ecological risks

populations. Marine ﬁsh can escape from farms forof aquaculture.

a variety of reasons including: when improper mesh

OMEGA characterizes the aquaculture program

size is used or when holes in webbing develop fromby brood source, size and growth of cultured

normal wear and tear, during transfer from cage tofish, and frequency and magnitude of escape

cage or while grading or harvesting ﬁsh, from highevents. Genetic and ecological interactions arewind and high sea conditions during severe storms,

calculated from assumptions regarding survival ofor when net cages are breached by large predators.

escapees in nature, their likelihood of encountering

Potential effects include the introduction ofconspeciﬁcs, breeding success, and the consequence

maladaptive genes and reduced ﬁtness, competitionof interbreeding on the fitness and abundance offor food and space, and predation on native stocks.

wild conspecifics. The 100-year model simulation

Several factors merit consideration whendescribes the influence of aquaculture escapees on

evaluating the likely ecological and genetic impacts

survival and genetic ﬁtness of the natural population.

cultured ﬁsh may have should they escape. Among

By evaluating different aquaculture operation

them are: the wild population genetic structure andscenarios and wild stock population dynamics,

phenotypic variability of a species or among stocks

OMEGA allows the user to compare differences inwithin a species, the size of the local (or affected)

total abundance trends of escapees and wild fish,

population relative to the estimated number andas well as the effects of aquaculture program on

frequency of escapement, the type of breeding

survival and long-term sustainability of wild ﬁsh (ICF

program to be used, including selection of the2014).

founding stock, and the likelihood that unintentional

The modeling concepts of OMEGA have been

genetic drift (domestication) related to hatcherysuccessfully applied over the past decade using

practices will occur.

a similar model, the All-H Analyzer (AHA). AHA

In reality, there is little evidence-based information

has been successfully used by the Hatcheryavailable that can reliably assign risk to escapes of

Scientific Review Group (HSRG) to evaluate

ﬁnﬁsh from aquaculture facilities, therefore, escape

178 salmon and trout hatchery programs in thestandards are, out of necessity, more preventative

Columbia River Basin (Paquet

than prescriptive. Because of uncertainty, locallymodel simulation results, the HSRG formulated aadapted indigenous species will probably beworking hypothesis for baseline conditions fromencouraged over the use of nonnative and geneticallywhich they evaluated strategies to better achieve

modiﬁed species unless a compelling argument canstated goals for hatcheries and wild populations.

., 2011). Usingbe made that the genetic and ecological risks are

The HSRG concluded that through following

demonstrably low.

recommended management and harvest practices,

The Offshore Mariculture Escapes Genetic

such as broodstock selection and selective harvest,

Assessment (OMEGA) Model was developed byhatcheries can serve dual goals of contributing

ICF International (ICF) and NOAA as a tool for use

to harvest while remaining compatible with orby scientists and resource managers to help with

contributing to conservation goals. The guiding

understanding the potential negative impact ofprinciples of the HSRG are core to the purpose of

farmed escapees on their wild counterparts.

the OMEGA model.

The purpose of OMEGA is to identify and weighenvironmental risks of escapes of marine aquacultureﬁsh to their wild conspeciﬁcs. OMEGA is intendedModeling Intraspeciﬁc Genetic Eﬀects for Management of Aquaculture Programs
OMEGA Model Structure91

to year, assuming there are a certain amountof fish lost due to routine factors such as

As shown in Fig. 1, the OMEGA input data

small tears in nets, transfer between cages, or

structure is organized around three components:▪

▪

the biology and operations of the cultured

veterinary maintenance;▪

population, including number of fish by size

defect in a cage containing one size class of ﬁshclassification, broodstock source (domesticated(this can also occur because cage farms attract

or wild), maturation and growth factors;

predators);

the wild conspecific population, including▪

due to failure of multiple

assumptions of abundance, distribution, survival,

cages in the farm structure, resulting in a

age and size at maturity, age composition, andmassive loss of ﬁsh, such as from a major storm

age speciﬁc harvest rates; and▪

due to a mechanical failure or

event; andfactors affecting the potential for interaction▪

spawning in cages, leading to release of.

between farmed fish escapees and their wild

Once cultured fish escape into nature, theirconspecifics, assumptions of frequency andsurvival is described by a number of factors, suchmagnitude of fish escaping from the pens,

as size at time of escape, level of domestication andsurvival of escapees in nature, location of thetheir ability to survive in nature, and environmental

mariculture pens relative to the wild population,

factors. An attrition of the escapes occurs in thepotential movement patterns of escapees andmodel simulation based on these factors, resulting in

breeding success of escapees in nature.

a fraction of escapees surviving to encounter wild-

OMEGA accounts for the different factors byorigin fish. If escapees successfully reproduce inwhich ﬁsh can escape from cages, such as:

the wild, there are spawners in nature persisting▪

from cages, which can bewith potentially maladapted traits. There leads

considered a baseline level of escape from year

to an effect on genetic fitness of wild fish, which

Fig. 1. OMEGA Model Components

92

Jason D. VOLK, Michael B. RUST, Gregory R. BLAIR, Lars E. MOBRAND,
Conrad V. W. MAHNKEN, and Walton W. DICKHOFF

consequently aﬀects wild recruitment and harvest.
Included in this module are parameter inputs

they are provided only to describe the functions of
the model.

for the phenotypic fitness model in OMEGA. The

In nature, sablefish reach maturity at 6 years

equations are those described by Ford (2002) andof age and can live up to 80 years. Fish are fully

described in additional detail by Campton (2009)selected to the fishery at 4 to 5 years of age. In

for evaluating relative fitness effects of hatcheryculture, sableﬁsh can grow to a harvestable size of 1

salmonids interbreeding with wild populations.

kilogram in approximately 52 weeks. In this example,

The phenotypic trait model is described by two

the culture program is assumed to consist of 50

environmental optimum values, an aquaculture

separate offshore pen operations, each containing

optimum and a natural optimum, with selection

multiple pens with different size classifications

forcing the respective populations towards theiras fish grow to harvestable size. Total culture

environmental optimum (Fig. 2). The model

production is assumed to be 10,000 metric tons per

calculates the shift in phenotype trait value ofyear. For the two scenarios described below, natural

the wild population based on assumed gene flow

production, harvest, culture production, encounter

between escapees and wild ﬁsh. The consequence israte, and fitness and interactions including genetic

a loss in ﬁtness and survival of the wild population

effects are the same. Only escape rates wereas a result of this mixed wild population (thechanged between scenarios (chronic losses, cageprogeny of escapees and wild parents) phenotype.

failures, and catastrophic events).
In the “low escape scenario”, catastrophic eventsOMEGA Model Simulation Example

could occur with a probability of 5% in a given year
for the first 25 years. After year 25, it is assumed

This example uses a hypothetical commercial
aquaculture operation for sablefish (technology improvements would reduce theprobability of catastrophic events (1%) resulting in) along the coast of Washington, Oregon anda loss of up to 20% of cultured stock. The scenario

California. The purpose of this example is to show

assumes that chronic losses are the same across

how escapes can affect population abundance andall fish sizes (0.1% leakage). Cage failure rate islong-term viability, and how limiting escapes canassumed to be the same across all size categories

greatly reduce impacts to ﬁtness and abundance of(0.1%). Chronic losses and moderate events together

the wild population. These scenarios were developed

average about 200,000 ﬁsh escaping per year.

to demonstrate model capabilities and to test model

In the “high escape” scenario, catastrophic events

sensitivity to changes in parameter values, andare assumed to occur with a probability of 5‑10% on

are not intended to describe the operations of ana given year and each event results in up to a 60%

actual sableﬁsh aquaculture industry, or to suggest

loss of cultured stock. Chronic losses depend on ﬁshstandards that should be used to regulate operations;size classiﬁcation (3% for the smallest size class, 0.5%

Fig. 2. The Ford Model: Two Environments, Two Selective Forces

Modeling Intraspeciﬁc Genetic Eﬀects for Management of Aquaculture Programs

93

for harvest size ﬁsh). Cage failure rate is assumed torelative fitness also declined slightly, indicating

be 0.5% for smallest size class and 1.0% for harvest

effects of introgression. However, natural fitness

size fish. Chronic losses and moderate events

reaches an equilibrium value fairly quickly (~year

together average about 1.0 million fish escaping25). In contrast, under the “high escapes” scenario,

per year. This is an extremely high percent of total

natural fitness responded quickly to the effect of

production (10.0 million fish at harvest size) andescapes on the wild population and it appears ﬁtness

would represent an extreme example of escapesdoes not approach a lower equilibrium value until

due to chronic and moderate events. In the 100-year

year 75.

simulation, ﬁve random catastrophic events occurred
resulting in even larger losses.

The response of the wild population in termsof mixed population natural recruits under both

The model response in terms of natural population

scenarios is shown in Figure 4. The dark line

fitness under both scenarios is shown in Figure 3.

represents no-escape scenario (i.e. 0% change) as

Escape rates in “low escapes” resulted in a slightreference. Under the “low escapes” scenario, there isshift in the phenotypic trait value of the wild

an initial increase in recruitment (progeny of escapee

population toward the aquaculture optimum value

and wild parents) due to increases spawning biomass

over the 100-year simulation.

relative to no escapes, with the value approaching

Consistent with the slight shift in trait value,an equilibrium value of about +0.5% at year 100 of

Fig. 3. Natural Population Fitness, 100-year simulation

Fig. 4. Mixed Population Natural Recruits, 100-year simulation

94

Jason D. VOLK, Michael B. RUST, Gregory R. BLAIR, Lars E. MOBRAND,
Conrad V. W. MAHNKEN, and Walton W. DICKHOFF

the simulation.

analysis of scenarios for sablefish or other species,

The “high escapes” scenario describes the model
response if there was greater selective pressure
on the population due to a high level of escapes inand developing an economic cost/benefit analysis
module.
OMEGA is available at:nature. As in the previous scenario, there is an initial

http://www.nmfs.noaa.gov/aquaculture/science/26̲

increase in recruitment; more than in the previous

omega̲model̲homepage.html

scenario but the decline is steep and nearly constantafter year 30, approaching negative values in year

References100. This trend would likely continue for some years
due to a long-term loss in natural ﬁtness.
The results suggest a lag between natural

Campton D. E., (Hatchery Scientiﬁc Review Group),
2009: Columbia River hatchery reform system-

recruitment (Fig. 4) and ﬁtness (Fig. 3). Initially thewide report. February 2009 Appendix A. 278

trend is increasing recruitment while fitness was

p. Available: <http://www.hatcheryreform.us/

declining. This lag is likely due to the retention of

hrp̲downloads/reports/columbia̲river/system-

older, larger and ﬁt adults in the spawning biomass.

wide̲full/columbia̲river̲hatchery̲reform̲As more ﬁt ﬁsh die out over time the population issystem-wide̲report.zip>.

shifting to less fit adults and the consequence is a

Ford M. J., 2002: Selection in captivity during

decline in wild production. The results of the “highsupportive breeding may reduce ﬁtness in theescape” scenario indicate year-over-year declining

wild.

, 16, 815 825.

ﬁtness and reduced survival of wild ﬁsh. The long-

ICF International., 2014: Offshore Mariculture

lived nature of sablefish is a factor in the delayed

Escapes Genetic/Ecological Assessmenteﬀect on wild ﬁsh biomass.

(OMEGA) Model Version 1.0 Model Overview

OMEGA includes many other parameter inputs
that allow for exploration of different scenarios

and User Guide. August. Prepared for NOAA
Fisheries, Seattle, WA.

and evaluate model sensitivity due to combinations

Paquet P. J., Flagg T., Appleby A., Barr J.,of parameter value changes (culture and wild

Blankenship L., Campton D., Delarm M., Evelyn

population).

T., Fast D., Gislason J., Kline P., Maynard D.,

Balancing aquaculture goals with conservation

Mobrand L., Nandor G., Seidel P., and Smith S.,

goals will require careful evaluation of the risks

2011: Hatcheries, Conservation, and Sustainable

and benefits. The challenge of decision makers is

Fisheries̶Achieving Multiple Goals: Results

to ﬁnd solutions that serve both goals. The purposeof the Hatchery Scientific Review Groupʼsof OMEGA is to help identify possible solutions, orColumbia River Basin Review, Fisheries, 36:11,

if a range of solutions exists, how programs can be

547‑561.

managed for the best outcomes.
Annotated Bibliography
Next Steps
Jensen Ø., Dempster T., Thorstad E. B., Uglem
OMEGA is ready for general use. The modelI., and Fredheim A., 2010: Escapes of fishes

is available from NOAA along with a user guidefrom Norwegian sea-cage aquaculture: causes,that includes model background and instructions

consequences and prevention.

for using the model. The model also includes, 1, 71‑83.

the scenarios for sablefish discussed in thisThe authors broadly discuss the magnitude

presentation. We are interested in your feedbackof the problem of escapes from salmon and codand comments. At this time we seek collaborators

cage-farming aquaculture operations in Norway

to develop case studies for currently farmed or

and provide specific recommendations to prevent

considered aquaculture finfish species. Items forescapes. Current knowledge about the extent of

future development include completing a sensitivity

threats presented by escapes in terms of economic

Modeling Intraspeciﬁc Genetic Eﬀects for Management of Aquaculture Programs95

and ecological impacts are discussed in the context

these differences, hybridization is likely, especiallyof experiences in the Norwegian aquaculture

between farmed females and wild males. The

industry. While escapes occur due to several internal

results illustrate that behavioral diﬀerences between

and external factors, reports from fish farming

cultured and wild ﬁsh may not preclude spawningcompanies indicated that cage failure was by far theinteractions. The authors conclude there is a highmost common cause of large-scale escape in Atlantic

potential for farmed cod to hybridize with wild

salmon farming operations. They report studies thatfish. They recommend further research should beshow the mechanisms of escape are not the samea priority to further understand the consequences

across species. Atlantic cod may cause more wearof interbreeding and to identify methods for escapeto nets and be more likely to escape through tears

prevention.

in the net. Consequences of escape, such as diseasetransfer, interbreeding, competition, and predation

Ford M. J., 2002: Selection in captivity during

are generally discussed as areas for further research.

supportive breeding may reduce ﬁtness in the

The main message of this paper is that prevention

wild.

, 16, 815 825.

is the best tool to reduce the risk of escapes. The

Much of the concern surrounding effects ofauthors report evidence that the level of escapes

escaped cultured fish involves interbreeding withfrom cages was greatly reduced in Norway following

wild conspecifics and potential loss of genetic

legislation that has speciﬁc requirements for design

fitness of the wild population. Ford presents aof farms and the handling and use of equipment.

single trait phenotypic model that assumes diﬀerent

They recommend countries develop similar

optimum trait values for the culture and natural

measures to reduce escapes such as mandatoryenvironments. The Ford model describes how mean

reporting of escapes, a process to use these reports

phenotype values of captive and wild fish shift

to develop better standards, mandatory technicalrelative to optimum values for the environments

assessments following reported “large” escapebased on gene flow between escapees (or captive

events, technical standards for equipment, andbreeding) and wild fish. The results suggest thatﬁnally, identiﬁcation of key operational components

cultured fish can have a strong influence on thethat have a higher potential to cause an escapefitness and sustainability of wild populations

event, including training of staff to reduce humandepending on the amount of interbreeding. The

errors. This paper was used to identify mechanisms

level of effect depends on the details of the modelof escape through cage failure and operationalsuch as diﬀerences in optimum trait value, selection

scenarios, which are central to development of thepressure, and trait heritability. Controlling geneescapes component of the OMEGA simulation model.

ﬂow between wild and cultured ﬁsh can potentiallyreduce the domestication eﬀect in wild populations.

Meager J. J., Sklæraasen J. E., Fernö A., andOverall outcomes of ﬁtness in the wild also depend

Løkkeborg S., 2010: Reproductive interactions

heavily on habitat capacity and population dynamics.

between fugitive farmed and wild Atlantic cod

This model has been used in several other studies(

to explore the potential consequences to wild) in the ﬁeld.
, 67, 1221‑1231.

The authors present a study of spawninginteractions between cultured and wild Atlantic

population ﬁtness from captive breeding to increase
the size of wild populations, and from the unintended
straying of cultured ﬁsh to wild populations.

cod and tested the potential for hybridization
between farm escapes and wild conspeciﬁcs. Using

Paquet P. J., Flagg T., Appleby A., Barr J.,a spatial and temporal analysis of wild and farmed

Blankenship L., Campton D., Delarm M., Evelyn

cod tracked through biotelemetry, positioning ofT., Fast D., Gislason J., Kline P., Maynard D.,

ﬁsh based on sex and origin indicated that farmed

Mobrand L., Nandor G., Seidel P., and Smith S.,

fish behave differently from wild fish relative

2011: Hatcheries, Conservation, and Sustainable

to spawning ground location. However, despite

Fisheries̶Achieving Multiple Goals: ResultsJason D. VOLK, Michael B. RUST, Gregory R. BLAIR, Lars E. MOBRAND,
Conrad V. W. MAHNKEN, and Walton W. DICKHOFF

96

of the Hatchery Scientific Review Groupʼs
Columbia River Basin Review.

, 36: 11,547‑561.
The Hatchery Scientific Review Group (HSRG)
was established by the U.S. Congress to review
salmon and trout hatchery programs in the Paciﬁc
Northwest with the goal of recommending hatcheryreform guidelines while still retaining the goal ofproviding fish for harvest and conservation goals
for natural populations. This paper presents theapproach used and recommendations that included
an assessment of 178 hatchery programs and 351
salmonid populations within the Columbia River
Basin. This approach included a scientiﬁc frameworkand three principles to guide their assessment: 1)“clear and speciﬁc quantiﬁable goals for harvest andconservation,” 2) “be scientiﬁcally defensible,” and 3)“include monitoring and evaluation of benefits andrisks.” HSRG used the All-H Analyzer (AHA) model
to evaluate dynamics of populations in the ColumbiaRiver system through an integrative analysis ofseveral factors related to hatchery operations,and productivity and capacity of wild populations.
Using model simulation results, HSRG formulateda working hypothesis for baseline conditions
and to evaluate strategies to better achieve
stated goals for hatcheries and wild populations.
The HSRG concluded that through following
recommended management and harvest practices,such as broodstock selection and selective harvest,
hatcheries can serve dual goals of contributing
to harvest while remaining compatible with orcontributing to conservation goals. The guiding
principles stated by the HSRG are the core purposeof the OMEGA model.

水研センター研報，第40号，97−107，平成27年
Bull. Fish. Res. Agen. No. 40，97−107，2015

97

Application of Physiological Tests to Determine Speciﬁc Monovalentand Divalent Ion Supplementation for Culture of Marine Species
Calvin FISHER＊1, Charlotte BODINIER＊2, Adam KUHL＊3, and Christopher C. GREEN＊1

Abstract: The culture of marine and euryhaline ﬁshes in low salinity or ion deﬁcient waters has
been an area of interest for many aquaculturists due to the expense of marine salt mixes. The
Gulf killiﬁsh () is a euryhaline teleost found abundantly in coastal marshes along

the Gulf of Mexico. The ability to investigate the molecular underpinnings of speciﬁc ions in thismodel species could have a number of implications for other commercially important marine ﬁnﬁsh
species. Although studies have examined the inﬂuence of salinity on adults and juveniles, few haveinvestigated the role of salinity or speciﬁc ion concentrations in larvae. These investigations utilize
a model teleost to determine the role of speciﬁc monovalent and divalent ions at biochemical andmolecular levels.
Separate four week trials were conducted exposing newly hatched Gulf killiﬁsh to concentration
gradients of potassium (K+), calcium (Ca2+), and magnesium (Mg2+). The K+ supplementationconsisted of 0.3, 1.3, and 2.9 mM. Treatment groups for Ca2+ consisted of 0.2, 1.1, 1.5, and 2.1 mM
Ca2+, while trials using Mg2+ consisted of 0.1, 2.7, 5.1, and 10.4 mM Mg2+. All treatments weremaintained at a salinity of 9.5‑10‰ using crystal salt (99.6% NaCl). Each investigation consistedof four 50-L aquariums stocked at 7 larvae per liter for each concentration. Fish were sampled
at 0, 1, 3, 7, 10, 14, and 28 d post hatch (DPH). Upon each sampling the standpipe was adjusted
to maintain a constant density for each treatment. Collected samples were analyzed for wholebody ion concentrations (K+, Na+, Mg2+, Cl−), Na+/K+-ATPase (NKA) activity, dry weight, andexpression/localization of ion transport proteins (NKA, Na+/K+/2Cl− cotransporter (NKCC) andcystic ﬁbrosis transmembrane conductance regulator (CFTR)).
Mortality and growth was signiﬁcantly inﬂuenced by K+ concentration (< 0.05). No diﬀerenceswere observed among treatment groups for NKA enzyme activity, however at 28-d post hatch (dph)there were signiﬁcant diﬀerences in dry weight among K+ treatment. At seven dph diﬀerences inintestinal NKA and CFTR staining were observed, and NKA mRNA expression was also foundto be higher in the 0.3 mM [K+] group than in other treatment groups. Survival was signiﬁcantly
inﬂuenced by both Mg2+ and Ca2+ concentration (侑 0.05). Highest survival (71.1%) in the Ca2+

2+

trial was noted in the 0.2 mM [Ca ] treatment. In the Mg2+ trial, highest survival was noted in
the 2.7 mM [Mg2+] treatment (82.9%). NKA enzyme activity was reduced and delayed peaks wereobserved in whole body ion composition in the Mg2+ treatments. In addition, decreased CFTR
intensity was observed at the gill and intestine epithelium for the 0.05 mM [Mg2+] treatment at 1
dph, indicating that Mg2+ deﬁciency has a possible eﬀect on larval osmoregulatory capability. The
role of intestinal epithelium in ion uptake not only allows the potential for uptake and maintenanceof ion balance from dietary sources, but also demonstrates the potential to modulate concentrationsof speciﬁc ions in prepared waters for euryhaline and marine teleosts.
Key words: Gulf killiﬁsh, osmoregulation, potassium, magnesium, euryhaline
2015年 1 月30日受理（Received on January 30, 2015）＊1
Aquaculture Research Station, Louisiana State University Agricultural Center, Baton Rouge, LA, USA＊2
Rosenstiel School of Marine & Atmospheric Science, Miami, FL, USA＊3
Huntsman Corporation, Houston, TX, USA
Corresponding author:Christopher C. GREENE-mail: cgreen@agcenter.lsu.edu

98Calvin FISHER, Charlotte BODINIER, Adam KUHL, and Christopher C. GREEN

The major ions in seawater are; chloride (Cl−),

Grosell, 2005). The apically located NKCC2 most

sodium (Na +), magnesium (Mg 2+), Calcium (Ca 2+),

likely plays an active role in the reabsorption of−+

sulfate (SO4 ), and potassium (K ), with a variety

ions (Lorin-Nebel., 2006). The exact mechanismof other ions present in low concentrations. The

by which Mg

2+

uptake occurs has yet to be

three ions Ca 2+, Mg 2+, and K + are necessary fordescribed in detail. It is suggested that significant

maintaining electrolyte and acid-base homeostasis,

diﬀerences in Mg2+ regulation exist among species,

and the regulation of osmolality and intracellularknowledge of Mg2+ transport across epithelia andﬂuids (Fielder

cell membranes is still limited (Bijvelds., 2001). The high cost of synthetic., 1998).

marine salt mixes has facilitated interest in

By exposing euryhaline teleosts to varying salinities

alternative low-cost salt sources and supplementing

or individual ion concentrations, it may be possible

physiologically important ions. Potassium is involved

to immunolocalize specific osmoregulatory proteinsin ion regulation of intracellular fluids and can bein the gill filaments and intestinal epithelium to

added directly to the water, usually in the form

better understand the physiological adaptationsof KCl, or supplemented in the diet (Wilson andthese species undergo to cope with environmental

El Naggar, 1992).Calcium and magnesium bothchallenges.

influence the permeability of osmoregulatory
membranes making them critical ions in teleostionic regulation (Silva., 2005). Magnesium is

The Gulf killiﬁsh,, inhabits the estuarine

waters of the Gulf of Mexico and Atlantic coast of
Florida where it is commonly used as a baitﬁsh byresponsible for the activation of numerous enzymes

anglers for speckled trout,making it a necessary cofactor to the transfer

flounder,, and red drum,. From Texas to Florida it isof phosphate groups and the activation of ATP-referred to by many regional names including

dependent ion pumps such as NKA (Bijvelds., 1998). In freshwater fish, Mg

2+deficiency is

associated with increased Na+ and Ca2+ levels as well+

as low K levels (Bijvelds,., 1997).

but not limited to: mudminnow, mudfish, cocahoe
minnow, and bull minnow.

are closely

related to the mummichog,, whichIon regulation is controlled by ionocytes in which

inhabits the Atlantic coast and is similarly utilized bythere are several proteins involved in ion andanglers as a live bait. Both species are characterizedwater exchange (Evans

as a “hardy” bait tolerant of wide swings in salinity,., 2005; Lorin-Nebel., 2006). Previous investigations have focused on
+

+

+

+

Na /K -ATPase (NKA), Na /K /2Cl−

cotransporter

temperature, and other conditions encountered bylive marine bait.

occupy coastal marshes(NKCC), and the cystic fibrosis transmembrane

where salinity can change dramatically over a short

conductance regulator (CFTR) as the three primarytime period. These fish have the ability to live inproteins involved in ionoregulatory processes

salinities that range from freshwater (0 ‰ ) to near

(Hirose., 2003). The ionoregulatory process

double the concentration of seawater (up to 70

is initiated by a basolaterally located NKA whichppt) for several days. Exploitation of this salinity

couples two extracellular K+ with three intracellular

tolerance in+allows investigators the

Na generating an electrochemical gradient thatability to illicit physiological responses across a wide

drives the ions according to expression, location,

range of ion concentrations.

and abundance of NKCC and CFTR (Bodinier.,

Several studies have investigated the use of low., 2008). NKA is responsible for thesalinity inland seawater or water from brackish

lowering of intracellular Na+ concentrations allowing

aquifers as a source for marine aquaculture. Much

2009; Kang

+

+

−

the basolateral NKCC to import Na , K , and Cl ,of these inland saline waters were found to have ion

the excess Cl− is then secreted through the chloride

deﬁciencies, where speciﬁc manipulation of the major

channel CFTR. Additional water exchange and Cl−

ions in saltwater have signiﬁcantly increased growth

ion regulation occurs intestinally (Christensen.,

and survival (Doroudi., 2006; Fielder., 2001;2012) and it has also been suggested that substantial

FotedarMg 2+ uptake occurs intestinally (Marshall andthe effects of low salinity waters on juvenile and., 2008). Many studies have investigated99

Ion Supplementation for Culture of Marine Species
adult euryhaline species (Coulon., 2006; Fielder
Patterson., 2012; Doroudi., 2012; Roy

Newly hatched (< 4 h old)

larvae were., 2008;

stocked, in triplicate, at a density of 7 larvae per., 2007). Few studies

L into 50-L aquaria maintained on a common., 2001; Fotedar

have attempted to explore the importance ofrecirculating system utilizing biological and ultra

specific ion supplementation in euryhaline teleost

violet ﬁlters. Larvae were fed a diet of−1d ) for the ﬁrst 7 d at alarviculture. The objectives of this work were tonauplii (30demonstrate the use of physiological examinations

rate of three times per day followed by OtohimeTM

at biochemical and molecular levels to determine

B1 (Reed Mariculture Inc., Campbell, CA, USA) diet

+

ﬁsh

.

−1

specific supplementation of monovalent (K ) andfed to apparent satiation 4 times per day for thedivalent ions (Ca2+ and Mg2+) as replacements forremainder of the study. After 4 weeks, survival was

saltwater in the culture ofcalculated by draining all aquaria and counting eachlarvae.

individual.
Materials and Methods

Treatments were established by adding crystal
salt (Diamond Crystal Solar Salt, 99.6% NaCl) to 10‰

Three separate four-week experiments werewith ion supplementation.Potassium supplementation

conducted to investigate the impact of external K+,

consisted of 0.3, 1.3, 2.0and 2.9 mM (Table 1). ThisCa

2+and Mg2+

concentrations on larval.

investigation represents [K+] from values similar to

Table 1. Mean salinity and ion concentrations (± SEM) given for each
K+ ,Ca2+, and Mg2+ Concentration treatment group. Superscript lettersdenote statistically signiﬁcant diﬀerences in speciﬁc ion concentrations
among treatment groups ( < 0.05)
Parameter

1

K+ (mM)
0.33 ± 0.05A
Na+ (mM)
170.19 ± 3.40A
Mg2+ (mM)
0.04 ± 0.00A
Ca2+ (mM)
0.31 ±0.00A
Salinity(‰)
9.8 ± 0.28
pH
8.56 ± 0.05
TAN (mg/L)
0.03 ± 0.01
NO2 (mg/L)
0.00 ± 0.00
37.25 ± 1.55
Hardness (mg/L)**
184.00 ± 25.16
Alkalinity (mg/L)*
Reported as CaCO3**
Represents reference salt group

K+ Treatment group23
1.31 ± 0.04B
157.52 ± 6.08AB
0.12 ± .01A
0.43 ± 0.05A
9.4 ± 0.4
8.27 ± 0.10
0.02 ± 0.00
0.00 ± 0.00
39.17 ± 2.06
186.00 ± 6.55

2.06 ± 0.04C
151.56 ± 7.03AB
0.11 ± 0.01A
0.30 ± 0.03A
9.4 ± 0.4
8.24 ± 0.09
0.01 ± 0.00
0.01 ± 0.00
33.83 ± 2.20
182.50 ± 6.98

4**
2.96 ± 0.04D
138.01 ± 0.83B
11.55 ± 0.10B
2.25 ± 0.02B
9.6 ± 0.1
8.07 ± 0.11
0.04 ± 0.03
0.01 ± 0.00
1442.67 ± 11.04
209.83 ± 3.06

Parameter
K+ (mM)
Na+ (mM)
Mg2+ (mM)
Ca2+ (mM)
Salinity(‰)
TAN (mg/L)
NO2 (mg/L)
Hardness (mg/L)*
Alkalinity (mg/L)**
Reported as CaCO3

1
2.37 ± 0.03
165.31 ± 3.07
0.11 ± 0.01
0.20 ± 0.02A
10.3 ± 0.03
0.03 ± 0.02
0.05 ± 0.02
193 ± 32.76A
178 ± 17.97

Ca2 Treatment groups
23
2.32 ± 0.02
2.33 ± 0.02
166.72 ± 4.83
166.72 ± 4.83
0.16 ± 0.02
0.15 ± 0.01
B
1.08 ± 0.03
1.50 ± 0.04C
10.1 ± 0.07
10.1 ± 0.06
0.03 ± 0.02
0.01 ± 0.01
0.03 ± 0.02
0.02 ± 0.01
1090 ± 15.81B
1433 ± 32.24C
263 ± 57.64
140 ± 4.08

42.31 ± 0.05
165.84 ± 2.97
0.12 ± 0.01
2.07 ± 0.05D
10.0 ± 0.09
0.02 ± 0.02
0.01 ± 0.00
1740 ± 31.52D
170 ± 7.07

Parameter
K+ (mM)
Na+ (mM)
Mg2+ (mM)
Ca2+ (mM)
Salinity(‰)
TAN (mg/L)
NO2 (mg/L)
Hardness (mg/L)*
Alkalinity (mg/L)**
Reported as CaCO3

1
2.37 ± 0.03
168.48 ± 3.64
0.05 ± 0.01A
0.19 ± 0.02
9.9 ± 0.04
0.02 ± 0.01
0.01 ± 0.03
102 ± 25.82A
165 ± 19.53

Mg2+ Treatment groups
23
2.40 ± 0.04
2.29 ± 0.05
169.96 ± 4.11
162. 64 ± 4.83
2.88 ± 0.08B
5.76 ± 0.16C
0.24 ± 0.02
0.15 ± 0.01
10.2 ± 0.03
10.1 ± 0.03
0.01 ± 0.02
0.02 ± 0.02
0.03 ± 0.02
0.00 ± 0.00
320 ± 44.82B
530 ± 29.35C
160 ± 4.71
150 ± 10. 62

42.41 ± 0.02
170.55 ± 4.97
11.52 ± 0.19D
0.26 ± 0.02
10.0 ± 0.05
0.03 ± 0.02
0.02 ± 0.01
1040 ± 36.21D
185 ± 21.83

Calvin FISHER, Charlotte BODINIER, Adam KUHL, and Christopher C. GREEN

freshwater sources (0.33 mM) to concentrations ofA

Mean final mass

2.96 mM, found saline waters at 10 ‰. Potassium
were maintained at 2.35 mM, as determined by aprevious study (Fisher., 2013). Calcium was

supplemented to give final concentrations of 0.20,
1.08, 1.50, and 2.07 mM (Table 1), while Mg

2+

was

80

100

A

A

Mean final mass (mg)concentration in [Ca 2+ ] and [Mg 2+ ] experiments

Mean survival

W

60

B

80

B

X

60

Y

40

40

20

supplemented to give concentrations of 0.05, 2.88,

Z

0

20

Mean survival %

100

0
0.20

5.76, and 11.52 mM. The [Mg 2+] experiment also

1.08
1.50
[Ca2+] mM

received Ca 2+ supplementation to the water in

2.07

the form of CaCO2, as determined in the previous
experiment (Table 1). Weekly water samples wereanalysis by the LSU Agriculture Center; AgricultureB

post hatch (DPH). Upon each sampling the standpipe
was adjusted to maintain a constant density foreach treatment. Three replicates of 5 larvae wereused to establish an average desiccated dry massdue to the inability to accurately measure individual

Mean final mass (mg)Chemistry Department (Baton Rouge, LA, USA).
Fish were sampled at 0, 1, 3, 7, 10, 14, and 28 d

Mean final mass
100
80

Y
A

using the methods described by (Van Genderen,
2003; Fisher., 2013). Three replicates of 5 larvae

were collected at each time point to determine Na+/
K+-ATPase activityusing the protocol described in

A

100

B

80

YZ
A

60

Z

40

60
40

X

20

20

0

0
0.05

dry mass of larvae less than 7 dph. Whole body
ion concentration (Na+, K+ and Mg2+) was analyzed

Mean survival

Mean survival %collected to quantify ion concentrations via ICP

2.88
5.76
[Mg2+] mM

11.52

Fig. 1. Final mass and survival of Gulf killiﬁsh larvae
after 4 weeks in (A) [Ca2+] treatments or (B) [Mg2+]
treatments. Letters represent signiﬁcant diﬀerencesamong treatment groups. Letters denote signiﬁcantdiﬀerences among treatments.

McCormick (1993) and expressed as µmol ADP mg
protein−1 h−1.

5%); however the 2.9 mM [K+] treatment resulted in

Six larvae were collected per treatment group

signiﬁcantly greater survival after 4 weeks (survival

at 1, 3, and 7 dph and preserved in Z-ﬁx (Anatech,~60%). Final dry mass of the 2.9 mM [K+] reference

LTD, Battle Creek, MI, USA). Immunocytochemistrygroup was signiﬁcantly higher than both the 1.3 andreactions were performed according to methods

2.0 mM [K+] treatments (described in Bodinier. (2010) and Fisher

侑 0.05). The 2.9 mM [K+]reference group had a ﬁnal dry mass of 4.93 ± 0.54

. (2013) for NKA, CFTR, and NKCC1. Ionocyte

mg while the 1.3 and 2.0 mM [K+] treatments had

length measurements were taken using Image Tool®

dry mass of 2.39 ± 0.19 mg and 2.21 ± 1.55 mg,

version 3.0 (University of Texas Health Science

respectively.

Center, San Antonio, TX, USA). A minimum of 50

Survival and dry mass were significantly

cells were measured per sampling period for each

influenced by both [Ca2+] and [Mg2+] experiments.

treatment.

Supplementation of Ca 2+ showed a decrease inmean ﬁnal mass and mean survival with increasing
concentrations of Ca 2+ in the absence of Mg 2+

Results(Fig. 1A). Supplementation of Mg 2+ resulted in
Growth and survival: The 0.3 mM [K+] treatment

approximately a 5-fold increase in survival with Mg2+

resulted in 100% mortality within 24 h of hatch.

supplementation as compared to no supplementation,

No significant differences were seen in survival

no diﬀerences were observed among supplemented+

between the 1.3 and 2.0 mM [K ] groups (survival 侑

treatment groups. Final mass was not signiﬁcantly

101

Ion Supplementation for Culture of Marine Species
influenced until a concentration of 11.52 mM Mg2+

by a significant increase in activity throughout

was reached (Fig. 1B).

the experiment, however no treatment specificdifferences in activity were determined at++Whole-body ion concentrations & Na /K -ATPase+

any specific sample dates. The [Mg 2+ ] study+

activity: No differences were found in Na /K -demonstrated significant differences in Na + /K +ATPase activity among [K + ] treatment groups-ATPase activity between Mg2+ deﬁcient treatments

2+

and those with suﬃcient supply at 0 dph followed byconcentration showed no difference for treatment

minor, but not signiﬁcant, increases throughout theor treatment × dph interaction; however signiﬁcant

experiment (Fig. 2).

or among time (侒 0.05). Whole body Mg

diﬀerence was determined for dph (侑 0.05). There

was a signiﬁcant increase in [Mg2+] within the ﬁrst

Immunocytochemistry & cell volume: All negative+

week followed by a rapid decrease for all three[K ]slides, with no primary antibody, showed notreatment groups. Whole body K + concentration

immunostaining as expected (not illustrated). In

had no significant difference in treatment × dph[K+] treatments at 7 dph, no significant differencesinteraction, although significant differences were

were determined among treatments for gillobserved among treatments and for dph (侑 0.05).

ionocyte length. Gill ionocytes from the 1.3 mM

No diﬀerences were observed among treatments[K+] treatment were found to have a significantly

2+

for whole body ion composition in the [Ca ] study.

larger area than those in the 2.0 and 2.9 mM [K+]The [Mg2+] experiment showed delayed peaks intreatments. Intestinal CFTR and NKA were present

+

2+

+

whole body [Na ], [Ca ], and [K ] for the 0.05 mM
2+

at 7 dph in all surviving [K+] treatment groups. No[Mg ] treatment group, with ion concentrations

changes were observed in NKA or CFTR in thereaching their highest point 7 days later thangill ionocytes. Intestinal NKA and CFTR staining

other treatments. As expected, the Mg

2+

deficientappeared to show a decrease in intensity as K +2+concentration increased (Fig. 3). Very little staining

treatment groups (0.05 and 2.70 mM [Mg ]) also
2+

showed a signiﬁcant decrease in whole body [Mg ].
+

Na /K

+

was observed for NKCC both intestinally and at the2+-ATPase activity in the [Ca ] study

ATPase activity (μmol/mg protein/hr)

was significantly lower at 0 and 1 dph followed

0.14

gill epithelium.
No diﬀerences among treatments were observed[Mg2+] mM

0.12
0.10

0.05

2.88

5.76

11.52

0.08
B B

0.06
0.04

A A

0.02
0.00
0

1
3
Days post hatch

7

Fig. 2. Whole body NKA activity for larvae stocked in [Mg2+] treatments after 0, 1, 3,
and 7 dph. Letters represent signiﬁcant diﬀerences among treatment groups at that day.

102

Calvin FISHER, Charlotte BODINIER, Adam KUHL, and Christopher C. GREEN+[K ] Treatment
Group

Merged

v

CFTR

v

L

1.3

v

L

v

v

2.0

L

v
L

L

L

2.9

NKA

L

L

v

v

L
v

Fig. 3. Representative histological sections from larval intestines after one week in [K+] treatment groups
cut transversely at 5 µm intervals. Immunofluorescence of NKA (red) and CFTR (green). The 2.9 mmol K+
group represents a reference salt used andcontains other ions which may be essential for the function ofosmoregulatory proteins. L: lumen; v: villi

for NKA or NKCC staining intensity in the gills orby the chloride cotransporter, NKCC, to assist

intestine, nor were there any signiﬁcant diﬀerencesin the transport of chloride ions, an extracellularamong treatments for gill ionocyte length orK+ shortage could reduce a larval fishʼs ability toquantity at 1, 3, and 7 dph for both the [Ca2+] andregulate sodium and chloride ion concentrations at

2+2+[Mg ] experiments. In the [Mg ] experiment, acritical biological membranes. Despite the fact that

decrease in CFTR staining intensity was observed

there were no statistical diﬀerences in NKA activity

at 1 dph in both the intestine (Fig. 4) and gill inbetween any treatment groups, it is still conceivable2+

the 0.05 mM [Mg ] treatment, no other changes inthat suﬃcient K+ was not available for the function

CFTR staining intensity were observed.

of NKCC. If so then the inability of the larvae toregulate either Na+ or Cl− ions may possibly havebeen a factor in the low survival of the 0.3, 1.3 andDiscussion

2.0 mM [K+] groups. The reference group (marine
The chemical gradient generated by the activation

mix salt) was the only group that did not displayof NKA is one of the primary driving forces behindpoor survival and it is possible that the presence ofion regulation, thus the ability to measure wholeother essential ions such as Mg2+ and Ca2+ played

body NKA activity may allow for the evaluation ofa significant role in the survival of this treatment

larval fish osmoregulation capacity in hyper andgroup.

hypo-osmotic environments. It is possible that K+

Potassium supplementation across the gradientis a limiting factor in killiﬁsh larval osmoregulation

examined in the current study at a constant salinity

and a deﬁciency can restrict the ability of NKA to

not only altered survival, but affected differences

function. The current model for chloride secretion

among molecular components of intestinal

as described by McCormick. (2003) suggests aepithelium. Although potassium treatments inbasolateral NKA and NKCC coupled with an apicallythe current study did not appear to alter whole+

located CFTR-like protein. Because K is also usedbody Na/K ATPase activity, immunofluorescence103

Ion Supplementation for Culture of Marine Species[Mg2+]
TreatmentGroup

DIC with CFTR

CFTR

L
v

0.05

v
L

2.88

v
L

5.76

11.52

L

v

Fig. 4. Representative histological sections of the intestine from 1 day post hatch(dph) Gulf killifish larvae in [Mg2+] treatment groups, cut transversely at 5 µm
intervals. Diﬀerentialinterference contrast (DIC) images with immunoﬂuorescenceof CFTRL: lumen; v: vill
showed increased density of NKA and CFTR+

the potential for uptake and maintenance of ion

proteins as K concentration decreased. Whole bodybalance from dietary sources and thus important in

K+ concentration was greater in the reference asaquaculture, but also demonstrates the potential tocompared to treatments with 1.3 and 2.0 mM of K+

modulate concentrations of speciﬁc ions in preparedboth of which remained lower throughout the study

waters for euryhaline and marine teleosts.

as expected due to the inability to selectively retain+

Calcium and Magnesium are critical in teleost

K ions. It is important to note that the reference

ion regulation, varying concentrations of Ca2+ and+Mg2+ can impact the permeability of osmoregulatorygroup containing the highest concentration of K

was also comprised of greater concentrations of
2+

Mg

2+

+

and Ca , which could have modulated Na andepithelia to both ions and water (Silva., 2005). In2+

the sea water adapted eel, removal of Ca

from theCl − absorption in the intestine relative to lower K+

external medium diminished the active excretion oftreatments in the current study that were relatively

sodium by half, excretion returned to normal level

low in these divalent ions (Marshall and Singer,

after the reintroduction of Ca2+ to the medium (Isaia

2002; OʼGrady, 1989). Growth and survival metrics

and Masoni, 1976). Proper development of manyfor the reference treatment may reflect combined

euryhaline teleosts is related to environmental Ca2+

eﬀects of a full to partial complement of other ionsconcentration, for example red drum (in addition to the K+ gradient examined. The role of) fry displayed a drop in blood osmolarity

intestinal epithelium in ion uptake not only allows

when transferred from salt to freshwater, this104

Calvin FISHER, Charlotte BODINIER, Adam KUHL, and Christopher C. GREEN

decline in osmolarity was reduced by the addition

Bijvelds M., Velden J., Kolar Z., and Flik G., 1998:of Ca 2+ (Wurts and Stickney, 1989). Previous

Magnesium transport in freshwater teleosts.

2+

studies have determined that Mg

is critical in the., 201, 1981‑1990.

activation of ATP-dependent ion pumps (Bijvelds

Bodinier C., Boulo V., Lorin-Nebel C., and., 1998) making it critical for the activation ofCharmantier G., 2009: Inﬂuence of salinity on the2+

NKA. In this study, both the 0.05 and 2.88 mM [Mg ]localization and expression of the CFTR chloride

treatment groups displayed significantly reduced

channel in the ionocytes of

NKA activity at hatch.

during ontogeny.

., 214, 318‑329.

2+

Bodinier C., Sucré E., Lecurieux-Belfond L., Blondeau-

had effects on survival, growth, and molecular

Bidet E., and Charmantier G., 2010: Ontogeny

components at both the gill ﬁlaments and intestinalof osmoregulation and salinity tolerance inI n t h e cu r r e n t s t u d y b o t h Ca

2+epithelium. In the absence of Mg
larval2+

a n d Mg

supplementation

the gilthead sea bream Sparus aurata.

had reduced survival, decreased‑

Na /K -ATPase activity, and a delayed peak in., 157, 220‑228.

++

whole body compositions for several ions essential for

Christensen A. K., Hiroi J., Schultz E. T., andlarval development. A decrease in CFTR intensity

McCormick S. D., 2012: Branchial ionocyte

2+

was also observed in the gills and intestine of Mg

organization and ion-transport protein

deﬁcient treatment as seen by immunocytochemistry

expression in juvenile alewives acclimated tostaining at 1 dph. This is an indication that Mg2+ isfreshwater or seawater.

a necessity for larval

growth, survival,., 215, 642‑

652.

and ion regulation. However, the Mg 2+ of 10‰

Coulon M., Gothreaux C., and Green C., 2012:seawater is approximately 11.52 mM and the

Influence of substrate and salinity on air-greatest growth and survival were achieved in thisincubated Gulf killiﬁsh embryos.

study at a concentration of 2.88 mM Mg2+. These., 74, 54‑59.

results demonstrate that the potential for euryhaline

Doroudi M. S., Fielder S D., Allan G L., and Webster

teleost culture in prepared water may only require

G. K., 2006: Combined effects of salinity anda fraction of the ion concentrations found in 10‰

potassium concentration on juvenile mulloway

seawater. Also the role of intestinal epithelium in ion(

uptake allows for the potential for ion maintenance

Schlegel) in inland saline groundwater.

through dietary sources.

, Temminck and., 37, 1034‑1039.

Evans D. H., Piermarini P. M., and Choe K. P., 2005:
Acknowledgements

The multifunctional fish gill: Dominant siteof gas exchange, osmoregulation, acid-baseThis work was supported, in part, through grants

regulation, and excretion of nitrogenous waste.

from the Southern Regional Aquaculture Center., 85, 97‑177.

and Louisiana SeaGrant. The authors would like

Fielder S. D., Bardsley W. J., and Allan G. L., 2001:

to acknowledge the faculty, students and support

Survival and growth of Australian snapper,

staff that assisted with these investigations: Paige, in saline groundwater fromOʼMalley, Emily McArdle, Chris Mariani, Mike

inland New South Wales, Australia.

Coulon, Josh Patterson, Charlie Brown.

201, 73‑90.

.,

Fisher C., Bodinier C., Kuhl A., and Green C., 2013:
References

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survival and ion regulation in Gulf killifish

Bijvelds M. J. C., Flik G., and Bonga S. E. W., 1997:
Mineral balance in Oreochromis mossambicus:
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larvae reared in ion deﬁcientsaline waters.
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Fotedar R., Harries S., and Savage S., 2008: Survival,

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Ion Supplementation for Culture of Marine Species
growth and osmolality of greenlip abalone

C142-C146.

(Donovan 1808) when exposed

Patterson J., Bodinier C., and Green C., 2012: Eﬀects

to diﬀerent ionic proﬁles of inland saline water.

of low salinity media on growth, condition, and., 39, 441‑448.

gill ion transporter expression in juvenile Gulf

Hirose S., KanekoT., Naito N., and Takei Y., 2003:
Molecular biology of major components ofchloride cells.

killifish,. Comp. Biochem.

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Roy L. A., Davis D. A., SaoudI P., and Henry R. P.,

Isaia J., and Masoni A., 1976: The effects of

2007: Supplementation of potassium, magnesium

calcium and magnesium on water and ionicand sodium chloride in practical diets for thepermeabilities in the sea water adapted eel,

Pacific white shrimp,L.

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221‑233.

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reared in low salinity waters.
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Kang C. K., Tsai S. C., Lee T. H., and Hwang P. P.,

Silva L. V. F. D., Golombieski J. I., and Baldisserotto

2008: Diﬀerential expression of branchial Na+/B., 2005: Growth and survival of silver catfish

K+-ATPase of two medaka species, Oryzias

larvae, Rhamdia quelen (Heptapteridae), at

latipes and Oryzias dancena, with different

diﬀerent calcium and magnesium concentrations.

salinity tolerances acclimated to fresh water,brackish water and seawater.

, 3, 299‑304.
Van Genderen E. J., 2003: Inﬂuence of soft surface‑., 151,water conditions on copper toxicity to

566‑575.

Rafinesque: responses atLorin Nebel C., Boulo V., Bodinier C., and Charmantiermechanistic-, population-, and watershed- levels

G., 2006: The Na+/K+/2Cl- cotransporter in theof organization. Clemson University, Clemson,sea bass

SC, USA.

during ontogeny:

involvement in osmoregulation.

.,

209, 4908‑4922.

Wilson R. P., and El Naggar G., 1992: Potassium
requirement of fingerling channel catfish,

Marshall W. S., and Grosell M., 2005: Ion transport,
osmoregulation, and acid-base balance,. Aquacult., 108, 169‑175.
Wurts W. A., and Stickney R. R., 1989: Responses of., 177‑230.

Red drum (Marshall W. S., and Singer T. D., 2002: Cystic ﬁbrosis
transmembrane conductance regulator in teleostfish.

) to calcium andmagnesium concentrations in fresh and salt
water. Aquacult., 76, 21‑35.

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Annotated Bibliography

McCormick S. D., 1993: Methods for nonlethal gillbiopsy and measurement of Na+, K+-ATPase
activity.

., 50, 656‑

658.

Fisher C., Bodinier C., Kuhl A., and Green C., 2013:
Effects of potassium ion supplementation on
survival and ion regulation in Gulf killifish

McCormick S. D., Sundell K., Bjornsson B. T., Brown
C. L., and Hiroi J., 2003: Influence of salinity+++

on the localization of Na /K -ATPase, Na /larvae reared in ion deﬁcientsaline waters.

.,

164, 572‑578.

K+/2Cl− cotransporter (NKCC) and CFTR anion

This study encompasses results pertaining tochannel in chloride cells of the Hawaiian goby+
the K ion manipulation portions of the abstract(

presented above for the UJNR Scientiﬁc Symposium.

).

., 206,This investigation increased [K+] from values similar

4575‑4583.
OʼGrady S. M., 1989: Cyclic nucleotide-mediatedto freshwater sources (0.33 mM) to concentrations

effects of ANF and VIP on flounder intestinalof 2.96 mM, found in saline waters at 10‰. A

ion transport.

number of biochemical and molecular techniques., 256,

106

Calvin FISHER, Charlotte BODINIER, Adam KUHL, and Christopher C. GREEN

were performed to examine the effect of thisBodinier C., Sucré E., Lecurieux-Belfond L., Blondeau-

K + ion gradient, which included: whole body ion

Bidet E., Charmantier G., 2010: Ontogeny of++composition, Na /K -ATPase (NKA) activity, gillosmoregulation and salinity tolerance in theionocyte morphometrics, relative gene expression

gilthead sea bream Sparus aurata. Comp.

(NKA, NKCC, and CFTR), and corresponding

Biochem. Physiol. ‑ Part A: Mol. Integrative

immunocytochemistry at the gill and intestinal

Physiol., 157, 220‑228.

epithelium. Results more tangible to aquaculturist,

The gilthead sea bream () is asuch as growth and survival, indicated the presence

commercially important aquaculture species, whichof a threshold within the gradient examined whereby

spawns in the ocean and whose resulting larvae andsurvival was not different between 1.3 to 2.9 mMjuveniles migrate to lower salinity estuaries and+[K ]. Utilizing immunocytochemistry, the diﬀerences

lagoons. These authors investigated the development

between these seemingly similar treatment groupsof salinity tolerance in gilthead sea bream from 3,

indicated that the treatments groups between 1.3 to30, 75, 96, and 300 d post hatch (dph) by challenging+

2.9 mM [K ] was diﬀerent in terms of gill ionocyte

them with 9 salinities ranging between freshwater

area, NKA and CTFR localization.

and 45.1‰. Utilizing immunohistochemistry, theseauthors localized the NKA throughout these

Ostrowski A. D., Watanabe W. O., Montgomery F.

challenges to document location of ion regulation

P., Rezek T. C., Shafer T. H., Morris Jr J. A.,within respect to this ionoregulatory protein. Initially,

2011: Eﬀects of salinity and temperature on the

immunopositive NKA ionocytes were laocted in thegrowth, survival, whole body osmolality, andintegument along the yolk sac and integumentary

expression of Na+/K+ ATPase mRNA in red

folds representing the branchial slits. A functional

porgy (

shift from integument to gills was demonstrated) larvae.

., 314,

193‑201.

30 and 70 dph, when both the integument and gills

These authors investigated a number ofwere observed to locally express NKA, whereby

physiological parameters pertaining to salinity andfrom 70 to 300 dph the gills remain the main site

temperature in embryos and larvae of red porgyof osmoregulation. Increases in osmoregulatory(), which is viewed as a high-market

capacity for this species at the intervals examinedvalue marine species with good potential as anwithin this study related to the shifts and patterns

aquaculture species. Embryos and resulting larvae

observed through immunohistochemistry.

were reared at four temperatures (17, 19, 21, 23℃)
and two salinities (24 and 34‰). Larvae (16 dph) were

Christensen A. K., Hiroi J., Schultz E. T., McCormick

transferred from their respective salinities to 44‰

S. D., 2012: Branchial ionocyte organization andto represent a sublethal hyperosmotic challenge.

ion-transport protein expression in juvenile

These authors demonstrated signiﬁcant increases inalewives acclimated to freshwater or seawater.

NKA mRNA expression in individuals acclimated to24 ‰ at 24 h after transferred to increased salinity,., 215, 642‑652.
In gill ionocytes, ion transport is activated bywhile individuals from 34‰ exhibited no signiﬁcantthe basolaterally located NKA which generates

changes in NKA expression. Temperature was not

an electrochemical gradient by coupling two

observed to influence expression of NKA, while

extracellular K

metabolic parameters related to growth were

driving the ions according to expression, location,

influenced by the temperature gradient in theirand abundance of other proteins such as NKCC

study. The authors utilized traditional growthand CFTR. Christensen

metrics including molecular tools to anticipate

in alewife physiology and branchial epithelium

optimum salinity (24‰) and temperature (23℃)

as individuals were acclimated to freshwater or+with three intracellular Na + ,. investigated changes

conditions that are optimum for larval rearing of thissaltwater and represents the ﬁrst study of its kind

species.

to characterize multiple ion-transport proteins ina non-salmonid anadromous fish. Corresponding

Ion Supplementation for Culture of Marine Species
increases in NKA, NKCC1, and CFTR abundance at
the gill epithelium with increasing salinity was used
to establish a gill model for hypo-osmoregulation.
In gill ionocytes, NKA is responsible for lowering
intracellular Na+ allowing the basolateral NKCC1 toimport Na+, K+, and two Cl− ions. Excess intracellular
Cl− is then secreted through the CFTR chloride
channel. NKCC1, the secretory isoform is expressedbasolaterally in the gill ionocytes, while NKCC2 is
identiﬁed as an absorptive isoform and is expressedapically along the intestinal and urinary bladderepithelium of saltwater and euryhaline teleosts. The
authors determined the key differences between
freshwater and seawater acclimated alewives in thecontext of ion transporters at the gill epithelium.
The implications of these investigation has assisted
in increasing the information multicellular complexesof mature ionocytes and the role of salinity andspeciﬁc ion in the maintenance of homeostasis.

107

水研センター研報，第40号，109−110，平成27年
Bull. Fish. Res. Agen. No. 40，109−110，2015

109

Genetically Modiﬁed Salmon in Aquaculture: Well Regulated and Safe
Paul G. Olin＊1

Abstract: A Massachusetts company, Aquabounty Technologies, submitted an application to the
U.S. Food and Drug Administration (FDA) in 1995 to grow a genetically modiﬁed AquAdvantage®
Atlantic salmon to be marketed as a food product. Aquabounty proposed to raise the broodstockﬁsh on Prince Edward Island, ship their eggs to a contained inland recirculating production system
in Panama to grow, harvest and process the ﬁsh, and then ship food grade product back to theUnited States for sale. The ﬁsh for this physically secure production system would be at least 99
percent triploid and all-female, as an additional reproductive-containment measure.

The AquAdvantage® Atlantic salmon carries aheld in September 2010. This briefing packet

Chinook salmon growth hormone gene that resultssummarized their scientific review, and the basicin production of growth hormones that enable theconcl usi ons were that the AquAdvantage ®

fish to grow to market weight in half the time it

salmon are safe, nutritionally comparable to othernormally takes (Fig. 1) This gene is regulated by a

Atlantic salmon, and when produced as describedsegment of DNA from the ocean pout, a blenny-likein the application do not pose a threat to theﬁsh found in frigid waters of the Northwest Atlantic.

environment. The review process used by FDA on

The review process used by FDA on thethe AquAdvantage® salmon was rigorous, detailed

AquAdvantage® salmon involved a team ofand extensive, spanning more than 15 years. Speciﬁcscientists and subject-matter experts on the

conclusions from the report stated that “Food from

Veterinary Medicine Advisory Committee who

AquAdvantage® Salmon is the same and as safe toadvise the FDA and the general public on scientiﬁc

eat as food from other Atlantic salmon.”issues as they relate to ensuring public and animal

In reference to concerns about adverse

health. The FDA released a 172 page briefing

environmental impacts the report states “There

packet and an 84 page environmental assessment

is substantial, reliable information available incontaining information relevant to the application

the environmental assessment document toin advance of a public advisory committee meetingconclude that AquAdvantage® Atlantic salmon
are not expected to have a significant impact on
the environment when raised and reared under
the current conditions of physical, biological andgeographical/geophysical confinement present at
hatchery and grow-out facilities in Canada andPanama. We have a high degree of certainty in ourconclusions regarding AquAdvantage® Salmon.”
Despite this rigorous scientific review, there
remains considerable public controversy regarding

Fig. 1. AquAdvantage® and non-GM Atlantic
salmon of similar age.

the potential for FDA approval of this Aquabounty
application to produce and market a genetically

2015年 1 月30日受理（Received on January 30, 2015）＊1
UC San Diego, Scripps Institution of Oceanography, La Jolla, CA 92093‑0232
E-mail: polin@ucsd.edu

110Paul G. Olin

modified salmon.

As a result, 11 senators signed

salmon farming and world markets and then analyze

a request that FDA stop the process for approving

three scenarios based on the level of acceptance ofgenetically modified AquAdvantage® Atlantic

GM salmon in the marketplace. The three scenarios

salmon. The manner in which this review for

were named as 1) ʻno market for GM ﬁsh,ʼ 2) ʻGM

approval of a transgenic animal for agricultural

salmon for dinner,ʼ and 3) ʻGM salmon doesnʼt takeproduction and marketing in the United States hasoff.ʼ The authors provide a summary of the main

progressed has stymied American research in thisoutcomes for each scenario.

promising sector of animal biotechnology.
New technologies to genetically improve food andFDA 2012.AquAdvantage® Salmon, Draft

animal crops are one tool to supply the additional

Environmental Assessment.Center forfood people will need in the future, improving human

Veterinary Medicine, United States Food andhealth, reducing the use of pesticides and fertilizers,

Drug Administration, Department of Health andand lessening the carbon footprint of animal and

Human Services. Washington D.C.

plant agriculture. A 2008 scientiﬁc review publishedThis document provides a detailed environmental

in the Journal of the Royal Society of Medicine noted

assessment of the transgenic AquAdvantage®

that genetically modified foods had been eaten by

Salmonmillions of people worldwide for 15 years, with noreports of ill eﬀects.
Annotated Bibliography
Van Eenennaam, Alison L., and Muir William M.,
2011: Transgenic salmon: a final leap to thegrocery shelf? Nature Biotechnology, 29, 706‑
710.
This paper presents a detailed analysis of theregulatory and review process that FDA used for
the AquAdvantage® salmon.
Entis, Elliot and Greenberg, Paul. 2013: The Salmon
Dialogue: Whatʼs the future of sustainable,
genetically engineered foods? Genetic Literacy
Project.http://www.geneticliteracyproject.
org/2013/09/17/the-salmon-dialogue-whats-thefuture-of-sustainable-genetically-engineeredfoods/#.UjpTgtLBOSo
This online debate features, Elliot Entis, whose
company has created a genetically modified
salmon that may soon be for sale in the U.S., who
discusses the environmental and health impactsof this controversial technology with author PaulGreenberg, a critic of GM ﬁsh.
Menozzi, Davide, Mora, Cristina and Merigo, Alberto.
2012. Journal of Agrobiotechnology Management& Economics, Volume 15, Number 3.
In this paper the authors provide an overview of水研センター研報，第40号，111−119，平成27年
Bull. Fish. Res. Agen. No. 40，111−119，2015

111

Reproductive Dysfunction in Cultured Sableﬁsh ()

José M. GUZMÁN＊1, J. Adam LUCKENBACH＊1,2, Frederick W. GOETZ＊1,
William T. FAIRGRIEVE＊1, Mollie A. MIDDLETON＊3, and Penny SWANSON＊1,2

Abstract: Sableﬁsh

is a ground ﬁsh native to the North Paciﬁc Ocean that is

considered a promising new species for marine aquaculture in the US. However, eﬀorts to establish
sustainable production of sablefish have been constrained by the reproductive performance offemales from the ﬁrst-ﬁlial (F1) generation. Although some F1 females may mature after 5+yearsof age, some others fail to initiate puberty in captivity. Development of methods to unblock/induce
early puberty are necessary to reduce costs associated with rearing F1 female broodstock andreduce generation times for selective breeding.
Current research at the Northwest Fisheries Science Center (NOAA, Seattle, USA) integrates
basic and applied biology to gain knowledge on the reproductive endocrine system of sableﬁsh anddevelop approaches to unblock or reduce the age of puberty in F1 female sableﬁsh. As part of ourbasic line of research, we compared the pituitary gonadotropin-ovary axis in wild-caught, maturing
females and 8 year-old F1 females that had never shown signs of sexual maturation. Wild-maturing
females had higher levels of pituitary gonadotropin subunit and ovarian gonadotropin receptor
mRNAs and plasma sex steroids compared to F1 females, which were holding at the immature,perinucleolus ovarian stage. Anecdotal evidence from sablefish farms indicates that F1 female
broodstock maintained in ~4℃ seawater mature in captivity.

We hypothesize that culture

conditions that use warmer water (10‑15 ℃) suppress the pituitary gonadotropin-ovary axis insableﬁsh, and ultimately block the onset of puberty.
As part of our applied line of research, we conducted a series of studies to determine the abilityof exogenous hormones to stimulate the reproductive axis in prepubertal F1 females. Treatmentswith testosterone or estradiol 17-beta (E2) increased the expression of pituitary luteinizing hormone
beta subunit 40- and 185-fold, respectively, relative to control. This ﬁnding suggests that pituitaries
from immature females are responsive to exogenous hormones, and that sex steroids may be an
important part of a hormone therapy to stimulate the reproductive axis of F1 females. In addition,using anovarian tissue culture system, we demonstrated that fragments of prepubertal

sableﬁsh ovaries incubated with human-chorionic gonadotropin increased the secretion of E2. This
indicates that ovaries of prepubertal females are equipped to synthesize and release sex steroids
critical for vitellogenesis under the appropriate hormone stimulation, and that the failure to
initiate puberty is likely due to a lack of adequate gonadotropin signaling. These data provide thefoundation for the development of hormone treatments aimed at inducing puberty in prepubertal
F1 female sableﬁsh.
Key words: sableﬁsh, marine aquaculture, reproduction, gonadotropins

2015年 1 月30日受理（Received on January 30, 2015）＊1
Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic andAtmospheric Administration, Seattle, WA 98112, USA＊2
Center for Reproductive Biology, Washington State University, Pullman, WA 98164, USA＊3
School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA
Corresponding author: José M. GUZMÁN, E-mail: jose.guzman-jimenez@noaa.gov

José M. GUZMÁN, J. Adam LUCKENBACH, Frederick W. GOETZ, William T. FAIRGRIEVE,
Mollie A. MIDDLETON, and Penny SWANSON112

In ﬁshes, as in other vertebrates, reproduction isstaying in the US market. Due to its rapid growth

primarily controlled by the hypothalamic-pituitary-rate (1.5 kg after 12-months of growout) and highgonadal axis. The hypothalamic neuroendocrine

market value (22 USD/lb. approx. market price),

system regulates synthesis and release of thesableﬁsh has been identiﬁed as an excellent marine

pituitary gonadotropins, follicle-stimulating

aquaculture species in the US. However, efforts tohormone (Fsh) and luteinizing hormone (Lh). Bothestablish sustainable and efficient production havegonadotropins are essential in the endocrine control

been constrained by the reproductive performanceof reproduction by regulating steroidogenesis andof females from the first filial (F1) generation (i.e.,

germ cell development through interactions withproduced and maintained in captivity). Although

their respective receptors in gonadal tissues (Levavi-

some F1 females may mature at 5+ years old in

Sivan

the industry setting, others never initiate puberty., 2010; Zohar., 2010).

Optimal function of the reproductive endocrinein captivity (B. Campbell, Sablefish Canada Inc.,system is critical for fish to complete gonadal

personal communication). This situation compromises

development, maturation and spawn successfully.

the development of selective breeding programs

Under culture conditions, however, many ﬁsh species

and increases costs associated with rearing female

exhibit some degree of reproductive dysfunction.

sableﬁsh broodstock.

These can vary from inconsistent spawning to a

Our approach to this issue integrated a series

complete failure to undergo puberty (i.e., the timeof basic and applied studies aimed at gaining aduring which an individual becomes capable ofbasic understanding of the reproductive physiology

reproducing for the ﬁrst time) (Mylonas., 2010;of sablefish and developing a method that would., 2010). Such a delay in age of puberty

eliminate the block or reduce the age of puberty in

Tarangerposes a major problem for selective breeding in

F1 female sableﬁsh.

ﬁnﬁsh aquaculture and necessitates further research.
Hormone therapies are widely used to control

Materials and methods

reproduction in cultured fish species. During thepast two decades, hormone therapies have been

Study I: Assessment of the pituitary gonadotropin-

developed to induce puberty in striped bass (ovary axis in wild maturing and F1 non-maturing) (Holland(., 2002), European sea bass
) (Zanuy

eel (grey mullet (female sablefish: To understand the endocrine., 1999), European

differences that underlie the reproductive., 2004), andimpairment of female sablefish propagated) (Vidal., 2005).

in captivity, we first compared the pituitary

Therapies for inducing puberty or to accelerate) (Aizen

gonadotropin-ovary axis of two stocks of sablefish

this process were based on an understanding

with different reproductive status: wild-caught

of how the endogenous reproductive system ismaturing females and 8 year-old F1 females that had

hormonally regulated and where the insufficiency/

never shown signs of sexual maturation.

failure occurs in the reproductive system (e.g., brain,

Wild female sablefish were caught using

pituitary gland and/or gonad) that delays or blocks

sportfishing gear near the mouth of the Quinault

puberty onset. Therefore, characterization of theRiver (Washington, USA) in October 2010. The ﬁshreproductive dysfunction in a given species helps towere transported to the Manchester Research Station

tailor speciﬁc strategies to optimize its reproduction(Port Orchard, Washington, USA) and maintained inin captivity.

tanks supplied with ﬂow-through, sand-ﬁltered andSableﬁsh (), known as

UV-treated seawater. During February 2011, thein Japan, is a groundﬁsh native to the North Paciﬁcpeak spawning period along the Washington coast

Ocean ranging from Baja California to Alaskaʼs(MasonBering Sea and Japan. The primary market forof sexual maturation by ultrasound. F1 sablefish

sableﬁsh is in Japan, where demand and prices are(broodyear 2003) were reared at the Manchester

high, but an increasing amount of the wild catch is

Research Station in net-pens until fall 2010 when., 1983), the wild females showed signs

113

Reproductive Dysfunction in Cultured Sableﬁshthey were transported to the Northwest Fisheriesin ethanol: 0.9% NaCl (1:7) or vehicle alone (control),

Science Center (NWFSC, Seattle, Washington, USA)every other day. Two days after the last injection,

where they were reared on recirculated seawater.

fish were euthanized and pituitaries removed

These females were maintained under similar

to determine gonadotropin beta subunit generearing conditions as the wild-caught fish at theexpression. Doses of T and E2 were selected from

Manchester Research Station. In contrast to theprevious studies in other ﬁsh species (Guzmán

wild females, the F1 females did not exhibit signs of

2008; Holland

ovarian maturation when assessed by ultrasound.

Study III: Effect of gonadotropins on ovarian

Wild and F1 females were sampled on 28., 2002; Vidal

secretion of estradiol.,., 2004): To determine

February 2011 and 8 March 2011, respectively.

whether immature ovaries of F1 female sablefish

Three females from each broodstock group wereare responsive to gonadotropin stimulation,

deeply anesthetized with MS-222 and body weight

we evaluated the effect of two gonadotropin(BW) and fork length (FL) recorded (wild, 6413.0

preparations, recombinant coho salmon Fsh analog± 712.9 g BW and 81.3 ± 3.1 cm FL; F1, 4160.0(sFsha) and human chorionic gonadotropin (hCG), on± 303.3 g BW and 69.8 ± 2.7 cm FL). Plasma was

E2 production.

obtained by centrifugation of whole blood and stored

For this, a 3-year old prepubertal F1 femaleat − 20 ℃ for sex steroid analyses. The pituitary

sableﬁsh (2910 g BW, 67.6 cm FL) maintained at thegland and a small piece of ovary (~80 mg) were also

NWFSC was euthanized and the ovaries removed.

collected from each fish, frozen in liquid nitrogen

Ovarian fragments (~60 mg each) were cultured

and stored at −80 ℃ until RNA extraction. Genes ofin Cortlandʼs solution alone (control) or containing

interest included the pituitary gonadotropin subunitssFsha (provided by Dr. W.R. Moyle, Robert Wood(

) and ovarian gonadotropin receptors

Johnson Medical School, New Jersey, USA) at doses(). For histology, a middle portion ofof 50 and 500 ng/ml or hCG (Sigma) at doses of 1,

the ovary was preserved in Bouinʼs ﬁxative for 48hand

10 and 100 IU/ml. After 24 h incubation at 11 ℃,

prior to storage in 70% ethanol and processing as

the culture medium was collected and stored at ‑20

described elsewhere (Campbell℃ for E2 analysis. Doses of sFsha and hCG were., 2006).

Study II: Effect of sex steroids on pituitary

selected based on previous studies in other species

gonadotropin gene expression(Luckenbach: To determine., 2011; Sorbera., 2001).

potential effects of sex steroids on pituitary

RNA isolation, cDNA synthesis and quantitative

gonadotropins and whether sex steroids will be an

PCR: Total RNA from sablefish pituitaries andimportant part of hormone therapies to reduce theovarian tissue was isolated with Tri-Reagent

age of puberty in sableﬁsh, we evaluated the eﬀect(Molecular Research Center) using a TissueLyserof testosterone (T) and estradiol-17beta (E2) on

II (Qiagen). An aliquot of total RNA was diluted topituitary gonadotropin beta subunit (andmRNA levels in prepubertal F1 females.

)~250 ng RNA/µl in nuclease-free water and DNase
treated using the DNA Free kitʼs ʻʻrigorousʼʼ protocol

Twelve 2-year old prepubertal F1 female sableﬁsh(Ambion). RNA yields and quality were assessed(1187.4 ± 49.4 g BW and 469.9 ± 5.3 cm FL)

by NanoDrop (ND-1000 Spectrophotometer) and gel

maintained at the NWFSC on 12 ℃ recirculated

electrophoresis. For reverse transcription, 1 µg ofseawater were distributed into three groups (n=4

total RNA of each sample was reverse transcribedsablefish/group) and implanted with cholesterol-

in a 20-µl reaction with the Superscript II kit

based pellets (0.2 x 0.5 mm) containing no hormone(Invitrogen). Quantitative PCRs were conducted(control) or containing T at doses of 0.75 or 3.75 mg.

as previously described (Luckenbach

Twenty-eight days after implantation, fish were

Reactions consisted of 1x Power SYBR Green PCR

euthanized and their pituitaries removed and snap

master mix (Applied Biosystems), 150 nM of gene-., 2011).

frozen for later RNA isolation. In a parallel study,

speciﬁc primers (see Guzmán

6 females from the same cohort of fish received 6

sequences) and 0.5 ng of pituitary or ovarian cDNA

intramuscular injections of 2 mg E2/kg dissolved

template. Assays were run on an ABI 7700 Sequence., 2013 for primer

114

José M. GUZMÁN, J. Adam LUCKENBACH, Frederick W. GOETZ, William T. FAIRGRIEVE,
Mollie A. MIDDLETON, and Penny SWANSON

Detector using standard cycling conditions.

with normality and homogeneity of variance, which

Sex steroids: Plasma levels of E2 were measured bywere tested by Kolmogorov‒Smirnov and Bartlett

radioimmunoassay and levels of T were quantified

methods, respectively. Data are expressed as mean

by enzyme-linked immunosorbent assay using± standard error of the mean (S.E.M.).

protocols previously validated for sableﬁsh (Guzmán., 2013).

Results

Statistical analyses: Statistical analyses were
performed using Prism 5 software for Mac OSX

Study I: Assessment of the pituitary gonadotropin-(GraphPad Software) with the minimum level ofovary axis in wild maturing and F1 non-maturingsigniﬁcance set tofemale sableﬁsh<0.05. Diﬀerences were examinedusing t-test or one-way ANOVA followed by a

Endocrine parameters indicated a more active

fshb

1.0
0.00500.0000

*

fshr

1.0
0.0250*0.0000

Wild

C.

1.5
1.0
0.5

*

0.0

Wild

F1

Wild

F1

Wild

F1

1.5
1.0
0.5

*

0.0
0.5

1.5
(ng/ml)

0.5

F1

2.0

Estradiol

1.0

0.0

1.5

0.0125

1.5

F1

1.0
0.5

*

0.0

Wild

F1

0.4
(ng/ml)B.

lhcgr
relative expression

Wild

relative expression

0.0025

2.0

cga

1.5

Testosterone

lhb

A.

relative expression(Fig. 1). At the pituitary level,

relative expression

reproductive axis in wild versus F1 female sableﬁshdata were log or Ln transformed in order to comply
relative expression

Tukey multiple comparisons test. When necessary,

0.3
0.2
0.1
ND

0.0

Wild

F1

D.

Fig. 1. Relative gene expression of pituitary gonadotropin subunits (A)and ovarian gonadotropin receptors (B), plasma sex steroid levels (C), andrepresentative ovarian histological sections (D) of wild maturing and F1 female
sablefish. Gene expression levels were determined by qPCR and normalizedto. Levels of E2 were determined by RIA, whereas levels of T were
determined by ELISA. Data are expressed as the mean ± SEM ( =3). Asterisks
indicate signiﬁcant diﬀerences (t-test, <0.05). Maturing follicles with the nucleus
migrating to the periphery predominated in the ovaries of wild maturing
females, whereas only follicles at the perinucleolus stage were found in ovaries
of F1 females (D). Scale bars: 100 μm. ND, non detectable.

and

mRNA

115

fshb

A.

relative expression

Reproductive Dysfunction in Cultured Sableﬁsh

5
4
3
2
1
0
CNT

0.75

CNT

3.75

lhb

B.

relative expression

Testosterone（mg/imp）

300

*

200

b

100

a

3
2

2
Estradiol（mg/kg）

a

1
0
CNT

0.75

3.75

CNT

Testosterone（mg/imp）

2
Estradiol（mg/kg）

Fig. 2. Relative gene expression of pituitary follicle-stimulating
hormone beta subunit (A) and luteinizing hormone beta
subunit (B) in prepubertal F1 female sableﬁsh treated with T(left graphs) or E2 (right graphs). For T treatments, females
received a single implant containing hormone at doses of 0
(control), 0.75 and 3.75 mg for 28 days. For E2 treatments,
females received 6 intramuscular injections of 2 mg hormone/
kg dissolved in ethanol: 0.9% NaCl (1:7) or vehicle alone (control,
CNT), every other day. Gene expression levels were determined
by qPCR and normalized to. For T treatments, bars with
diﬀerent superscript letters are signiﬁcantly diﬀerent (ANOVA,<0.05). For the E2 treatment, an asterisk indicates a signiﬁcantdiﬀerence relative to control (t-test, <0.05).

levels were signiﬁcantly higher in wild ﬁsh than in

the perinucleolus stage. In contrast, ovaries from F1F1 fish. Levels of

females were characterized by the sole presence ofwere also elevated in wildﬁsh relative to F1 ﬁsh (7.1‑fold), although diﬀerencesfollicles at the perinucleolus stage.

were not statistically significant due to the highvariance across individuals. In the ovary, transcripts

Study II: Effect of sex steroids on pituitary

for both gonadotropin receptors,

gonadotropin gene expression

and,were signiﬁcantly higher in wild than in F1 females.

The eﬀect of treatment with T or E2 on

Plasma levels of E2 were also signiﬁcantly higher inpituitary gonadotropin beta subunit (wild than in F1 females, whereas only wild females

mRNA levels is shown in Fig. 2. Transcripts forhad detectable levels of T in plasma.

were not significantly affected by treatment with

The ovary of wild females consisted of post-

T or E2. In contrast, transcripts forand)increasedvitellogenic preovulatory follicles characterized bysignificantly in fish treated with the highest dose

the nucleus migrating to the periphery and yolkof T or multiple injections of E2 (40- and 185-fold,coalescence, as well as another clutch of follicles atrespectively, relative to control).

José M. GUZMÁN, J. Adam LUCKENBACH, Frederick W. GOETZ, William T. FAIRGRIEVE,
Mollie A. MIDDLETON, and Penny SWANSON

116

Interestingly, ovarian（ng/ml）

Estradiol

0.25
0.20

b*

b*

b*

was observed for the pituitary

0.15mRNA levels.

Little is known about specific roles of Fsh during

0.10
0.05

mRNA levels were also

higher in maturing females and a similar tendencyaagametogenesis in ﬁshes, although it is accepted thata*

Fsh is important for early gonadal development andvitellogenesis (Lubzens

0.00
CNT

50

500

1

10

100

sFsha（ng/ml） hCG（IU/ml）
Fig. 3. Eﬀect of sFsha or hCG on E2 production by
ovarian fragments obtained from prepubertal F1
female sablefish. Ovarian fragments were cultured
in Cortlandʼs solution alone (control, CNT) or
containing sFsha at doses of 50 and 500 ng/ml or
hCG at doses of 1, 10 and 100 IU/ml, at 11 ℃ for
24h. Levels of E2 were determined by RIA. Bars
with different superscript letters are significantly
different (ANOVA, <0.05), whereas asterisks
indicate significant differences relative to control(t-test, <0.05).

., 2010; Luckenbach., 2011). The elevated levels of pituitaryovarianandthat we observed in wild females may

indicate that a population of perinucleolar oocytes
within the ovary were preparing for the transition tovitellogenic growth, or that Fsh also plays a role inﬁnal oocyte maturation. Since the ovary of the wild
mature females had a mixed population of oocytes it
is not possible to distinguish these two possibilities.
Plasma levels of E2 and T were also higher inwild females than F1 females as expected fromthe observed differences in pituitary gonadotropin
subunit and ovarian receptor mRNA levels. This
demonstrates classical gonadotropin-mediated sex
steroid secretion (Lubzens., 2010) by the ovaries

Study III: Effect of gonadotropins on ovarianof wild females and indicates a lack of gonadotropin

secretion of estradiol

signaling in F1 females.

The eﬀect of sFsha or hCG on ovarian secretion of
E2

Although F1 females from Study I were 8 years

is shown in Fig. 3. Incubation with sFsha

old, they remained arrested in a prepubertal stage.

had a subtle eﬀect on the secretion of E2 with onlyInterestingly, it has been recently demonstrated

the highest dose of sFsha having a signiﬁcant eﬀect

that some F1 females can initiate vitellogenesis (i.e.,compared to control. In contrast, treatment withonset of puberty) if they are maintained in coldhCG regardless of dose significantly stimulated E2

water (~4 ℃) after 5 years of age (B. Campbell,secretion (>4‑fold).

personal communication). It is generally acceptedthat every fish species has an optimal range ofDiscussion

water temperatures for vitellogenin synthesis, andboth higher and lower temperatures affect theCurrently, the emerging aquaculture industry fornormal progression of vitellogenesis (Guzmán

sablefish is mostly dependent on spawning wild-., 2008; Kim and Takemura, 2003). Consideringcaught individuals. The expansion and optimizationthat sableﬁsh is a deep-water species and the onsetof this industry requires a closer understandingof vitellogenesis may naturally occur at very lowof the reproductive physiology of this species andtemperatures, it is possible that the temperature at

the development of protocols to control the age of

which fish were maintained (fluctuated between 8

puberty in cultured broodstock.

and 15 ℃ throughout the year) had a deleterious

In Study I, we demonstrated that transcriptlevels of pituitaryand ovarianwereeﬀect on reproductive axis function, and ultimately
on the onset of vitellogenesis in F1 females.

higher in wild-caught maturing females than in non-

Although some F1 females maintained in cold

maturing F1 females. This is in accordance with thewater can achieve puberty after 5+ years, thepredominant role of Lh in the regulation of gonadal

development of methods to reduce and synchronize

maturation, ovulation and spawning in ﬁshes (Gothilf

the age of puberty is essential for reliable and cost., 1997; Swanson., 2003; Yaron., 2003).

effective sablefish breeding programs. Hormone

117

Reproductive Dysfunction in Cultured Sableﬁshtreatments are widely used in aquaculture to(Ohta and Tanaka, 1997), while multiple injections

improve the reproductive performance of captiveof hCG can induce testicular development andfishes, including activation of the reproductive

spermiation in males (Dou., 2007). Our data

endocrine system and promotion of gonadal

suggest that, as in anguillid eels, immature ovaries

development in immature stages (Holland., 2002;of F1 female sableﬁsh are equipped with the ability., 1999). We conducted Studies II and III

to synthesize and release sex steroids (criticalto determine the ability of exogenous hormones tofor vitellogenesis) under appropriate hormone

stimulate the reproductive axis in prepubertal F1

stimulation, and that treatment with gonadotropin

sableﬁsh.

preparations may effectively stimulate ovarian

Zanuy

In Study II we demonstrated that treatment
with T or E2 significantly elevated transcripts
for pituitary, but did not affect levels offunction in this species.
In conclusion, wild-maturing females had elevatedgonadotropin signaling (pituitary and ovarian gene

mRNA. Effects of sex steroids on levels of theexpression and plasma steroid levels) compared topituitary gonadotropin subunit mRNAs have been

8 year-old F1 females, which were holding at thedemonstrated in a number of ﬁshes, although results

immature, perinucleolus ovarian stage. Anecdotalvary among species. For example, treatment withevidence indicates that F1 females maintained in ~4

T elevated pituitary transcripts of℃ seawater may mature in captivity after 5 years ofin European

sea bass during the period of “sexual resting,” whileage. It might be possible that culture conditions that

T and E2 reduced pituitary

mRNA levels

use warmer water (10‑15 ℃) suppress the pituitary., 2002). In immature European eel

gonadotropin-ovary axis in sableﬁsh, and ultimately), treatments with E2 but not T

block the onset of puberty. Our results indicate(Mateos(elevated transcripts forobserved on, whereas no eﬀect was

mRNA levels (Aroua

that hormone treatments, such as sex steroids., 2007).

and gonadotropin preparations, can stimulate theLastly, treatments with T and E2 increased pituitary

reproductive endocrine axis in prepubertal females

transcript levels forand therefore should be considered as candidates toandin zebraﬁsh () (Lin and Ge, 2009). Our results suggest that

reduce the age of puberty in this species.

pituitaries from prepubertal female sablefish areresponsive to exogenous hormones, and that sex

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水研センター研報，第40号，121−128，平成27年
Bull. Fish. Res. Agen. No. 40，121−128，2015

121

Induced Spawning in the Sea Cucumber
Neuropeptide, Cubifrin

byKeisuke YAMANO＊1, Atushi FUJIWARA＊2, Michiyasu YOSHIKUNI＊3

Abstract: In hatcheries the induction of spawning in sea cucumbers has been typically carried out
by regulation of rearing conditions such as temperature and light intensity. However, this method
is relatively ineﬀective and the rate of spawning is unpredictable. In this study we established aneﬃcient method for inducing spawning in the Japanese sea cucumber

byinjecting a neuropeptide, cubifrin.
We purified peptides that can induce oocyte maturation from the buccal tissues containing
the nerve ring, using liquid chromatography andassay with ovarian fragments incombination. The effective dose of each peptide was evaluated with chemically synthesizedpeptides. Consequently, the most potent peptide was identiﬁed as NGIWY-amide. We also foundthat synthetic derivatives replaced the third amino acid, isoleucine, with a diﬀerent basic aminoacid could be 10‑100 times more potent than the natural hormone.
When injected into the body cavity of sexually matured individuals, NGIWY-amide, or its
derivative, induced spawning in both males and females. NGIWY-amide was then named cubifrin
after the Japanese word “kubifuri” meaning waving head, which is a reproductive behavior of sea
cucumbers. Gamete shedding started about 60 min and 80 min after the injection in males andfemales, respectively, and was completed almost simultaneously in the two sexes about 2 hours
after the administration. The

responsiveness of biopsied ovarian fragments was wellcorrelated with the spawning success induced by an injection. Therefore, cubifrin can be used as
an excellent detector of maturity as well as an inducer of spawning inin a hatcherysetting.
Key words: reproduction, hormone, aquaculture, echinoderm

The Japanese common sea cucumber

In hatcheries, the induction of spawning in sea

is the most commercially-important

cucumbers is typically carried out by regulation

holothurian species in Japan. Until recent years theof rearing conditions such as temperature, water

annual catch had been about 5,000‑10,000 tons (wetexchange, and light intensity (Battaglene., 2002;weight) but fishing pressure has been increasingSui, 2004; Wang and Cheng, 2004; Liu., 2004;

mainly because of a growing demand for export to

Shao

China. Therefore, the sustainable production of thebroodstock are induced to spawn in the dark in., 2006). In Japan, wild-caught

sea cucumber is crucial. For over 30 years, cultured

tanks of seawater at temperatures that are ~ 5℃

juveniles of this species have been released intohigher than natural seawater (Ito, 1995). However,fishing grounds to supplement natural stocks. The

these methods are relatively ineffective and thecurrent situation concerning sea cucumber ﬁsheriesrate of spawning is unpredictable. Therefore, more

makes this activity more important than ever.

effective method of spawning induction has been

2015年 1 月30日受理（Received on January 30, 2015）＊1
National Research Institute of Aquaculture, Fisheries Research Agency, Minamiise, Watarai, Mie 516‑0193, Japan
E-mail: yamano@fra.aﬀrc.go.jp＊2
National Research Institute of Fisheries Science, Fisheries Research Agency, Fukuura, Kanazawa, Yokohama, Kanagawa 236‑8648, Japan＊3
Fishery Research Laboratory, Kyushu University, Fukutsu, Fukuoka 811‑3304, Japan

122

Keisuke YAMANO, Atushi FUJIWARA, Michiyasu YOSHIKUNI

desired.

liquid chromatography (RP-HPLC) using aStarﬁshes have long been used as model animals

Develosil RPAQUEOUS-AR-5 column (10 x 250

for the study of reproductive biology after themm) with a linear gradient of acetonitrile fromfinding of spawning-inducing activity in a nerve

10 to 40 % with 0.1 % TFA. Each fraction was

extract in the middle of the 19th century (Chaetlyophilized, then dissolved in 0.5 ml MilliQ waterand McConnaughy, 1959). At long last, the nervous

to detect the gonadotropic activity by thesubstance was purified and identified as a relaxin-

maturation assay. The fractions containing thelike protein (Mita., 2009). Since both starﬁshes

gonadotropic activity were separated by a Develosil

and sea cucumbers belong to the Echinodermata,

RPAQUEOUS-AR-5 column (10 x 250 mm) with

their reproductive mechanism might be driven bya linear gradient of acetonitrile from 10 to 30 %similar endogenous factors. Thus, we started thecontaining 20 mM ammonium acetate (pH 6.0). The

study to discover a spawning-inducing substance infractions were lyophilized and assayed as mentioned, following the strategy employed in theabove. The active fractions were separated by astudy in starﬁsh.
Here, we report a native neuropeptide potent
in inducing oocyte maturation and spawning in, and propose a procedure of using the
peptide in a hatchery setting.

Develosil RPAQUEOUS-AR3 column (2 x 250 mm)
with a linear gradient of acetonitrile from 15 to
22 % with 0.1 % TFA. The gonadotropic activity
was detected in fractions 30 and 33 of the lastchromatography step.
Determination of chemical structure ofMaterials and Methods

gonadotrophic substances: The fractions 30 and 33
were analyzed for accurate mass values and amino

Preparation of neural extract for purification ofacid sequences by liquid chromatograph-tandema spawning-inducing substance: Buccal tissues

mass spectrometers (Quattro Premier, Waters;

containing the ring nerve were homogenized in

nanoFrontier LD, Hitachi High-Technologies) and athe same volume of ice-chilled 4 N acetic acidprotein sequencer (491 cLC, Applied Biosystems).

containing 0.4 mM 㸥-aminoethyl benzensulfonyl

Preparation of peptides: NGIWY-amide (cubifrin)

fluoride, 10 µg/ml leupeptin and 4 µM pepstatin

and its derivatives were chemically synthesized. A

A. The homogenates were centrifuged at 15,200

stock solution of 1 mM dissolved in MilliQ waterx g for 15 min. at 4℃. The resulting supernatants

was stored at ‑80℃. Prior to use, it was diluted with

were then ultracentrifuged at 45,000 x g for 1 hr

ﬁltered seawater to the desired concentrations.

at 4℃. These supernatants of ultracentrifugation

maturation assay: A portion of ovary waswere kept as a crude neural extract in aliquots at

excised through a short incision in the body wall.

‑80℃. The crude extract was separated by serial

The excised ovarian tissue was cut into small

ultrafiltration with AmiconUltra YM30 and YM10

fragments about 3 mm long. For the puriﬁcation of(Millipore). The gonadotropic activity determined bya maturation-inducing substance, ovarian fragmentsthematuration assay was mainly recovered

were incubated with each fraction for 1.5 hr atin the fraction of less than 10K daltons. After YM10

20℃. The gonadotropic activity was determined byﬁltration, the ﬁltrate was lyophilized and preserved

germinal vesicle breakdown (GVBD) and also by

for a subsequent puriﬁcation process.

the incidence of ovulation of oocytes from ovarian

Liquid chromatography: The lyophilized preparation

fragments.

was dissolved in MilliQ water. After centrifugation,

For the detection of the responsiveness tothe supernatant was applied to a Develosil C8-

peptides, ovarian fragments were incubated either

UG-5 column (20 x 50 mm) and eluted with 80

with the peptide solution at 100, 10, 1 nM, or 100% acetonitrile containing 0.1 % trifluoro acetic

pM in filtered seawater or with filtered seawateracid (TFA) for desalting. The desalted effluent

alone. The test was duplicated for each individual.

was lyophilized. The lyophilized extract was

The incidence of GVBD were scored on a scale offractionated by reversed-phase high performance0 3 in which 3 indicates GVBD in over two-thirds

123

Spawning of Sea Cucumber by Neuropeptideof oocytes in the fragment; 2, in over one-third ofoocytes; 1, in up to one-third of oocytes; 0.5, in a few
percentage of oocytes; 0, no response of oocytes.
The sum of scores of all treated ovarian fragmentsfrom one individual (8 fragments for each individual)
was regarded as the oocyte maturation score of that
individual. After the assay, oocytes in the control
ovarian fragments were mechanically separated
by gentle pipetting and the diameters of 20 welldeveloped oocytes were measured.
Injection of cubifrin and observation of reproductive
behavior: Cubifrin solution (10 µM) or filteredseawater was injected into the body cavity ofthesea cucumbers; the injection volume was 0.1%
of body weight (v/w). Each sea cucumber was thenseparately placed on the bottom of a 21-l tank, andits behavior was continuously monitored until thecompletion of spawning, or for 2 hr if no spawning
occurred. We recorded the times at which theunderside of the water surface was reached, whenhead waving commenced, and when spawning wasstarted and completed. After the completion of
spawning, the number of spawned eggs in 10 mlof tank water was counted to estimate the total
number of spawned eggs for each female.
Results and Discussion
Purification of an oocyte maturation-inducing
substance: The neural extract was separated
by three serial HPLCs (Fig. 1). On the third
chromatography, the gonadotropic activity was
separated into two independent fractions, Fractions
30 and 33 (arrowheads in Fig. 1c). Both fractions
were analyzed by mass spectrometry and protein
sequencing. The amino acid sequence of Fraction
30 was NGIWY, and Fraction 33ʼs sequence was
QGLFSGV. While the calculated monoisotopic mass
values of these sequences were 651.320 and 706.365,

Fig. 1. Separation of the gonadotropic activity
by RP-HPLC. Dotted lines in the chromatograms
indicate changes of programmed concentrationsof acetonitrile in the eluents. Hormonal activity
in each fraction is shown with the help of bars
hanging from the top side of the chromatograms (a,b). Arrowheads in (c) indicate fractions 30 and 33
containing the bioactivity. Reproduced from Kato
(2009) with permission.

analyses by mass spectrometry indicated masssignals of 650.35 and 705.42, respectively. Further

30 and 33 were NGIWY-amide and QGLFSGV-

sequencing analyses of these mass signals

amide, respectively. Unexpectedly, both of thesesuggested that these had the same sequences as

peptides completely differ from a relaxin, which isthose given through protein sequencing except

an oocyte maturation-inducing substance in starﬁsh.

for anamidation at each C-terminal end of the

NGIWY-amide has been reported as a peptide

peptides. With these analyses we determined thatassociated with contractions of muscle, intestine

the sequences of the components in Fractions

and tentacles in(Inoue., 1999),

124

Keisuke YAMANO, Atushi FUJIWARA, Michiyasu YOSHIKUNI

whereasQGLFSGV-amide has not been reported

peptide is indispensable for the bioactivity.

ever.

Effect of NGIWY-amide and NGLWY-amide inmaturation-inducing activity of peptides: Two

inducing spawning: NGIWY-amide and NGLWY-

peptides, NGIWY-amide and QGLFSGV-amide, wereamide were injected into sexually matured males

chemically synthesized and GVBD-inducing activity

and females to examine the effect on inducing

was estimated with

spawning behavior. Injections of NGIWY-amide

maturation assay.

NGIWY-amide was extremely potent even at 1 nM

induced spawning in males and females at 10 nM.

or less (Fig. 2). QGLFSGV-amide was less potent in

NGLWY-amide more effectively induced spawninginducing GVBD, with eﬀective concentrations being

in males at 1 nM and in females at 100 pM.NGLWY-

1 µM or more. Thus, NGIWY-amide is strongly

amide was therefore at least ten times more potent

suspected to be involved in the regulation of oocyte

than NGIWY-amide, as in the case of

maturation. In contrast, QGLFSGV-amide does notexperiments. Sea cucumbers injected with NGIWY-

appear to be the primary endocrine regulator ofamide or NGLWY-amide exhibited reproductive

oocyte maturation.

behaviors that were independent of sex and

The activities of the two derivatives, NGLWY-

typically included: (1) climbing up the side wall

amide and NGIWY-COOH, were compared with thatof the tank to the underside of the water surface;of the natural NGIWY-amide (Fig. 3). NGLWY-amide(2) throwing back and swaying of the head; (3)was eﬀective at 10 pM or less, a hundred times more

shedding gametes from the gonopore in the headpotent than the natural peptide, NGIWY-amide.

region. Gamete release occurred 45‑60 min after

However, NGIWY-COOH barely induced GVBD at
100 nM. Accordingly, C-terminal amidation of theFig. 2. Concentration dependence of NGIWY-amide
and QGLFSGV-amide to induce GVBD in ovarianfragments. Ovarian fragments were incubated withNGIWY-amide (white bars) and QGLFSGV-amide(gray bars) at various concentrations. Bars represent
percentages of GVBD (means ± SD of duplicate
determinations of four separate experiments).
Bars with different labels differ significantly (＊< 0.01 ＊＊). The grouping symbol at< 0.05 ,the top side of the graph indicates a significant
difference between NGIWY-amide and QGLFSGVamide at each concentration. Signiﬁcant diﬀerenceswere determined using Wilcoxonʼs rank sum test.
Reproduced from Kato
. (2009) with permission.

Fig. 3. Concentration dependence of NGIWY-amide
and its derivatives to induce GVBD in ovarianfragments. Ovarian fragments were incubatedwith synthetic analogues at various concentrations.
NGIWY-amide (white bars), NGLWY-amide(gray bars) and NGIWY-COOH (black bars) were
examined. Bars represent percentages of GVBD
(means ± SD of triplicate determinations of six
different experiments), and bars with different＊
labels diﬀer signiﬁcantly in each peptide ( < 0.05 ,< 0.01＊＊). The grouping symbol at top side of
the graph indicates a significant difference among
two peptides at each concentration. Significant
diﬀerences between means were determined using
Steel-Dwassʼs pair-wise comparisons. Reproduced
from Kato. (2009) with permission.

125

Spawning of Sea Cucumber by Neuropeptide
the peptide injections in males and 70‑90 min inwith oocytes <155 µm failed to respond to thefemales, and was completed almost simultaneously

treatment. The injection of cubifrin-L into the bodyin the two sexes about 2 hr after the administration

cavity induced spawning in all females with ovaries(Fig. 4). This series of actions from climbing tothat responded to cubifrin-L

spawning observed in the experiment seemed to bewith unresponsive ovaries

a typical behavior of spawning by sea cucumbers

following the injection, with the exception of two. Females
did not spawn(McEuen, 1988; Battaglene, 2002). NGIWY-amide andfemales (nos. 12 and 13). The number of spawned

NGLWY-amide were named cubifrin and cubifrin-L,

eggs per 100 g body weight varied from 765 torespectively, after the Japanese word “kubifuri”

1,826,000, and there was a significant correlation

meaning waving head, which is a reproductive

between this value and the oocyte maturation scorebehavior of sea cucumbers.

( = 0.84). The number of eggs spawned by the two

Association between oocyte size andnon-responsive females in

responsiveness to cubifrin: The competence ofespecially low (765 eggs per 100 g body weight inovarian fragments to undergo

female no. 12 and 23,700 in female no. 13).

oocytematuration and the induction of spawning by

experiment wasThese results indicate that potential spawners incubifrin-L stimulation were examined in relationbroodstock can be selected by

to oocyte size. All ovarian fragments with oocytesof the cubifrin or cubifrin-L responsiveness of>155µm in diameter (animal nos. 17 25 in Fig. 5)

ovarian fragments or by measuring oocyte size.

responded to cubifrin-L

Furthermore, the number of spawned eggs can be, as indicated byexamination

their oocyte maturation score, which was a measureroughly estimated byof their ability for ovulation and GVBD. In contrast,

Procedure of spawning induction in a hatchery: In

all fragments but one (animal no. 11 in Fig. 5)

conclusion, spawning induction by cubifrin is an

examination.

Fig. 4. Progress of spawning behavior in females (A) and males (B). Open and closed circles
indicate the data point of each NGLWY-amide-injected male and female ( =14 each) and control sea
cucumber ( =4 each). Vertical bars show the averageofthe NGLWY-amide-injected group.

126

Keisuke YAMANO, Atushi FUJIWARA, Michiyasu YOSHIKUNI

Fig. 5. Association between oocyte size and responsiveness to cubifrin.
Responsiveness to NGLWY-amide was determined by spawning success, as
indicated by the number of spawned eggs per 100 g body weight (a) and by
maturation assay, evaluated from the oocyte maturation score (b).
See the Materials and methods for a description of the oocyte maturation
score. Oocyte sizes (mean diameter ±SD, = 20) of each female are shownin (c).
eﬀective and simple method for practical application
during the artificial culture of

Acknowledgements. We

propose the procedure of spawning induction as

This work was supported by grants from thefollows:

Program for Promotion of Basic Research Activities(1)preparation of sea cucumbers during the spawningfor Innovative Biosciences (PROBRAIN).

season; (2)sex determination and gross evaluation of
maturity from the appearance of biopsied gonad; (3)References

evaluation of deﬁnite maturity with biopsied ovarybytest; (4) Rearing mature individuals below

Battaglene S., Seymour J., Romafaﬁa C., and Lane I.,

15℃ to suppress unanticipated natural spawning

2002: Spawning induction of three tropical sea

until spawning induction; (5)spawning induction

cucumbers, Holothuria scabra,

andby the injection of 10µM cubifrin solution (0.1% of.

body weight (v/w)); (6) standard fertilization and

Chaet A. B., and McConnaughy R. A., 1959:

cultivation of fertilized eggs.

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Y., Muneoka Y., and Motokawa T., 1999:

127

Spawning of Sea Cucumber by Neuropeptide
Localization of the neuropeptide NGIWY-amide

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McEuen F., 1988: Spawning behaviors of northeast

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128

Keisuke YAMANO, Atushi FUJIWARA, Michiyasu YOSHIKUNIof the peptide for induction of spawning in thehatchery. Ovarian fragments with oocytes larger
than 155 µm in diameter responded to cubifrin-L. The

responsiveness of ovarianfragments was well correlated with the spawningsuccess induced

by a cubifrin-L injection.

Mature sea cucumbers injected with cubifrin-L
displayed sequential reproductive behaviors, which
comprised climbing the side wall of the tank toward
the water surface, waving of the head, and sheddingof gametes. Gamete shedding started about 60
min and 80 min after the injection in males andfemales, respectively, and was completed almost
simultaneously in the two sexes about 2 hours after
the administration. Repeated injections of cubifrin-L
at intervals of about 10 days successfully induced
multiple spawnings in males and females. This study
demonstrated that cubifrin-L is an eﬀective inducerof spawning in Japanese sea cucumber cultivation.

水研センター研報，第40号，129−134，平成27年
Bull. Fish. Res. Agen. No. 40，129−134，2015

129

Mass Production of Artiﬁcial Seed of the Japanese Common Sea
Cucumber () in Hokkaido, Japan
Yuichi SAKAI＊1

Abstract: Seed production and release ofis a key approach to increase andmaintain their natural stocks in Japan. Nevertheless, in many trials conducted mainly in Honshu
and Kyushu by the end of the 1990ʼs, the amount of seed production ﬂuctuated annually, and thesurvival rate of released artiﬁcial juveniles was uncertain because of the diﬃculty of distinguishingthem from wild ones. Due to these issues, some hatcheries discontinued

enhancement

projects. However, due to increase in Chinese demand, the price ofdramaticallyincreased after 2003 in Hokkaido. Facing the decline in the price of sea urchin and abalone, which
are major high-value catches in the coastal areas, ﬁshermen were very interested instock enhancement by releasing artiﬁcial seeds. Accordingly, sea urchin and abalone hatcheries in
Hokkaido began to produce

seed after 2006. This paper will introduce the recent mass

production techniques developed in Hokkaido.
Key word:, artificial seed production, larval rearing, post-larval rearing,

control of predation

Japanese common sea cucumber
is found in shallow coastal bottoms ofaround 2‑3 million year−1 (Fig. 1).
Mass mortality during post-larval rearing

Japan, Kuril Islands, Sakhalin, northeastern China

resulted in fluctuations in the number and size

and Korean peninsula. Among these countries,

of seed produced. Furthermore, the survival rate

Japanese and Korean people prefer to taste thisof released artificial seeds had not been properly

mainly in raw or picked as a seafood, but Chinese
people prefer dried sea cucumber as a tonic rather
than a seafood. Due to the economic development ofChina, demand for this species has increased. Unit
price in Hokkaido peaked at 43.2 USD/kg in 2010.
Facing the decline in the price of sea urchin andabalone, fishermen were very interested in stockenhancement by releasing artiﬁcial seeds.
After Imai. (1950) made the first trial of

artificial seed production in Japan, many trials
were conducted in Honshu and Kyushu. Small
juveniles (<2mm) were mainly produced by 1990,−1
and the productions peaked at 26.6 million year .

Thereafter, due to increase of production of larger
juveniles, the total number of juveniles decreased toFig. 1. Number of seed produced in Japan. Drawnfrom the statistical data published by Fisheries
Agency, Fisheries Research Agency and National
Association for Promotion of Productive Seas.

2015年 1 月30日受理（Received on January 30, 2015）＊1
Fisheries Research Department Mariculture Fisheries Research Institute, Hokkaido Research Organization, 1‑156‑3, Funami-cho, Muroran,
Hokkaido, 051‑0013, Japan
E-mail: sakai-yuichi@hro.or.jp

130

Yuichi SAKAI

evaluated, because of difficulty in distinguishing

their gametes into the container ca. 0.5‑1.5 hours

cultured from wild individuals. Therefore, someafter the stimulation. Alternatively, broodstock

hatcheries in Honshu and Kyushu discontinued thisinjected with gonadotrophic hormones (CUBIFRIN)

stock enhancement project. However the demand

will also begin to release their gametes.

for artiﬁcal seed production by ﬁshermen, began to

For insemination, 10‑20 ml of sperm is added toincrease after 2006 in Hokkaido and the production

a liter of seawater containing 0.1 ‑ 0.2 million eggs.

of artificial seed increased again and reached to

To prevent polyspermy, sperm concentration should

12.3 million in 2011. From this experience andnot exceed 1,000,000 sperm ml−1(Sakai and Konda,

background, the improvement of artificial seed2006). After insemination, eggs are transferred onto

production is necessary for stock enhancement to bea 45μm mesh sieve immersed in a shallow tray andsuccessful.

gently rinsed with filtered seawater to wash theSeed production ofis divided into 5

excess sperm through the sieve.

processes: broodstock collection, fertilization, larval
Larval rearing

rearing, settlement and post-larval rearing. I review
the recent mass production techniques mainly
developed in Hokkaido for each process.

Hatched blastulae swim to the surface and aretransferred to larval rearing tanks at a density ofBrood stock collection

1‑2 individuals ml − 1. About 2 days after hatch, thelarvae grow to the auricularia larvae and begin to

Genetic impacts due to release of artificiallyeat, unicellular phytoplankton.

produced organisms are considered in broodstock

Daily feeding rate is near to 10 000 cells individual−1.

collection. Polymorphic microsatellite allele frequency

The fully developed auricularia larvae grow to ca.

of artiﬁcial seed tends to decrease to compare with

1 mm body length in 5 to 10 days. After this, theywild ones due to small number of their parents (Sakai

begin to shrink to 0.4‑0.5mm doliolaria larvae inand Kanno, 2013). So to keep enough broodstock

2 days. Then about 1 day after, doriolaria larvae

from wild stocks is necessary especially in the casegrow to pentactula larvae which are ready toof survival rate of released seed is high enoughmetamorphose to young juveniles and begin to settle

to contribute its reproduction on the released

onto the tank bottom or collectors.

bottoms. Furthermore, to maintain genetic diversity,
broodstock should not be used repeatedly.

The growth rate of larvae is regionally different
in Hokkaido, fast in southwestern Sea of Japan areas

Maturation of this species begins after they growand slow in Pacific areas. It takes only 9 days in

to ca. 50g in body weight, so larger broodstock

southwestern Hokkaido and 14 days after hatch inare necessary. They are normally collected in thethe Paciﬁc area.

spawning season, late June to August in Hokkaido,
to avoid excess labor and cost to rear them.

Larval rearing tanks are static through the rearing
period to minimize the loss of food and satisfy theirfood requirement. However, to avoid precipitation oflarvae, food and feces on the bottom, aeration is very

Fertilization

important; the optimum rate is 1.5‑2.0 L min−1. The
Gametes are obtained from fully matured

water temperature should be kept at 18‑20℃ .

broodstock by two methods: thermal stimulation
or injection of gonadotrophic hormone (CUBIFRIN)
through the body wall (Kato

Settlement., 2009; Fujiwara., 2010). To induce to release gametes by

Doriolaria and pentacutula larvae are transferred

thermal stimulation, broodstock are placed in 15 L

to post-larval rearing tanks. They settle on

volume containers individually and the seawater

corrugated polyvinyl chloride (PVC) plates or balled

temperature is raised by 5‑7℃ above ambient

1mm mesh polyethylene screens of which surface

temperature. Mature broodstock begin to releaseare covered with natural attached diatoms or algal

131

Mass Seed Production of Sea Cucumber in Hokkaido
powder which are used as the juvenile diet. To avoid

fed LIVIC-BW grow to 6.0 mm in about 3 months

the loss of these larvae, water exchange is initiated(Sakai10 days after the larvae are stocked, by which timedensity (Hatanaka, 1996; Sakai

most larvae complete settlement.

densities (Ito and Kawahara, 1993; Sakai., 2009). Growth rate affects rearing., 2009) and food., 2009).

During the Great East Japan Earthquake, thePost-larval rearing

factory of LIVIC-BW was destroyed. Argin-Gold(Andes-Trading Co., Ltd.) made from dried powder

Settled juveniles are reared up to 5 to 10 mmofbegan to be used. Thisbody length on corrugated PVC plates and/or

diet compensates the same growth of juveniles atpolyethylene screens without temperature control.

least 3 month (Sakai, unpublished data). Further

Daily water exchange rate varies from 1‑7 times

experiments are necessary to ﬁnd the proper diet to

−1

day

with aeration at the center of the tank to stir

the rearing water well.

In this stage, the predation of settled juveniles

Various diets during this post-larval rearing havebeen examined (Yanagibashi

increase the growth rate of artiﬁcial seeds.
by, which is commonly found., 1984; Yanagibashi

in the coastal area of western north-Pacific Ocean,and Kawasaki, 1985; Ueki and Ikeda, 1989; Konda

was a major problem to produce the artiﬁcial seeds

and Sakai; 2005). The naturally occurring attachedin Japan (Kobayashi and Ishida, 1984). This species

diatoms and LIVIC-BW, a commercially sold dietgrow to only ca. 1 mm in its body length as adults

made from a mixture of dried algal powder ofand produce more than 100 eggs at each spawn

and

are

(Fig. 2). Even just hatched out nauplius larvae, 50

used as inducers for larval settlement and as foodμ m in its body length, begin to eat

for post-larval stages. Phytoplankton

juveniles.

isalso a good initial food for juveniles for the first

The predatory impacts byaremonth (Sakai and Konda, 2008). Proper feeding ofserious during post-larval rearing. The predation

LIVIC-BW has been examined according to juvenile

begins after the density of this animals increase tosize (Ikeda

ca. 10‑20 individuals 10ml., 1992; Sakai., 2009). Juveniles−1(Sakai and Konda, 2008;

Fig. 2. Development of. a: eggs in the sac, b: higher
magnification of fertilized nauplius larva, c: copepodid stage larva, d: adultfemale holding egg sac, e: pradation ofjuvenile. bar：250μｍ.

132

Yuichi SAKAI

Kobayashi and Yamaguchi, 2011; Noguchi and Noda,

from the plates, thereby decreasing predation (Sakai2011). To prevent predation of artiﬁcial juveniles, it

and Konda, 2008). Daily observation to conﬁrm theis necessary to decrease the density ofincrease of.

and to determine the timing

Trichlorphon, organo-phosphorous compound at aof this treatment is necessary. Furthermore, a newconcentration of 1‑2 ppm has been used to control

tank free of copepods to set the collectors in is also

this animal (Kobayashi and Ishida, 1984). However,necessary.

after 2006, this medicine was prohibited by the

Pumping up to filter copepods: To reduce labor,

Pharmaceutical Aﬀairs Law in Japan.

I set an underwater pump on the bottom of theAfter this, I developed 3 methods to prevent therearing tank to pump the copepod rich rearing

predation: to immerse the plates into salt enrichedwater. The pumped water discharged into a 45 μm

seawater (ca.50‰), pumping the rearing watermesh net set at the surface of the rearing tank.

containing this predatory animal and filtered out

were ﬁltered out by this method and thewith a 45μm mesh net to decrease copepod density

density of copepods in the rearing tank decreasedin the water, and using a balled 1 mm mesh as theautomatically.

settlement material which allows the juvenile sea

Balled screen collector: The balled polyethylene

cucumbers to escape from the copepods (Fig. 3).

screen is also available as the collector and rearing

Immersing salt enriched water: Hypersaline seawater

device to prevent predation. Sea cucumber juveniles

induces paralysis of the copepods and removes themcan escape into the center of the balled screen if the

a

b

P
c

N

P

Fig. 3. Three main methods to control copepod density in the post-larval rearing tanks. a: Immersing the plates
into salt enriched sea water, b: Pumping up and ﬁlter out the copepods, c: Balled screen collector. N: 45μm meshnet, P: under water pump.

133

Mass Seed Production of Sea Cucumber in Hokkaido
density of copepods increases and allows them toescape from predation.

(in Japanese).
Imai T., Inaba D., Sato R., and Hatanaka S.,
1950: Mushoku Benmouchu niyoru Namako

Release of artiﬁcial seeds() no Jinkoushiiku (Artificial

seed production of sea cucumber, Stichopus
Juveniles are released onto the reefs by divers.

japonicus, using non-pigmented ﬂagellates).

Mitsunaga and Matsumura (2004) reported thatlarge juveniles (26mm) survive better than smaller., 2(2), 269‑
277 (in Japanese).

ones (14mm). However, the survival rate one year

Ito S., and Kawahara I., 1993: Manamako noafter releasing was only 3.8% and 0.1%, respectively.

futyakukeisouban siiku niyoru tairyouseisan

Sakai and Kanno (2013) estimated the residual rate

(Mass production of sea cucumber juveniles

in an experimental area using msDNA marker. The

using benthic diatoms).

residual rate of 8mm juveniles was only 3.9% four

, 2, 1‑11 (in Japanese).

years after releasing, but the released juveniles had

Kato S., Tsurumaru S., Taga M., Yamane T.,

also observed in the catches from the fishing area

Shibata Y., Ohno K., Fujiwara A., Yamano K.,neighbor to this experimental area.

Yoshikuni M., 2009: Neuronal peptides induce
oocyte maturation and gamete spawning of sea

Future perspective

cucumber,. Dev. Biol.,

326, 169‑176.
To succeed in increasing the stock of sea

Kobayashi M., and Ishida M., 1984: Chinamako

cucumbers, further improvement in seed production,

no genmouyouin ni kansuru nisan no jikken

especially improving feeding to promote growth(Some experiments on the cause of death of sea

during post-larval rearing, is necessary. In addition,

cucumber juveniles).

evaluating the eﬀectiveness of artiﬁcial seed release(in Japanese).

strategy is indispensable.

, 13(1), 41‑48

Kobayashi T., and Yamaguchi H., 2011: Effects of

The cheapest juvenile for release onto theﬁshery ground is the newly settled size, 0.4 mm infor seedling production ofJapanese sea cucumber

body length. Fishermen can produce these small
juveniles themselves even in the corner of the port
without many devices, technicians or long term

, 7, 19‑24 (InJapanese with English abstract).
Konda Y., and Sakai Y., 2005: An Experimentallabor. We can now distinguish even such a small

study to observe the gut contents of juvenile

seed from wild ones by msDNA markers. We willsea cucumber,now determine if this small seed contributes to, 68,

increase the natural stocks. The size dependence of

71‑74 (in Japanese with English abstract).

the recapture rate should be evaluated as soon as

Mitsunaga N., and Matsumura Y., 2004: Growth andpossible for this project to succeed.

survival of hatchery-produced juveniles of sea
cucumber

Referencesin different

release size.
, 30, 7‑12 (in Japanese with English

Hatanaka H., 1996: Density effects on growth ofartificially propagated sea cucumber
juveniles.

abstract).
Noguchi K., and Noda S., 2011: Inﬂuence of Copepods, 44(2),141‑146 (in Japanese with English abstract).
Ikeda Z., Ueki N., and Kusaka K., 1992: On theproper volume of the diet LIVIC-BW given to
the juvenile sea cucumber

which occur during seed production of the sea
cucumber,, 3(2), 131‑135 (In Japanese withEnglish abstract).
Sakai Y., and Kanno M., 2013: DNA kaiseki niyoru., 7, 53‑55

manamako no houryukouka suiteigijyutsu no134Yuichi SAKAI
kaihatu to keigunkouzou no kaimei (Estimation

Rikujyoikusei Manyuaru (Manual for theof the effectiveness of artificial seed releasing

seed production ofand population structure oflandbased hatcheries).

inusing DNA analysis).
6‑18 (inJapanese).

, p.48‑51 (in Japanese).
Ueki N., and Ikeda Z., 1989: Rearing experiment

Sakai Y., and Konda Y., 2006: Kairui syubyouseisanof newly settled sea cucumber

gijyutsukaihatsu-Manamako- (Developmentof mass production technique of shellfish-)

using freezed benthic diatoms as foodorganization.
., 7, 53‑55.

16‑38. (inJapanese)

Fujiwara A., Yamano K., Ohno K., and Yoshikuni
M., 2010: Spawning induced by cubifrin in the

Sakai Y., and Konda Y., 2008: Manamako

Japanese common sea cucumber

Jinkousyubyou no Syokugai Boujyogijyutsu., 76, 795‑801.

nituite (New mass production technique to

Yanagibashi S., Yanagisawa T., and Kawasaki K.,

prevent the predation of artificial juveniles

1984: A study on the rearing procedures forfrom copepods)

the newly settled young of a sea cucumber,

76, 8‑14 (inJapanese).

, with special reference to

Sakai Y., and Konda Y., 2008: Manamako
jinkousyubyou no rikujyou ikuseigijyutsu
kakuritusiken (Experimental study of rearing
techniques ofthe supplied food items.

, 32(1),

6‑14 (in Japanese with English abstract).
Yanagibashi S., and Kawasaki K., 1985: Manamako

in land

Syubyou Seisan (Seed production of, 81‑93

Houkoku Aichiken Suisansikenjoh (The 1984base hatcheries)

Selenka). In: Syouwa 59-nendo Gyoumu(in Japanese).
Sakai Y., Konda Y., Nakajima K., and Gohda
H., 2009: Manamako Jinkousyubyou no

Technical report of Aichi Fisheries ResearchInstitute), p.16‑19. Aichi Prefecture, Japan (inJapanese).

水研センター研報，第40号，135−143，平成27年
Bull. Fish. Res. Agen. No. 40，135−143，2015

135

Fluorochrome Marking of Out-planted Green Sea Urchins,, for Sea Ranching and Restocking
Programs in the Gulf of Maine, USA
Pamelia Fraungruber＊1, Stephen D. Eddy＊1, Nicholas P. Brown＊1, and Larry Harris＊2

Abstract: Marking calciﬁed structures with ﬂuorochromes is done in a variety of vertebrate andinvertebrate species to tag individuals for growth, population, and ecological studies. Here, we
describe the use of the ﬂuorochrome tetracycline to identify hatchery reared green sea urchins
released on-bottom onto two aquaculture leases known as Job and Sloop, located in the Gulf ofMaine, USA. This was done to examine the viability of sea ranching and stock enhancement bylooking at recovery rates and growth of reseeded juveniles over the course of two years. 21,000
hatchery reared green sea urchin juveniles

were marked

with the ﬂuorochrome tetracycline when they were at 10 ‑ 20 mm test diameter, and released
onto 400m2 study areas located at each lease. Juveniles from the same hatchery cohort were
simultaneously reared in a land-based recirculating aquaculture system so that sea ranching could
be compared with tank farming. The release areas were surveyed by SCUBA divers at 3‑5 monthintervals for over two years. Urchins were collected from the ﬁeld, measured, and dissected toremove the jaw structures, which were then examined with ﬂuorescence microscopy. Tetracycline
fluorescence was detected for up to 27 months post-release in recaptured urchins. Numbers ofrecaptured marked urchins ﬂuctuated over time, causing large variability in population survival
estimates for each site at each sample interval. Size measurements of recaptured urchins showeda decline in average test diameter at the Job site, but at the Sloop site average test diameterincreased during the two year study. Green sea urchins from the same hatchery cohort reared
in a land-based tank system had signiﬁcantly better growth than those recaptured from either
lease site. Environmental factors, rather than genetic factors (hatchery source), were likely thecause of the size diﬀerences observed between hatchery seed recaptured from the lease sites andthose reared in tank culture. Site factors may have resulted in size dependant mortality and/or
out-migration of larger urchins. One of the limitations of the mark/recapture approach with sea
urchins is that dive surveys need to expand over time to account for urchin movement away fromthe release area. Given the high cost of such eﬀorts, this may not be practical or cost eﬀective.
Because the marked jaw structures were internally located, it was not possible to identify marked
sea urchins in the field, and the animals had to besacrificed for laboratory analysis. Recent
advances in fluorochrome marking and visualization could allow field identification of marked
urchins. This would enhance the ability of resource managers to evaluate restocking programs in
the Gulf of Maine, as well as to assign provenance or ownership of sea ranched urchins.
Key Words: green sea urchins, ﬂuorochromes, sea-ranching

2015年 1 月30日受理（Received on January 30, 2015）＊1
University of Maine, Center for Cooperative Aquaculture Research, Franklin, Maine, USA＊2
University of New Hampshire, Durham, NH, USA Larry.Harris@unh.edu
Corresponding author: Stephen D. EddyE-mail: steve.eddy@maine.edu

136Pamelia Fraungruber, Stephen D. Eddy, Nicholas P. Brown, and Larry Harris

The green sea urchinincreased recruitment, or intensified fishing effort

has been an economically important

can also lead to improved stocks or increased catch

fisheries species in the Gulf of Maine, USA (GOM)

levels. Measures of survival, growth, and return tosince the 1980ʼs. Catch levels peaked in 1993 at

the fishery are needed to assess the cost/benefit

19,050 metric tons, and the ﬁshery value peaked inof stock enhancement. This can only be done if

1995 at $35,604,275. However, these large annual

released stock can be diﬀerentiated from wild stock,

harvests couldnʼt be sustained, and ecologicalbut there are no discernible external differenceschanges have contributed to a steep decline in wild

between hatchery and wild urchins (Agatsuma., 2004). Since 2008 the annual., 2004). However, sea urchins can be internallycatch in Maine has averaged about 1,300 mt, withstocks (Steneck

marked with ﬂuorochromes, either through injection

an average value of about $5.5 million.Althoughor bath immersion. Kobayashi and Taki (1969) werethe ﬁshery may not return to 1990 levels, resource

the ﬁrst to use tetracycline to mark the sea urchinmanagers and ﬁshermen believe that wild stocks canS.

and should be rebuilt to allow for increased harvest

variety of studies have used ﬂuorescent markers tofor growth studies. Since that time, alevels. Several management approaches have been

identify sea urchins in the lab or in the ﬁeld (Ellers

taken to help rebuild natural stocks, mostly based

and Johnson, 2009). This paper describes the use of

on ﬁshing restrictions. Stock enhancement throughfluorochrome marking to identify hatchery seed ofrelease of hatchery seed has also been discussed, but

released onto ocean bottom leases

uncertainty regarding its ecological and economic

in the GOM in order to evaluate sea ranching. Sea

viability has discouraged public funding of any

ranching is similar to reseeding, but in this casesustained stock enhancement programs in the GOM.

the juveniles were released onto privately held

Evaluating the effectiveness and benefits ofaquaculture leases. The project was carried out by

sea urchin reseeding programs is an essential

the Center for Cooperative Aquaculture Researchbut complex task. The economic return will be(CCAR), working with industry partner Friendshipa function of seed production costs, growth andInternational (FI), a sea urchin trading company

survival of out-planted seed, and market prices atbased in Maine. We were interested in ascertaining

the time of recapture. In Japan, sea urchin stock

whether this privatized mode of reseeding could beenhancement, known as reseeding, has been done

a viable model for the ﬁshery. To do this we needed

at large scale (>50 million seed annually) for over 20

to determine if released seed would remain within

years (Agatsuma., 2004). In

lease site boundaries, and whether growth andHokkaido it cost 4‑10 JPY (4‑8 US cents) to produce

recovery rates would be suﬃcient to realize a return

one seed of 5 mm test diameter, and nearly 3x thatto the lease site operator or to the ﬁshery.

., 2004; Sakai

for 20 mm seed (Sakai., 2004). The economic

beneﬁts of reseeding to the Japanese ﬁshery remain

Materials and Methods

uncertain; in some cases catches have declined
or remained static despite widespread reseeding(e.g.

Hatchery: The CCAR is a multi-species aquaculturein Hokkaido) ,research and development facility operated by thewhereas in other cases reseeding is correlated with

University of Maine (http://www.ccar.um.maine.edu/

improved catch levels (e.g.

at Esan nearindex.html). Hatchery production for the project was

Hakodate City) (Agatsuma 2014, in publication).

carried out at the CCAR in the spring of 2009 (Feb.‑

Ultimately, the costs of seed production versus theJune). Green sea urchinswere induced to spawn

economic return to the ﬁshery must be considered(N=39 females and 30 males) to provide gametes

in the context of cultural values and ecologicalthat were fertilized for larval rearing. Laboratory

consequences.

spawning, fertilization and larval rearing methods

Although increased catch levels might imply thatforare similar to those described

restocking has been successful, it is not evidence offor many other sea urchin species (McBride, 2005).

a cause and effect relationship. Ecological changes,

Larvae were reared in conical bottom 230 L clear

137

Mark Recapture of Green Sea Urchins in the Gulf of Maine
fiberglass vats continuously supplied with freshseawater at about 12 ℃.
were the primary algal feeds.
The larvae were competent for settlement at 24‑30
days post-fertilization. Following settlement, juveniles
were reared for an additional period of 8‑10 months
in a land based nursery system. During the nursery
period they were held in plastic hydroponic plantbaskets in shallow fiberglass raceways, and fed
with freshly harvested.

Fluorochrome marking and visualization: The
juveniles were marked (tagged) with tetracycline
about four months before release onto the lease sites,
using methods adapted from Ellers and Johnson(2009). Juvenile urchins were graded into perforated
baskets, and immersed for 24 hours in tanks filled

Fig. 1. A tetracycline marked green sea urchin
demipyramid
viewed using a WIB filter on a fluorescencecompound microscope.

with 0.2 µm ﬁltered seawater and 37.5 mg per L−1
tetracycline (Sigma-Aldrich Tetracycline T3258).

juveniles were transferred in plastic bags by divers

Urchins were fed to satiation before and during

onto the bottom and distributed along transect

tagging to ensure active growth and uptake of thelines laid out to 15 m in all four compass directions,fluorochrome into the calcareous exoskeleton. Two

encompassing a total area of 400 m . Between 1,000

weeks following marking twenty-ﬁve urchins wereand 1,500 juveniles were released at 5 m and 10

examined using a ﬂuorescence microscope. The jaws

m markers along the transects to ensure an evenof each individual were removed and placed in a

distribution. The juveniles were not enclosed andsodium hypochlorite solution to dissolve all organic

therefore were free to move. No feeding or any

material, leaving only the calcareous jaws behind.

other husbandry activity was conducted during the

These structures were then examinedthrough a

two years following the release.

2

GIB ﬁlter using a Zeiss Axio Imager Z1 ﬂuorescence

Site surveys: The sites were characterized in a

microscope. Oxytetracycline goes through excitation

previous study (Kirchhoff

at 390nm and emission at 560nm. Tags appear

to out-planting an initial transect dive was done., 2008), but prioras a bright line of fluorescence spanning the jaw

to estimate the extent of existing sea urchins,

horizontally, and for the most part were easily

predators, and bottom cover. At each release

identiﬁable (Fig. 1). 100% of those examined directlyarea a baseline was laid out in a North-Southafter tagging had clearly visible tags.

orientation and ﬁve transect lines were laid out on

Sea ranching: The tagged juveniles were releaseda perpendicular (East-West) bearing extending to 10

at two aquaculture leases located in Penobscot Bay,

m. Sample quadrats consisting of a 1 m2 PVC frame

Maine. Site 1 (Sloop) was located oﬀ of Northaven,

were placed at the 10 m marker in each direction, at

Maine near Sloop Island (44°12.2ʼN 68°50.1ʼW)

the center of the transect, and just over the baseline

and Site 2 (Job) was oﬀ of Camden, Maine near Job(0 m on transect), for a total of 15 quadrats per site.

Island (44°13.5ʼN 68°50ʼW). Each site comprised

During the pre-release survey the bottom substrate

two acres (0.81 ha) of sea bottom, with a mean water

was characterized and the numbers of predators

depth of about 2‑5 m. The leases were marked(crabs, sea starts, etc.) and naturally occurring (pre-with buoys to indicate that harvesting urchins byexisting) urchins were counted. The extent anddragging nets across the bottom was prohibited. In

composition of algal feed was also observed for eachFebruary of 2010, 10,500 juveniles were released

site. The out-planted areas were then dive surveyed

at each site onto a small study area locatedon six more occasions at 3‑5 month intervals overapproximately within the middle of each lease. The

the course of 27 months. All urchins within each

138

Pamelia Fraungruber, Stephen D. Eddy, Nicholas P. Brown, and Larry Harris

sample quadrat were enumerated and those between

Urchins reared in the land-based tank system

4‑30 mm TD were collected in numbered meshwere sampled at intervals coinciding with the leasetubes to be taken to the laboratory for measurementsite surveys. Thirty urchins from each tank wereand identiﬁcation (absence/presence of ﬂuorochrome

randomly removed and measured for weight to themarker). Urchins smaller or much larger than thenearest 0.1 g, and TD to the nearest 0.1 mm using

original release size were not collected in early

digital calipers.

surveys, but during later surveys larger urchins

Data analysis: The average number of total urchinswere collected to account for any growth.

(tagged and untagged) per square meter was

Tank culture: During the two year sea ranchingcalculated for each study area and survey datestudy 9,500 green sea urchins from the sameas the total number of urchins collected per site

hatchery cohort as the lease site urchins weredivided by the number of sample quadrats (usually

reared in a tank system at the CCAR, to compare15). The number of released seed remaining at eachgrowth and survival of lease site urchins withsite and survey date was estimated as the average

juveniles reared on land. The juveniles were stocked

number of recaptured (tagged) urchins per sample

into raceways assembled to form a slanted V interior22
quadrat (m ) x 400 m (the size of the release area

proﬁle (V-trough), with a perforated bottom plate to

as a whole). The mean, minimum and maximum test

remove wastes. The V-troughs were plumbed into a

diameter of recaptured urchins was calculated forrecirculating seawater aquaculture system equippedeach site and survey date. Chi squared tests werewith a parabolic ﬁlter for solids removal, moving bed

used to determine whether or not the numbers of

biofilter, foam fractionator, oxygen injection, 3 hp

tagged and untagged urchins were significantly

chiller, and UV sterilizer. Rearing temperatures weredifferent from each other. The standard deviation

held between 6‑16 ℃ year round and the juvenilesof the mean test diameter was determined to see if

were fed high quality formulated diets (Noﬁma dietthe average size of recaptured urchins signiﬁcantly

from Norway). It was anticipated that sea urchins

diﬀered (±1 SD) between the two sites and from thereared under these conditions would have goodtank reared urchins. Data were plotted to display

growth and survival, to provide a benchmark bytrends in numbers, average TD, and maximum TD

which the lease site urchins could be compared.

of recaptured urchins at each site over time, and theSpecimen analysis: Specimen bags containing

TD of lease site urchins was compared with that ofurchins from the sample quadrats were broughttank reared urchins.

back to the lab, drained and frozen until analysis.
Results

These were later (within 2‑6 weeks) thawed inseawater, and all individuals were blotted dry andweighed to the nearest 0.1g. Test diameter (TD) was

Site characteristics: The two sites were less than

measured to the nearest 0.1mm with digital calipers

six nautical miles apart and of comparable depth(model CD-6PMX Mitutoyo Corporation, Kawasaki,(2‑6 m mean water), but they differed in terms ofJapan). Each sea urchin collected from the lease sites

exposure, current, bottom substrate, and population

was analyzed for the presence of oxytetracycline

density of naturally occurring (pre-existing) urchins.

marking, as described above. In some cases multiple

The Job Island site had relatively uniform depth,

or single bands of auto-ﬂuorescence were seen thatbut was subject to periods of extreme slack tide andappeared atypical or ambiguous (e.g. diﬀuse). Sources

periods of strong current. The bottom substrate atof ambiguity and therefore error in identification

Job was 80% rock cobble with several small boulders

included size of the jaws, intensity of the light usedthroughout, which were populated with macroalgae,

to make the tags ﬂuoresce, and ambient light frombut relatively little drift algae was found. Predators

the surrounding room. If the results were uncertain,

were not found in abundance, with only one large

then the jaws were either reevaluated or marked as

Jonah crab (“untagged”. Urchins that were clearly tagged were

initial population density of pre-existing urchins at) observed, and theconsidered as recaptured (hatchery origin).

the Job site was 2.25 animals/m . At the Sloop site,

2Mark Recapture of Green Sea Urchins in the Gulf of Maine139

the bottom substrate was 80% shell hash, which

previous levels, and there was evidence (disturbed

provided abundant refuges for small and medium-grounds, gear tracks, and broken tests) that the site

sized urchins. The study area was on a sloped ledge,

had been recently ﬁshed by a dragger boat.

so the depth varied across the area compared with

Population estimates: Population estimates ofJob, which was more ﬂat. A few small boulders were

hatchery origin urchins remaining within thefound on the Sloop site with macroalgae growing on

400m 2 release areas at each survey varied inthem, and drift algae, mostly kelp, were abundant.

direct proportion with the recapture rates (Fig. 3).

The Sloop Island site had an abundance of large

Originally, 10,500 urchins were released at eachurchins and sea stars present on it at out-planting.

study area. Extrapolation from dive surveys

The initial population density of pre-existing urchinsindicated that the number of hatchery origin urchinsat the Sloop site was 4.5 animals/m2.

remaining within the Job release area at each

Recapture rates: Sea urchins were found on bothsurveyranged from 45 to 36,894; with 3,306 projectedstudy areas at every survey for over two years.

as still remaining at 27 months post-release. At the

The total number (tagged and untagged) found at

Sloop site, population estimates of hatchery urchinseach survey ranged from 4 ‑ 674 at the Job site andremaining at each survey ranged from 3,680 tofrom 194 ‑ 397 at the Sloop site. Tagged urchins

18,165; with 7,360 projected as still remaining at the(hatchery origin) were recaptured at both sites andﬁnal survey, 27 months post-release (Fig. 3).

at every dive survey up to the last, 27 months post-

Average and maximum size: The average test

release. Recapture rates declined in the first year

diameter (TD) of hatchery origin urchins recaptured

but then signiﬁcantly spiked in the summer of theat the Job site declined to 5.1 mm over the course offollowing year at both sites, before again declining

the study, which was the minimal release size, but

in subsequent surveys (Fig. 2). At the Job site 10%

TD increased at the Sloop site (Fig. 4). At Job, theto 100% of the urchins collected during each diveaverage TD declined from 10.6 mm at release to 5.1

survey were determined to be of hatchery origin,

mm 27 months post-release, whereas at Sloop theand at Sloop 35% to 71% of collected urchins wereaverage TD increased from 11.3 mm at release toof hatchery origin. Itʼs important to note that on the18.3 mm at 27 months. The largest marked urchin

one occasion when 100% of the animals collected

recaptured from any of the surveys at Job during

at Job were tagged, the entire sample population

the course of the study was 19.7 mm (Aug 2010),

consisted of just four animals, all very small (<7and at Sloop it was 49.3 mm TD (Sept 2011, 19

mm TD). At the ﬁnal survey a total of 107 urchinsmonths post-release) (Fig. 5). The Job site had awere collected from Job and about 30% of these

disproportionate number of small urchins remaining

were tagged. At the final Sloop survey the urchinon it at every survey throughout the course of the

population showed a significant decline from40,000# of tagged urchins

35,000

450
400
350
300
250
200
150
100
50
0

Job tagged

30,000Job

25,000Sloop

20,000Sloop tagged

Fig. 2. Total numbers of hatchery origin
recapturedfrom two release sites in
Penobscot Bay, Maine at each survey.

15,00010,0005,000
0Fig. 3. Estimated population of hatchery origin
remaining within the study area
at each lease site and sample date. Calculated as
average number of recaptured urchins per sample
quadrat (m2) x 400 m2 (total study area).

Pamelia Fraungruber, Stephen D. Eddy, Nicholas P. Brown, and Larry Harris

Average test diameter（mm）

140

50
Job

45
40

Sloop

35
30

Tank culture

25
20
15
10
5
0

Maximum test diamter（mm）

Fig. 4. Average test diameter of hatchery origin
recaptured at two release sites in Penobscot Bay, Maine at each dive survey,
and in tank culture at the CCAR. Error bars = ±1 standard deviation fromthe mean.
Job
60
Sloop
50
40
30
20
10
0

Fig. 5. Maximum test diameter of hatchery origin
recaptured at two release sites in Penobscot Bay, Maine at each divesurvey.

study. Of the total number of urchins (sum of six

Discussion

surveys) recaptured from Job, 84% were 侑6mm. At
the Sloop Island site, only 1% of the total recaptured
urchins were 侑6mm.
Growth rates diverged between land and sea

Few previous studies in North America havemonitored survival and growth of tagged sea urchins
released into the ﬁeld. Dumont. (2004) released

based hatchery urchins within the ﬁrst year of thethree size groups of green sea urchins tagged withstudy. Sea urchins reared in the land-based culture

tetracycline onto a small study area. Similar to thesystem were much larger on average at the end

present study, they found that recapture ratesof the two year study than those recaptured fromwere size and time specific: 69% for <10 mm andeither ocean lease site (Fig. 4). After 27 months the

2% for >15 mm urchins after nine days, and 25%

largest urchin sampled in the tank culture system

and 0% respectively after forty days. In a study by

was 53.4 mm, and 㲔 1/3 of the tank reared urchins

Rogers-Bennett

were 侒40 mm.

(. (1994), red urchin juveniles) were tagged andMark Recapture of Green Sea Urchins in the Gulf of Maine141

released onto study areas that varied in depth.

it could have been a sampling artifact. Presumably,

Recovery rates after 12 months were 21% frommisidentification would have occurred equally atshallow habitats and 11% from deep habitats.

Sloop at this survey date, and it did not. Although

The present study provides evidence that

Sloop had higher recapture numbers (per quadrat

hatchery reared green sea urchins can be

and total) at this survey date than at other surveys,

successfully out-planted for reseeding or sea

they were not signiﬁcantly diﬀerent from the otherranching in the Gulf of Maine. Success is defined

Sloop surveys. Also, we were concerned about thisas the ability of seed to survive and grow to legal

issue and any specimens with atypical ﬂuorescenceharvest size (52 mm) within 5 years of release. We

patterns were considered as unmarked. For thesesaw that released juveniles survived and remained

reasons, we believe that the spike in recapture

for an extended period (27 months) within eachnumbers observed at Job during the June 2011

release area. However, recaptured juveniles weresurvey was a sample artifact. Every sample quadrat

disproportionately smaller at one site (Job) than at

had to be thoroughly and equally searched, often by

the other (Sloop). This suggests that site factors

overturning rocks and shells to ﬁnd hidden urchins.

modified the size distribution of surviving orThis effort had to be consistent between sites

remaining out-plants in different ways at the twoand survey dates, which in practice was difficult

sites. The Sloop Island site may have had a habitatto accomplish. Under varying field conditions ofmore favorable for sea urchins, with more and larger

bottom substrate, current, turbidity, ambient light,

refuges, and greater feed abundance.

and temperature, itʼs likely that the success rate

Following settlement, juvenile green sea urchinsfor finding urchins would vary between sites andtake refuge under rocks, in crevices, or under debris

dates. In addition, random movement patterns ofas an adaptation to escape predation (Cameronurchins onto and off of the study areas probably

and Schroeter, 1980; Dumont., 2004). Hereoccurred, because urchins move in response to foodthey graze on diatoms, coralline algae and detritus

availability and the presence/absence of predators(Raymond and Scheibling, 1987). While both sites(Dumont

in the present study supported urchins, the shelljuveniles are less vulnerable to predation, and a shift

hash at the Sloop Island site was full of cracks, holes

from cryptic to active foraging occurs (Dumont., 2007). At about 15mm TD sea urchin

and larger spaces, providing refuges for a broader., 2004). Migration of urchins larger than 10 mmsize range of juveniles. The rock cobble at the Job

TD away from the release area in search of feed orIsland site was, for the most part, flat against the

refuge might explain the disproportionate numbers

sediment, with fewer and smaller hiding places forof small tagged urchins seen at the Job site.

juveniles. The rock cobble had small interstices that

Active foraging enhances the availability andwere well suited for juveniles at or below 5‑6 mm,

quality of macroalgae, increasing the growth rate.

but too small for larger urchins. Most of the urchins

When there is abundant food sea urchins will

recovered from the Job site surveys, whether wild

aggregate in high densities, and they can remain

or tagged, were < 15 mm TD.

stationary for several months or longer (Dumont

The Job site generally had lower recapture rates., 2007). In the present study, both sites providedof tagged seed than the Sloop site, indicating thatfeed in the form of encrusting algae and particulate

it was less hospitable for out-planted sea urchins.

macro-algae. However, the Sloop site was more

The notable exception occurred in the summer

exposed and had greater currents (Kirchhoﬀ

of the second year, when a large and significant

2008), and urchins at this site thus had access to.,number of small urchins were captured at the Junelarge pieces of drift algae, mostly kelp, that were2011 Job site survey, and subsequently identiﬁed as

carried onto the site by the current. This greater

hatchery origin due to presence of the ﬂuorochromefeed availability might explain why recaptured

mark. This spike in recapture numbers could haveurchins had a larger average and maximum TD

been due to misidentiﬁcation (e.g. detecting auto or

at Sloop than at Job. The lack of a substantial foodpseudo-ﬂuorescence and attributing it to the tag), or

source at the Job site might have encouraged

142

Pamelia Fraungruber, Stephen D. Eddy, Nicholas P. Brown, and Larry Harris

more of the larger urchins to leave the site, whilegreen sea urchins in the Gulf of Maine.

also causing slow growth of the small urchinsthat remained, due to low food intake. Green sea

References

urchin growth rates can be highly variable in thenatural environment, primarily in response to feed

Agatsuma Y., Sakai Y., and Andrew N. L., 2004:

availability and type (Nestler and Harris, 1994;

Enhancement of Japanʼs sea urchin fisheries.

Brady and Scheibling, 2006). Growth can be very
slow and rates of侑 0.25 mm per year have been

documented for urchins found in tide pools (Russell,1998). We observed that green sea urchins fromthe same hatchery cohort reared in the land-based: J. M. Lawrence and O. Guzman (eds.) Sea
Urchins: Fisheries and Ecology. DEStech
Publications, Inc., Lancaster. pp. 18‑36.
Agatsuma Y., 2014: Reseeding of sea urchins inJapan.

: Brown N. P., and Eddy S. D. (eds)

culture system had signiﬁcantly better growth thanAquaculture of Echinoderms,

those recaptured at either lease site. This is further

Wiley and Sons, New York, NYJohnevidence that growth potential at the lease sites

Brady S. M. and Scheibling R. E., 2006: Changes in

was limited more by environmental factors thangrowth and reproduction of green sea urchins,

by genetics or by the fact that the urchins were of, during

hatchery origin.

repopulation of the shallow subtidal zone after

In the present study we were able to diﬀerentiate
hatchery origin from wild urchins for up to 27
months in the field. Johnson

mass mortality.

., 335,277‑291.

. (2013) reported

Cameron R. A., and Schroeter S. C., 1980: Sea urchinthat tetracycline fluorescence could be detected

recruitment: Effect of substrate selection on

for at least two years in green sea urchins held injuvenile distribution.

the lab, when tetracycline was administered via., 2,

243‑247.

injection. The fact that ﬂuorochromes can persist for

Dumont C., Himmelman J. H., and Russell M. P.,

such extended periods makes this marking/tagging

2004: Size-specific movement of green sea

method invaluable for long term lab and ﬁeld studies

urchinsof sea urchins, and was essential to carrying out

urchin barrens in Eastern Canada.

the research described above. Recent advances inon., 276, 93‑101.

the application and visualization of fluorochromes

Dumont C. P., Himmelman J. H., and Robinson S.

offer further advantages, which could bring downM., 2007: Random movement pattern of the sea

costs and improve the effectiveness of sea urchinurchinmark/recapture studies. Ellers and Johnson (2009)., 340, 80‑89.

describe methods to create multiple marks on the

Ellers O., and Johnson A. S., 2009: Polyﬂuorochrome

demipyramids (e.g. at intervals or with multiple

marking slows growth only during theﬂuorochromes), which would allow for diﬀerentiation

marking month in the green sea urchinof multiple year classes released into the field.

.,

The same authors also describe visualization of128, 126‑144.

fluorochromes on external structures such as

Johnson A. S., Salyers J. M., Alcorn N. J.,

the skeletal plates (test) and spines, which allows

Ellers O., and Allen J. D., 2013: Externallytagged individuals to be identiﬁed without sacriﬁce

visiblefluorochrome marks and allometrics of(Johnsongrowing sea urchins.

., 2013). Ultimately, development of afield portable device for visualizing fluorochromes
seems feasible, to allow reliable identification ofstockswhile minimizing adverse impact on

the population (Johnson

269.
Kirchhoff N., Eddy S., Harris L., and Brown N.,
2008: Nursery-phase culture of green seaurchin., 2013). These methods

provide powerful tools for evaluating the results offuture restocking and sea ranching programs for., 132(3), 251‑

using “onbottom” cages.

., 27(4), 921‑927.

Kobayashi S., and Taki J., 1969: Calcification inMark Recapture of Green Sea Urchins in the Gulf of Maine

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sea urchins. A tetracycline investigation ofnorthern California. Final Report. Contract FG-

growth of the mature test in

2230-MR., Bodega Marine Laboratory, California.
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McBride S., 2005: Sea urchin aquaculture.
., 46, 179‑208.

pp. 1‑10.
Russell M. P., 1998: Resource allocation plasticity in sea
urchins: rapid, diet induced, phenotypic changes

Nestler E. C., and Harris L. G., 1994: The importancein the green sea urchin,of omnivory in., 220, 1‑14.

(Muller) in the Gulf of Maine. In: B. David (ed.)

Sakai Y., Tajima K. I., and Agatsuma Y., 2004: Mass

Proceedings of the 8th International Echinoderm

production of seed of the Japanese edible sea

Conference, Dijon, France. A. A. Balkema, NewurchinsandYork. pp. 813‑818.

: J. M.Lawrence

Raymond B. G., and Scheibling R. E., 1987:

and O. Guzman (eds.) Sea Urchins: Fisheries andRecruitment and growth of the sea urchin

Ecology. DEStech Publications, Inc., Lancaster.

(Muller)following mass mortalities oﬀ Nova Scotia,., 108: 31‑54.
Rogers-Bennett L., Fastenau H. C., Hibbard-Robbins
T., Kain Z., and Dewees C. M., 1994: Culturing
red sea urchins for experimental out planting in

pp. 287‑298.
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Accelerating trophic-level dysfunction in kelp
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水研センター研報，第40号，145−153，平成27年
Bull. Fish. Res. Agen. No. 40，145−153，2015

145

Improving the Food Quality of Sea Urchins Collected from Barren
Grounds by Short-Term Aquaculture under Controlled Temperature
Tatsuya UNUMA＊1, Yuko MURATA＊2, Natsuki HASEGAWA＊1, Sayumi SAWAGUCHI＊1,
and Kazuhiro TAKAHASHI＊3

Abstract: In west Hokkaido, there are many barren grounds whereisabundant. These sea urchins have poor commercial value due to their thin gonads, but can becomemarketable by intensive feeding for gonad enhancement. In general, the quality of sea urchin
gonads as food products decreases as gametogenesis progresses. Mature ovaries and testes are
not suitable as food products because of the unpleasant taste caused by gamete content and themelting appearance caused by gamete ﬂow via breakage of the gonoduct. Immature to pre-mature
gonads that contain predominantly nutritive phagocytes (somatic nutrient storage cells) and notcopious gametes have a higher commercial value. Thus, enhancing the nutrient accumulation into
nutritive phagocytes plus suppressing gametogenesis is advantageous for sea urchin aquaculture.
We are developing short-term aquaculture techniques to improve the food quality ofcollected from barren grounds under controlled temperature to suppress gametogenesis. Rearing
under a low temperature between summer and autumn has proved to be eﬀective toincrease the gonad size without the quality deterioration caused by maturation.
Keywords: aquaculture, food quality, gametogenesis, gonad, sea urchin, temperature

For decades, the loss of seaweed beds and the
expansion of barren grounds has been a serious
problem for coastal ﬁsheries in Japan (Fujita, 2010).
The bare grounds populated by sea urchins are
known as urchin barrens (Pearse, 2006; Fujita., 2008). In west Hokkaido, there are many urchinbarrens, whereis abundant(Fig. 1). The sea urchins in these barrens have poorcommercial value because they have thin gonads(edible portion) due to deﬁciency of their main food,macroalgae. However, they can become marketableby intensive feeding for gonad enhancement inshort-term aquaculture (Agatsuma and Nishikiori,
1991; Unuma and Kayaba, 2015). If the culturedof improved quality are sold in autumn, outof the ﬁshing season of this species, higher market
price can be achieved.

Fig. 1. An urchin barren in Suttsu, west Hokkaido.
The bare ground is populated bythat has poor commercial value because ofthin gonads. The photograph was taken by Kazuhiro
Takahashi.

Unlike the gonads of other animals, sea urchin
2015年 1 月30日受理（Received on January 30, 2015）＊1
Hokkaido National Fisheries Research Institute, Fisheries Research Agency, Kushiro, Hokkaido 085‑0802, Japan
E-mail: unuma@fra.aﬀrc.go.jp＊2
National Research Institute of Fisheries Science, Fisheries Research Agency, Fukuura, Yokohama 236‑8648, Japan＊3
Mariculture Fisheries Research Institute, Hokkaido Research Organization, Muroran, Hokkaido 051‑0013, Japan

146

Tatsuya UNUMA, Yuko MURATA, Natsuki HASEGAWA, Sayumi SAWAGUCHI,
and Kazuhiro TAKAHASHI

ovary and testis (both of which are equally preferrednutrients necessary for gametogenesis and supply it

as food) play a role as a nutrient storage organ

to GCs (Walker, 1982; Unuma, 2002).

(Walker, 1982; Walker., 2013; Unuma, 2002,

During the annual reproductive cycle, gonads of2015). A thorough understanding of the uniqueboth sea urchin sexes pass through a predictablecharacteristics of sea urchin reproduction should

series of structural changes (Unuma and Walker,

permit novel methods to improve the quality of

2009; Walker., 2013). Fig. 3 shows histologicalgonads as food products in its aquaculture. We arechanges in the ovary and testis ofcurrently conducting research and development for

gametogenesis as classiﬁed into ﬁve stages by Fuji

short-term aquaculture techniques to maximize the(1960a) with modiﬁcations (Unuma, 2002).

value ofduringcollected from urchin barrens

Stage 1. Immature gonad before gametogenesis: In

under controlled temperature and harvest themboth sexes each acinus is ﬁlled with NPs (eosinophilic

when wildis scarce in the market. In

cell populations). In ovaries, a few young oocytes arethis paper, we describe the theoretical backgroundpresent at the periphery of the acini. Hematoxylin-

to improve the food quality ofbystained round spots, residue from phagocytized ova

manipulating environmental conditions and give a(Masuda and Dan, 1977; Tominaga and Takashima,

brief overview of our ongoing research.

1987), are occasionally present centrally in the

Features of Gametogenesis

cells is sometimes difficult at this stage in paraffinovarian lumen. In testes, detection of spermatogenic
sections. Instead, many hematoxylin-stained
The sea urchin has ﬁve gonads attached internallyspeckles, residue from phagocytized spermatozoa

to the test (shell). A lobe of gonad consists of(Kato and Ishikawa, 1982; Reunov

hundreds of gonadal acini and resembles a bunch ofoften present in NPs. These speckles, which are., 2004), aregrapes (Fig. 2A). There are two major populations

amorphous unlike the round spots observed in theof cells inside the acinus (Fig. 2B): germ cells

immature ovary, are a useful feature to distinguish(GCs, from oogonia to ova in the ovary and fromtestes from ovaries.

spermatogonia to fully differentiated spermatozoa

Stage 2. Beginning of gametogenesis: Manyin the testis) and somatic cells called nutritive

developing oocytes or clusters of spermatogonia

phagocytes (NPs) that are present in both sexes

are present at the periphery of the acini, and the(Walker, 1982; Walker

gonadal lumina are still ﬁlled with NPs.

., 2013). NPs store

Stage 3. Middle of gametogenesis: NPs are replaced
A. Gonadov

B. Gonadal acinusszwith ripe ova or spermatozoa in the center of the
gonadal lumina. Numerous developing oocytes orclusters of spermatogonia and spermatocytes (Wardand Nishioka, 1993; Walker., 2005) are present

at the periphery of the acini. NPs are graduallyocdecreasing in size and are present between the GCs.

sg,sc
Ovary
NP

TestisStage 4. Fully mature gonads at the end ofGC

gametogenesis: The gonadal lumina are filled withripe ova or spermatozoa. Shrunken NPs, which

Fig. 2. Structure of sea urchin gonad. (A) A gonadof. A lobe of gonad consistsof hundreds of acini. (B) Schematic drawing of theinside of gonadal acinus. Each acinus contains two
main types of cell populations: germ cells (GCs) andnutritive phagocytes (NPs). NPs are somatic cells
that store nutrient necessary for gametogenesis.
oc = oocyte, ov = ripe ovum, sc = spermatocyte,
sg = spermatogonium, and sz = spermatozoon.

have already lost nutrients, are present only at the
periphery of the acini.
Stage 5. After spawning: The gonadal lumina havenumerous empty spaces and a few residual ova or
spermatozoa. NPs gradually phagocytize residual
gametes and begin to grow as they store nutrients.
After this stage, gonads return to Stage 1 and a newcycle starts.

147

Improving the Food Quality of Sea Urchins
Stage 1

Stage 2

Stage 3

Stage 4

Stage 5

np
Ovary

np

np

ov
oc

ro

ov

a

oc

sg
np

Testis
np

sz
np

b

rs

sz

100 μmNP
GC

Fig. 3. Histological changes in the ovary (upper panels) and testis (lower panels) ofduringgametogenesis. Paraﬃn-embedded sections are stained with hematoxylin and eosin. Schematic drawing below
the photomicrographs shows structural features that are common between the ovarian and testicular acini. At
stage 1, the gonadal lumina are ﬁlled with nutritive phagocytes. At stage 2, many developing oocytes or clustersof spermatogonia are present at the periphery. At stage 3, nutritive phagocytes are replaced with ripe ova or
spermatozoa in the center of the lumina. At stage 4, the lumina are ﬁlled with ripe ova or spermatozoa. At stage 5,
the lumina contain a few residual ova or spermatozoa. np = nutritive phagocyte, oc = oocyte, ov = ripe ovum, ro= residual ovum, sg = spermatogonium, sz = spermatozoon, and rs = residual spermatozoon. Inset a, round spot
derived from a phagocytized residual ovum. Inset b, amorphous speckles derived from phagocytized residual
spermatozoa. Scale bar represents 100 µm.
Seasonal Changes in Gonad Size and GCs/
NPs Proportion

gametogenesis increases the size or numbers of
GCs but also because NPs store extensive nutrient
reserves before gametogenesis (Unuma and Walker,
2009; Walker., 2013). Fig. 4 shows the seasonalchanges in the gonad index (the ratio of gonad
weight to total body weight) and in the proportionof GCs and NPs in. This species spawns

Spawning

20
Gonad index（%）

Sea urchin gonads grow in size not only because

Quality
gonad

Spawning

NP

15

GC
10
5
0

Oct.

Dec.

4

5

Feb.

Apr.

Jun.

1

2

Aug.

Oct.

3

4

5

Typical stage

around October and gonad indices rapidly decrease(Fuji, 1960b). After spawning, the gonad index
gradually increases until the next spawning. The
increase before gametogenesis is attributable to thegrowth of NPs. NPs accumulate various nutrients,
such as proteins (principally the major yolk protein;
Unuma., 2003, 2011), lipids, and carbohydrates(principally glycogen; Marsh., 2013) and increasein size. Proliferation and development of GCs begin

Fig. 4. Diagrammatic representation of the seasonalchanges in the gonad index and in the proportionof nutritive phagocytes (NPs) and germ cells (GCs)in. Typical gametogenic stages
are indicated below the graph. Spawning occurs
around October. The long-term increase in the
gonad index before gametogenesis is attributable to
the growth of NPs. The best season for eating gonad
is around July.

148

Tatsuya UNUMA, Yuko MURATA, Natsuki HASEGAWA, Sayumi SAWAGUCHI,
and Kazuhiro TAKAHASHI

three months before spawning in(Unuma,their gonads are at stage 1 (Murata 2009; Unuma2009). After gametogenesis begins, the size of the

2002, 2009). On the other hand, other Japanese sea

gonad continues to increase but the proportion ofurchins including

NPs in the gonad rapidly decreases.

their gonads are at stage 3, because the maturing

can be eaten until when

gonads do not produce a strong bitterness (Murata
The Best Season for Eating Gonads

2009; Unuma 2009). In every species, the best season
for eating gonads is rather short because of the

The best season for eating

gonads isproblems caused by gametogenesis. Low-quality or

around July, when NPs have grown suﬃciently but

small gonads can be obtained for a longer period of

the proportion of GCs is still smaller (Unuma, 2009).

time, but superior-quality and sizable ones can be

Their gametogenic stage is usually from stage 2 toharvested for only about one or two months (Unuma,

early stage 3 in July (our unpublished observation).

2002).

Before this period, the size of the gonad is too small
and the color of the gonad is not attractive (brownish).

Improving Food Quality by Short-term Aquaculture

After this period, the proportion of GCs becomes
higher and the quality as food gradually deteriorates.

As we have seen, NPs are more important as food

The most serious problem for food quality is thethan GCs. Thus, when sea urchins are cultured for

melting appearance caused by the ﬂow of gametes

gonad enhancement, not GCs but NPs should be(Unuma, 2002; Unuma and Walker, 2009). After ripe

increased in the gonads. The sea urchins dwelling

gonads are taken out of the test, eggs or sperm ﬂow

at urchin barrens cannot store much nutrient inout from the breakage of gonoduct, and the gonads

the gonads due to deﬁciency of food (Agatsuma andcannot maintain their consistency (Fig. 5). This

Nishikiori, 1991). However, the sea urchins produce

phenomenon considerably reduces the commercial

appropriate amount of gametes and spawn even if

value of the gonads. Additionally, the gametes cause

the food availability is low. Therefore, the schema

unpleasant tastes such as bitterness in the matureof gonad index and GCs/NPs proportion (Fig. 4)

gonads (Unuma, 2002; Unuma and Walker, 2009).

is turned to be depressed in the sea urchins at

Relationship between the gametogenic stageurchin barrens (Fig. 6A). We are trying to changeof gonads and the food quality varies among

this schema by short-term aquaculture as shown in

species. Ovaries ofs andFig. 6B. The aims of the culture are (1) to promote

produce an extremely

nutrient accumulation into NPs by intensive feeding

bitter taste after oogenesis begin (Murata., 2002;and (2) to suppress proliferation and development

Murata 2009, Unuma and Walker, 2009). In theseof GCs by manipulating environmental conditions.

species, the season for eating is limited only when

Combination of these two aims should prolong theseason for quality gonad until the spawning season

Ovary

Testiswhen wild

is not fished due to quality

deterioration as well as for resource management.
Environmental Factors Regulating Gametogenesis
Gametogenic cycles of sea urchins are regulatedby environmental factors, such as photoperiod
and water temperature. There have been many
Fig. 5. Gamete ﬂow from the mature ovary (left) andtestis (right) of. After mature
gonads are taken out of the test, they appear to bemelting because of ﬂowing eggs or sperm from thebreakage of gonoduct.

reports describing the effects of these factors on
gametogenesis of sea urchins as listed in Table 1.
Most of these researches were conducted as basic
biology to identify factors aﬀecting maturation, or as
applied biology to develop techniques for promoting

149

Improving the Food Quality of Sea Urchins
B. After culture
Gonad index（%）

Gonad index（%）

A. Before culture
20
15
10
5
0

Oct.

Feb.

Jun.

Oct.

Quality
gonad

20
15
10
5
0

Oct.

Feb.

1

NP

2

GC

Jun.

Oct.

Fig. 6. Purpose of the short-term aquaculture ofcollected from urchin barrens. Schema of gonad index and GCs/NPs
proportion can be changed from (A) to (B) by rearing under optimized
conditions. The aims of the culture are (1) to increase the nutritive
phagocytes (NPs) by intensive feeding and (2) to decrease the germ cells(GCs) by suppressing gametogenesis. Combination of these two aims should
prolong the season for quality gonads.

Table 1. Researches on environmental factors aﬀecting sea urchin gametogenesis)DFWRUVLQYHVWLJDWHG
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maturation to obtain gametes out of the spawning

1995; Sakai, 2015). However, for culturing adult sea

season for laboratory use or for seed production.

urchins to enhance the gonads, not promotion but

For example, in some sea urchin hatcheries insuppression of gametogenesis is required. To ourJapan, out-of-season maturation of brood stock

knowledge, only four studies have been conductedoffrom a view point of suppressing gametogenesis
andisto obtain the gonads of better food quality. In

promoted by manipulating water temperature (Ito., 1989; Masaki and Kawahara, 1995; Noguchi(Böttger.,2006),(Kayaba.,Tatsuya UNUMA, Yuko MURATA, Natsuki HASEGAWA, Sayumi SAWAGUCHI,
and Kazuhiro TAKAHASHI

150., 2012) and2000; Shpigel(Spirlet.,

Methods to Decrease Water Temperature., 2004), maturation was delayedby manipulation of photoperiod, water temperature

To put our findings into practice, economic

and both of them, respectively. However, the control

methods for decreasing the water temperature

regime of environmental conditions for culturing

in aquaculture farms are required. Using electric

is still to be clarified, because optimum

cooling devices is a classic method but has highconditions to suppress gametogenesis effectively

energy costs. It would not be practical to use it in avaries among diﬀerent species.

conventional ﬂow-through system. However, closed
or semi-closed recirculating system may minimize

Overview of Our Research

the problem of energy costs in electric coolingdevices.

Our final goal is to develop a land-based
aquaculture system, using

collected from

Deep seawater drawn from a depth of over 300 m
has a very low temperature (Nakasone and Akeda,

urchin barrens, controlling their gametogenic cycle,

2000). Large initial investment costs are required toand harvesting high-quality urchins when theirbuild a facility to draw the water. However, more

wild counterparts are scarce in the ﬁsh market. As

than ten facilities capable of drawing deep seawater

an initial step, we investigated the eﬀects of water

have been constructed in Japan, where it is usedtemperature on the size and quality of the gonads infor aquaculture, handling of captured fishes, the

.
Adult

food industry, medical treatment, and agriculturecollected from an urchin barren(Nakasone and Akeda, 2000; Kayaba., 2012);in Suttsu, west Hokkaido (Fig. 1), were reared from

including two in west Hokkaido. Therefore, the use

late June to mid October in three tanks maintainedof existing facilities is a practical way to adopt theat either a constant temperature (10℃ or 15℃)

deep seawater method in sea urchin aquaculture.

or a variable temperature, similar to their natural

The use of salty groundwater for aquaculture,environment (control group; 18.0℃ to 22.5℃ ). They

with a salinity comparable to seawater, is a recent

were given a surplus of kelp (development in Japan (Ebata).

., 2006; Imada

Over the experimental period, gonad indices., 2006). It can be obtained from wells bored

increased in all treatments from 7% initially to overnear to the shore and has a stable temperature

20% after three months. Histological observations

year round. In west Hokkaido, the temperature of

revealed that after three months, there were no

salty groundwater is about 10℃ even in summer,

urchins with fully mature gonads in the 10℃ group,

although that of surface seawater rises over 20℃.

but 6% in the 15℃ group, and 39% in the control

The advantage of this method is that it requires

group. Sensory tests of final gonads found that

small initial investment costs. However, this water

the quality of the control group was inferior tosometimes contains high levels of ammoniumthat of the 10 ℃ and 15 ℃ groups, in terms of bothand manganese ions (Ebata

appearance and taste. We conclude that rearing

groundwater requires treatment using aeration andunder a low temperature between

specified microbes to remove the ions before use

summer and autumn is effective to increase the(Ebata., 2006). Such., 2006).

gonad size without the quality deterioration caused

Thus, there are multiple methods for decreasingby maturation. These results are being prepared

the temperature of rearing water in summer. The

for publication in the journal. We are further

chosen method depends on the location and scale ofinvestigating the relationship between the rearing

the aquaculture farm.

conditions and the size and quality of the gonads todetermine the optimum control regime for culturing

Acknowledgments.
We are grateful to Ms. Rie Matsuda and Ms.
Suzuko Tanaka at the Hokkaido National Fisheries151

Improving the Food Quality of Sea Urchins
Research Institute for technical assistanceandthroughout the study. This study was financially

Southern Hokkaido.

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水研センター研報，第40号，155−165，平成27年
Bull. Fish. Res. Agen. No. 40，155−165，2015

155

Maturation Control of the Short-Spined Sea Urchin,, by Low Temperature Rearing Using Deep-Sea Water,
with the Aim of Extending the Market Season
Takaaki KAYABA＊1, Kohji TSUJI＊2, Hiroshi HOSHIKAWA＊3, Yaoki KIKUCHI＊4,
Kazuhiro KAWABATA＊4, Isao OTAKI＊4, Toru WATANABE＊5

Abstract: The short-spined sea urchin, is a valuable commercial

species and one of the most popular and expensive seafoods in Japan. Serving local short-spined
sea urchin to visitors during the summer tourist season in Rausu, located in a world natural
heritage site “Shiretoko,” has long been desired. However, it has not been feasible during the sea
urchin spawning season (July to September), resulting in quality degradation in gonads, the edible
part of sea urchins, due to maturation. Therefore, the Hokkaido Research Organization and theRausu Fishery Cooperative Associations cooperatively investigated the possibility of suppressing
gonadal maturation and maintaining high quality sea urchin gonads by low temperature rearing
using deep-sea water. Unripe sea urchins captured before the spawning season were reared under
two temperature conditions from June to September. In groups reared at ambient temperatures(2 18℃), gametogenesis in both sexes progressed rapidly with increased temperature, and almost
all sea urchins reached full maturity by late July. Whereas in groups reared at low temperatures(2 5℃), gametogenesis progressed slowly and over 60% of the sea urchins had not reachedmaturity even by early September. This result suggests that the progress of gametogenesis in the
short-spined sea urchin is effectively suppressed by rearing under low temperature conditions.
Additionally, we also examined the eﬀects of feeding on gonadal development in sea urchins reared
under low temperature conditions, and revealed that feeding with live

, whichwere cultured as food in Rausu, could increase the gonadal volume efficiently to commercially
preferable size, while suppressing the progress of gametogenesis. Moreover, the results of chemical
analysis and tasting test proved that the quality of gonads were very excellent in sea urchin fed
with live. Consequently, we demonstrate that low temperature rearing, supplemented

with feeding live

, is eﬀective in suppressing gametogenesis to allow for the harvestingof high quality sea urchins during the summer tourist season. At present, this aquaculture method
is being put to practical use by Rausu Fishery Cooperative.
Key words: short-spined sea urchin, gametogenesis, deep-sea water,

low temperature rearing, sea urchin aquaculture

The short-spined sea urchin, which is distributed on the northern

the country; more than 2000 tons of the sea urchinare caught annually in Hokkaido, the main harvest

coast of Japan, is a valuable commercial species andregion for this species.

is one of the most popular and expensive seafoods inorange gonads with a rich taste, and the gonads

2015年 1 月30日受理（Received on January 30, 2015）＊1
Kushiro Fisheries Research Institute, Hokkaido Research Organization, Kushiro, Hokkaido 085‑0024, Japan＊2
Abashiri Fisheries Research Institute, Hokkaido Research Organization, Monbetsu, Hokkaido 094‑0011, Japan＊3
Central Fisheries Research Institute, Hokkaido Research Organization, Yoichi, Hokkaido 046‑8555, Japan＊4
Department of Mariculture, Rausu Fishery Cooperative, Hokkaido Research Organization, Rausu, Hokkaido 086‑1893, Japan＊5
Hachinohe Scientiﬁc Museum MARIENT, Hachinohe, Aomori, 031‑0841, Japan
Corresponding author: Takaaki KAYABA E-mail: kayaba-takaaki@hro.or.jp

has bright156

Takaaki KAYABA, Kohji TSUJI, Hiroshi HOSHIKAWA, Yaoki KIKUCHI, Kazuhiro KAWABATA,
Isao OTAKI, and Toru WATANABE

are preferred especially as an attractive raw foodsummer, from July to September. In Rausu, there

material, such as “Sushi” or “Sashimi (Uni-don)”.

is a huge demand for serving

has also become one of the importanttovisitors during the summer tourist season. However,tourist resources of Hokkaido, because many tourists

it has been exceedingly difficult to supply highvisit Hokkaido in anticipation of the tastes of fresh

quality sea urchins during the summer, because

sea urchin every year.

the spawning season of sea urchins in Rausu is

Since the edible part of the sea urchin is usuallyfrom July to September and the quality of gonads

restricted to the gonads, its quality as food varies

degrades on maturation. Moreover, in Rausu, as

dramatically with gonadal growth and maturation.

a resource conservation measure, harvesting sea

In general, the superior quality of gonads, definedurchins from July to September is legally prohibitedby attractive taste, color, and shape, is only

by local government ﬁshing regulations. Therefore,

present during the term of the unripe phase when

it is necessary to develop a method for culturing

gametogenesis is limited. The mature gonad has ancaught before the closed season, so

unpleasant taste due to the gamete contents and aas to maintain their food quality by suppressing

melting appearance caused by the ﬂow of gametes

maturation.

(Unuma, 2002). Therefore, the best season for
harvestingis very short̶less thanAnnual reproductive cycles of sea urchins are
regulated by environmental factors, such as water

three months per a fishery region ̶in spite of an

temperature (Yamamoto

annual market demand. There would be considerable1989), photoperiod (Pearse

commercial value in developing an aquaculture

and Watts, 1990; Walker and Lesser, 1998; Böttger., 1988; Sakairi.,., 1986; McClintock

technique to control the timing of gonadal maturity., 2006), and lunar period (Horii, 1997; Coppard andso as to ensure a supply of high quality sea urchins

Campbell, 2005). Water temperature in particularover a longer period.

has been shown to have a strong effect on the

Rausu town, located on the Shiretoko Peninsula

progress of gametogenesis in sea urchins. In somein east Hokkaido, is a prominent production

Japanese urchin hatcheries, temperature control

region ofis used to induce broodstocks ofin Japan (Fig. 1). After

Shiretoko Peninsula was registered as a world, andnatural heritage site in 2005, increasing numbers of
tourists have visited Rausu, especially during theto mature out of the normal spawningseason (Ito., 1989; Masaki and Kawahara, 1995;

Noguchi., 1995). The gonadal development ofwild

tends to be correlated with arise in water temperature from spring to summer(Tomita., 1984). Accordingly, it may be possible

to suppress gametogenesis by rearing sea urchins
under low temperature conditions during that
period.
Generally, using electric cooling devices inaquaculture is limited because of its high costs.
Fortunately, a facility for collecting deep-sea wateris available in Rausu. The deep-sea water is deﬁned
as seawater that is pumped from a depth of greaterthan 200 m, and has certain advantageous properties:
stable low temperature, low in suspended particles
and bacteria, and rich in nutrient salts (Nakasone
and Akeda, 2000). More than ten facilities capable
Fig. 1. Location of Rausu town on the Shiretoko
Peninsula in east Hokkaido, Japan.

of drawing deep seawater have been constructedin Japan, where it is used for aquaculture, handling157

Maturation Control of the Short-Spined Sea Urchinof captured fishes, the food industry, medical

The eﬀects of rearing under low temperature

treatment, and agriculture (Takahashi, 2006). In

conditions on gametogenesis

Rausu, deep seawater is drawn from a depth of
350 m and has been used mainly as a raw material

To determine the eﬀect of water temperature on

for refining mineral water and as rearing watersuppressing gametogenesis, rearing experiments

to temporarily hold captured salmon. Additionally,

were conducted under two types of water

there is a suﬃcient supply of deep-sea water, suchtemperature conditions from 2008 to 2010 (Kayabathat it has been collected constantly and used for., 2012). Unripe sea urchins (weighing 54.0 81.3

aquaculture in Rausu. Fig. 2 indicates variationsg), were collected from the coastal fishery groundin water temperature between the deep-sea waterat Rausu in March ‑ May. They were divided into

and ambient sea water drawn from the surface. The

four experimental cages (100 × 100 × 80 mm) at

water temperature of deep seawater is stable atdensities of 150 individuals per cage, and transferred

lower degrees from March to September comparedto the experimental environment on June 1 of eachwith the ambient seawater, ranging from 2 to 5 ℃ .

year. Two cages of sea urchins were maintained

Therefore, taking advantage of the available facility,with running ambient sea water (2.8 ‑ 19.6℃ ) until

the Hokkaido Research Organization and the Rausu

September 1 (ambient temperature group), while

Fishery Cooperative Associations cooperatively

the other two cages were maintained with runninginvestigated the effects of rearing under low

deep-sea water (2.5 ‑ 4.9℃) over the same periodtemperature conditions on the gametogenesis(deep seawater temperature group). In addition, forof, in an effort to develop reliable

determining the eﬀects of feeding on gametogenesis

techniques for maintaining gonads in good quality

and gonad size, individuals in one cage of bothduring the summer tourist season in Rausu, as atemperature groups were reared without feeding

model (Kayaba., 2012). In this proceeding, we(non-feeding cage), while individuals in the otherdescribe new aquaculture technique of sea urchin

cages were given a surplus of commercial dry

and its possibility in the future, adding further data

wakame (to the results which have been reported in Kayaba. (2012).

., obtained from the Riken

Vitamin Company, Tokyo) every day (feeding cage).
Both groups of sea urchins were reared in thedark throughout the experimental period, except

Fig. 2. Variations in temperature of the ambient sea water drawn from thesurface of the Rausu coast and that of the deep-sea water drawn from a depthof 350 m oﬀ the coast of Rausu, in 2009.

158

Takaaki KAYABA, Kohji TSUJI, Hiroshi HOSHIKAWA, Yaoki KIKUCHI, Kazuhiro KAWABATA,
Isao OTAKI, and Toru WATANABE

for during feeding and cleaning of excreta. Twenty

testes progressed slowly in the deep seawater

individuals were sampled from each group monthly

temperature group, and about 60 % of the sea

for histological observation of gonads. Maturationalurchins were maintained at an unripe stage (stages

status of ovaries and testes was classiﬁed into ﬁve

1 3) until 31 August. In spite of distinct diﬀerences

stages according to Fuji (1960); stage 1 (before

in maturity among two rearing temperature

gametogenesis), stage 2 (early gametogenesis),conditions, signiﬁcant diﬀerences in the distribution

stage 3 (mid-gametogenesis), stage 4 (fully mature)of maturation stage could not be found between

and stage 5 (spent). Gonads at stages 1 to 3 aresea urchins in feeding cages and non-feeding cages

commercially favorable. On the other hand, thein both sexes. These results strongly suggest thatfully mature gonads (stage 4) were not suitable forthe progress of gametogenesis in

consumption because of the unpleasant taste caused

closely related to environmental temperature, and isisby the gamete contents and a melting appearance

eﬀectively suppressed by rearing sea urchins undercaused by the ﬂow of gametes via breakage of the

low temperature conditions. Following repeated tests

gonoduct (Unuma, 2002). The spent gonads (stage

over three years, it was proven that rearing at deep5) were also unsuitable for food, because they were

seawater temperatures could extend the harvest

shrunken.

season two months or longer beyond that of theTable 1 shows the frequency of maturation

wild population (Kayaba., 2012). Therefore, thestage of female and male sea urchins during thetechniques used in our study, for maintaining unripe

experiment in 2009. In the ambient temperature

sea urchins under low temperature conditions,group, gametogenesis progressed rapidly withare likely candidates for an efficient method forincreased temperature and almost all sea urchins

inhibiting gonadal maturation and serving sea

reached full maturity (stage 4) by 27 July, abouturchins with good quality during summer tourist

2 months after the initiation of the experiment.

season.

In contrast, gametogenesis in both ovaries andTable 1. The frequency of maturation stage of female and male sea urchins reared at ambient temperatureor deep seawater temperature during the experiment performed in 2009 (based on data from Kayaba.,2012).

Sex

Feeding
condition

Feeding
FemaleNon-feeding

Feeding
Male
Non-feeding

Ambient temperature group
Date

Favorable maturity

Unfavorable maturity

Deep seawater temperature group
Favorable maturity

Unfavorable maturity

Stage 1 Stage 2 Stage 3

Stage 4

Stage 5

Stage 1 Stage 2 Stage 3

Stage 4

Stage 527-May

0.00

0.07

0.00

0.00

0.07

0.00

0.71

0.21

0.71

0.21

1-Jul

0.00

0.50

0.50

0.00

0.00

0.00

0.67

0.33

0.00

0.00

27-Jul

0.00

0.00

0.22

0.78

0.00

0.00

0.29

0.43

0.14

0.14

31-Aug

0.17

0.00

0.00

0.33

0.50

0.10

0.00

0.70

0.20

0.00

27-May

0.00

0.71

0.21

0.07

0.00

0.00

0.71

0.21

0.07

0.00

1-Jul

0.00

0.20

0.60

0.20

0.00

0.00

0.33

0.50

0.17

0.00

27-Jul

0.00

0.00

0.30

0.70

0.00

0.00

0.27

0.45

0.27

0.00

31-Aug

0.00

0.00

0.00

0.33

0.67

0.00

0.20

0.40

0.20

0.20

27-May

0.00

0.38

0.38

0.25

0.00

0.00

0.38

0.38

0.25

0.00

1-Jul

0.00

0.00

0.38

0.63

0.00

0.00

0.43

0.57

0.00

0.00

27-Jul

0.00

0.00

0.00

1.00

0.00

0.00

0.08

0.77

0.15

0.00

31-Aug

0.00

0.00

0.00

0.75

0.25

0.00

0.00

0.60

0.40

0.00

27-May

0.00

0.38

0.38

0.25

0.00

0.00

0.38

0.38

0.25

0.00

1-Jul

0.00

0.00

0.50

0.40

0.10

0.00

0.00

0.80

0.20

0.00

27-Jul

0.00

0.00

0.00

0.71

0.29

0.00

0.00

0.67

0.33

0.00

31-Aug

0.00

0.00

0.00

0.50

0.50

0.00

0.00

0.53

0.33

0.13



159

Maturation Control of the Short-Spined Sea Urchin
Improvement in quality of short-spined sea urchins
reared under low temperature conditions; the former had the advantage
of low price and ready availability, while the later
was the primary food plant for wild

In general, the sea urchins with larger-sized unripe

and was flourishingly cultured as food in Rausu

gonads are commercially preferred for consumption.

(Kayaba

Therefore, for aquaculture of sea urchins, it is alsowith four feeding regimes at deep-sea water., 2012).

were reared

important to determine the rearing condition bytemperatures from June 1 to September 1 in 2009.

which gonads grow faster to a commercial size. For

In the first group, the sea urchins were reared

echinoderms, it has been established that nutritive

without feeding during the experimental period

condition is closely related to gonadal development.

as a control. In the second and third groups, theNutrients derived from ingested food are stored in

sea urchins were fed a surplus of dry wakame

nutritive phagocytes inside the ovary and testis, andfrom June 1 to September 1 (feeding for 3 months)

are utilized for production or growth of germ cells

and from August 1 to September 1 (feeding for(Unuma, 2002; Walker., 2006). In the rearing

1 month), respectively. In the fourth group, theexperiment mentioned above, obvious increases in

sea urchins were fed an unlimited amount of live

gonad volume were observed only in the sea urchinsbrown macroalgae,

reared with feeding: the gonad indexes (GIs) of

September 1 (feeding for 1 month). In comparing

from August 1 tosea urchins reared with feeding were significantly

GIs from each treatment, when sea urchins werehigher than those without feeding (Fig. 3). This

fed live

result indicates obviously that rearing with feeding

three times as fast as that when they were fed dry

under low temperature conditions is the best

wakame (Table 2). The daily food consumption forway to accelerate the accumulation of nutrientseach diet was almost the same, suggesting that forwithout proceeding gametogenesis in gonads of.
Accordingly, as a next step, we examined the, the growth rate of gonads wasmay be easier to assimilate.
Several feeding experiments have shown that theare the most nutritionally valuabledietary effects of two available diets in Rausu,

algae among several algae distributed along thecommercial dry wakame and live brown macroalgae,

coast of Hokkaido for promoting gonadal growth andFig. 3. Changes in the gonad index (GI) and histological structures ofreared at deep
seawater temperature under different feeding regimes during the experiment performed in 2009.
Values represent the mean ± standard deviation (based on data from Kayaba., 2012).

Takaaki KAYABA, Kohji TSUJI, Hiroshi HOSHIKAWA, Yaoki KIKUCHI, Kazuhiro KAWABATA,
Isao OTAKI, and Toru WATANABE

160

Table 2. Changes in the gonad index (GI) of
reared with four feeding regimes at low
temperatures during the experiment performed in 2009 (based on data from Kayaba., 2012).
Values represent the mean ± standard deviation. Mean values with different letters are significantly
different at the 0.05 level within each sampling day. (GIs were arcsine-transformed before analysis andreported as back-transformed values. When a treatment effect was significant, Tukeyʼs multiple range
comparison test was applied to identify diﬀerences among experimental groups.)

3
4
Dry wakame Live 6GLDEROLFD
1 month
1 month
1 Aug 䠉 1 Sep 1 Aug 䠉 1 Sep

1
Non-feeding䠉䠉

2
Dry wakame
3 months
1 Jun 䠉 1 Sep

27-May (Initial)

7.9 ± 0.7

7.9 ± 0.7

7.9 ± 0.7

7.9 ± 0.7

1-Jul

8.5 ± 0.8

9.7 ± 1.1

8.7 ± 0.8

8.7 ± 0.8

27-Jul8.4 ± 0.8 E

11.3 ± 0.8 D

8.2 ± 0.8 E

8.4 ± 0.8 E

20-Aug7.4 ± 1.9 F

14.1 ± 2.2 D

10.7 ± 0.9 E

9.9 ± 0.5 E

31-Aug7.4 ± 0.7 F

16.3 ± 1.4 D

11.0 ± 1.4 E

15.6 ± 1.6 D

7HVWJURXSSampling
date'LHW)HHGLQJWHUP)HHGLQJSHULRG

Fig. 4. Eﬀect of diet on the free amino acid content of gonads inreared under diﬀerent
feeding conditions. Wild: wild sea urchins captured from the fishing ground in Rausu,: seaurchins reared and fed live
for one month, Wakame: sea urchins reared and fed commercial
dry wakame for one month
improving the taste of gonads in the northern sea

comparing with those fed dry wakame (Fig. 4).

urchinFurthermore, tasting test proved that the taste, a sub-arctic species

like the(Agatsuma

Nabata., 1999). Accordingly, as demonstrated

in our experiments withbe possible, by feeding with live., 1993;, it may, toof gonads in sea urchin fed with live
were very preferable to customers (Fig. 5). These
results clearly indicate that live
essential diet for rearing

is an
to not only

promote gonadal growth efficiently even under

enlarge their gonad volume, but also to improve thelow temperature conditions, while suppressing thequality of gonads. Generally, it has been suggested

progress of gametogenesis.

that the largest limiting factor for the success ofMoreover, we also investigated the characteristicof taste in sea urchins reared with feeding liveby chemical analysis (free amino acids)

sea urchin aquaculture on a commercial scale isthe difficulty of obtaining sufficient macroalgae forfeeding. Fortunately, in the cultivation ofor tasting test directed to tourists in Rausu. As ain Rausu town, a large amount of surplus algae

result, the gonads of sea urchins fed live

is produced every early summer by thinning tocontained in rich amount of glutamic acid ﬂavoring

accelerate its growth. It will be capable of producing“Umami taste” and glycine flavoring “Sweetness” ,commercially viable sea urchins sustainably and at a

161

Maturation Control of the Short-Spined Sea Urchin

Fig. 5. A questionnaire to ask the tourists visited to Rausu in August 2010, forinvestigation of the practicability of low-temperature rearing system in.

Fig. 6. The best practical use ofin Rausu, with the aim of extending market season.

lower cost by using this source of unutilized algae as

extending the harvest season of sea urchins reareda diet.

with deep-sea water two months longer than that of
the wild population, and proved that serving them
In conclusion and future prospects

during the summer tourist season was certainly
possible. The best practical use of

Our present study was initiated to suppress thegametogenesis of unripe

capturedin Rausu would include the following steps: 1) alarge number ofare caught frombefore the spawning season by low temperature

the wild toward the end of the ﬁshing season (laterearing using deep-sea water. We succeeded inJune), 2) sea urchins are reared in tanks supplied

162

Takaaki KAYABA, Kohji TSUJI, Hiroshi HOSHIKAWA, Yaoki KIKUCHI, Kazuhiro KAWABATA,
Isao OTAKI, and Toru WATANABEwith deep-sea water to suppress their maturation

for Application in the New Policy of Agriculture,

until harvest, and 3) sea urchins are fed mainly on

Forestry and Fisheries,” funded by the Ministry of

live

Agriculture, Forestry and Fisheries, Japan.

for one month before harvest to

improve gonadal growth (Fig. 6). During August,the peak of the summer tourist market in Rausu,

Referencethe probability of harvesting sea urchins suitable for
consumption is approximately 70% per total reared

Agatsuma Y., Nakata A., and Matsuyama K., 1993:

sea urchins. Further detailed studies concerning the

Feeding and assimilation of the sea urchin,rearing conditions of sea urchins would increase thatprobability in the future.

., 40,At present, this aquaculture method is being put

21‑29. (in Japanese with English abstract)

to practical use by Rausu Fisheries Cooperative.

Agatsuma Y., Matsuyama K., Nakata A., Kawai T.,

There is a plan of serving sea urchins at seafoodand Nishikawa N., 1997: Marine algal succession

restaurants, hotels, and summer festivals that areon coralline ﬂats after removal of sea urchins invisited by many tourists, and they are expected toSuttsu Bay on the Japan Sea coast of Hokkaido,

be a new, attractive food item which will help toJapan.

promote the popularity of “Shiretoko” as a tourist

Japanese with English abstract), 63, 672‑680. (indestination. Should this trial be successful, it will

Böttger S. A., Devin M. G., and Walker C. W., 2006:

provide a good case for presenting the potential

Suspension of annual gametogenesis in greenof sea urchin aquaculture to expand not only local

sea urchins experiencing invariant photoperiodﬁshery production, but also tourism in the local area.

Applications for land-based aquaculture.

Recently, the extent of barren grounds has, 261, 1422‑1431.

increased in coastal areas around Hokkaido. An

Coppard S. E., and Campbell A. C., 2005: Lunar

excessive grazing of seaweeds by wild sea urchins

periodicities of diadematid echinoids breeding in

is suspected as one of cause of this phenomenonFiji.

, 24, 324‑332.

(urchin-dominated barren ground, called “uniyake”

Fuji A., 1960: Studies on the biology of the sea

in Japanese) (Fujita, 2008). To recover the luxuriant

urchin. I. Superficial and histological gonadal

growth of seaweed, large numbers of sea urchinschanges in gametogenic process of two sea

have been removed from these barren grounds andurchins,

destroyed, because they have poor gonads due toa shortage of food and have no commercial value(Agatsuma., 1997; Kuwahara.,
11, 1‑14.

., 2010). The

Fujita D., Kuwahara H., Watanuki A., Aota T., andpresent culture technique would make it possible

Yokoyama J., 2008: Kokunai no uniyake noto improve the quality of surplus wild sea urchins

genjou, in “Recovery from urchin barrens −removed from the barren ground so they areecology, fishery, and utilization of sea urchins”

preferred by customers. The technique may also(ed. by Fujita D., Machiguchi Y., and Kuwahara

become a useful method to create a balance betweenH.), Seizando-Shoten, Tokyo, pp. 14‑21. (inthe biomass of seaweed and the population of sea

Japanese)urchins in coastal ﬁshing grounds. In the future, it isHorii T., 1997: The annual reproductive cycle andexpected that this culture method will be a valuable

lunar spawning rhythms of the purple sea

tool for conserving the coastal fishing grounds inurchinHokkaido.

with English abstract).

(in Japanese

63, 17‑22.
Acknowledgment

Ito S., Shibayama M., Kobayakawa M. A., and TaniY., 1989: Promotion of maturation and spawningThis study was conducted as part of a research
project, “Research and Development Projectsof sea urchinbyregulating water temperature (in Japanese with

163

Maturation Control of the Short-Spined Sea Urchin
English abstract).

, 55,

757‑763.

deep seawater resources” (ed. by Fujita D., andTakahashi S.,), Seizando-shoten, Tokyo, pp. 1‑27.

Kayaba T., Tsuji K., Hoshikawa H., Kikuchi Y.,

Tomita K., Kishida M., Masusaki K., and Iisawa N.,

Kawabata K., Otaki I., and Watanabe T., 2012:

1984: Seasonal changes in gonado of short-spined

Effect of low temperature rearing, using

sea urchin

deep-sea water, on gonadal maturation of thecoast of East Hokkaido (in Japanese).

in theshort-spined sea urchin,, 41, 469‑

, in Rausu, Hokkaido.

., 78,

1263‑1272.

479.
Unuma T., 2002: Gonadal growth and its relationship

Kuwahara H., Hashimoto O., Sato A., and Fujita D.,

to aquaculture in sea urchins, in “The sea

2010: Introduction of isoyake recovery guidelineurchin: from basic biology to aquaculture” (ed.

(Fisheries Agency, Japan).

by Yokota., 32, 51‑60.

.), Swets & Zeitlinger, Lisse, pp.

115‑127.

Masaki K., and Kawahara I., 1995: Promotion

Yamamoto M., Ishine M., and Yoshida M., 1988:of gonadal maturation by regulating water

Gonadal maturation independent of photic

temperature in the sea urchin

conditions in laboratory-reared sea urchins,‑ I (in Japanese).

and, 4, 93‑100.

., 5, 979‑988.

McClintock J. B., and Watts S. A., 1990: The eﬀects

Walker C. W., and Lesser M. P., 1998: Manipulationof photoperiod on gametogenesis in the tropicalof food and photoperiod promotes out-of

sea urchinseason gametogenesis in the green sea urchin(Lamarck)(Echinodermata: Echinoidea).

: implications., 139, 175‑184.

for aquaculture.

., 132, 663‑676.

Nabata S., Hoshikawa H., Sakai Y., Funaoka T.,

Walker C. W., Unuma T., and Lesser M. P., 2006:

Oohori T., and Imamura T., 1999: Food value

Gametogenesis and reproduction of sea urchins,of several algae for growth of the sea urchin,(in Japanese withEnglish abstract).

in “Edible sea urchins: Biology and ecology”(Lawrence.), Elsevier, Amsterdam, pp. 11‑33.

., 54, 33‑40.
Nakasone T., and Akeda S., 2000: The application

Annotated Bibliographyof deep sea water in Japan. UJNR
, 28, 69‑75.

Unuma T., 2002: Gonadal growth and its relationship

Noguchi H., Kawahara I., Goto M., and Masaki

to aquaculture in sea urchins, in “The sea

K., 1995: Promotion of gonadal maturation byurchin: from basic biology to aquaculture” (ed.

regulating water temperature in the sea urchinby Yokota‒II (in Japanese).
, 4, 101‑107.

.), Swets & Zeitlinger, Lisse, pp.

115‑127.
The sea urchin gonad contains two main types ofPearse J. S., Pearse V. B., and Davis K. K., 1986:

cells: nutritive phagocytes (NPs) and germ cells (GCs).

Photoperiodic regulation of gametogenesis andNPs store nutrients necessary for gametogenesis,

growth in the sea urchinsuch as proteins, carbohydrates and lipids. The most., 237, 107‑118.

abundant protein in NPs is a glycoprotein called the

Sakairi K., Yamamoto M., Ohtsu K., and Yoshidamajor yolk protein (MYP), originally identiﬁed as theM., 1989: Environmental control of gonadal

most predominant component of yolk granules in thematuration in laboratory-reared sea urchin,eggs. The proportion of NPs and GCs varies withthe maturation of the gonads. Before gametogenesis,., 6, 721‑730.
Takahashi S., 2006: Deep seawater, in “Utilization of

NPs ﬁll the gonadal lumina and increase in size byaccumulating nutrients to the developing GCs and164

Takaaki KAYABA, Kohji TSUJI, Hiroshi HOSHIKAWA, Yaoki KIKUCHI, Kazuhiro KAWABATA,
Isao OTAKI, and Toru WATANABE

decrease in size. In fully mature gonads, a number

Böttger S. A., Devin M. G., and Walker C. W., 2006:of type ova or spematozoa fill the gonadal lumina,

Suspension of annual gametogenesis in green

whereas, NPs lose their nutrients and shrink. The

sea urchins experiencing invariant photoperiod

quality of the gonad as a food product usually

Applications for land-based aquaculture.

deteriorates while GCs proliferate and develop.

, 261, 1422‑1431.

Promotion of NP growth and suppression of

Sea urchin fisheries are valuable commercial

gametogenesis are prerequisites to achieving highresources in the United States with processed

proﬁtability in sea urchin aquaculture.

gonads sold in Japanese and American markets
and maximum U.S. sales of $150M US dollars in

Kayaba T., Tsuji K., Hoshikawa H., Kikuchi Y.,

1996. Wild populations of sea urchins on all coasts

Kawabata K., Otaki I., and Watanabe T., 2012:of the U.S. have been heavily fished. Aquaculture

Effect of low temperature rearing, usingof sea urchins in land-based facilities can help

deep-sea water, on gonadal maturation of therestore commercial populations and preserve thisshort-spined sea urchin,ecologically important herbivore. In this study, we, in Rausu, Hokkaido.

., 78,

1263‑1272.

used invariant summer photoperiod to prevent
gametogenesis in the North American green

It has long been hoped that sea urchins could

sea urchins ()

be served to visitors in seafood restaurants, hotels,

maintained in a land-based aquaculture system andand summer festivals in Rausu, located in a worldprovided a commercially available formulated feed

natural heritage site “Shiretoko,” during the summerthat promotes maximum growth of intra-gonadal

tourist season. However, to date this has not beensomatic nutrient storage cells called nutrient

feasible because of the sea urchin spawning season

phagocytes. Results were compared with individuals(July to September), during which the quality offed the same formulated feed under ambient

gonads, the edible part of sea urchins, decreases

photoperiod in cages in the ocean. Monthly samples

due to maturation. In this study, we examined theof the gonads from both treatments were evaluatedpossibility of suppressing gonadal maturation andfor gonad index, volume fractions of cellular

maintaining high-quality sea urchin gonads by low-

constituents of the germinal epithelium, oocyte

temperature rearing using deep-sea water. Unripe

diameter and taste. Over the 5 months of this study,

sea urchins captured before the spawning season

gonad indices increased significantly ( < 0.001) in

were reared under two temperature conditions

both treatments from 4.8% ± 0.9 (all values ± SE)from June to September. In those groups reared atinitially to 20.5% ± 2.1 under invariant and 23.2%

ambient temperatures (2.8 19.6℃), gametogenesis± 1.4 under ambient photoperiod with no signiﬁcantin both sexes progressed rapidly with increased

difference between treatments (temperature, and almost all sea urchins reached full

fractions of nutritive phagocytes increased to 80.3%

maturity by late July. In contrast, in groups reared± 5.9 (initial 37.9% ± 7.1) in males and 71.0% ± 6.7

at low temperatures (2.1 5.1℃), gametogenesis(initial 10.3% ± 4.0) in females ( < 0.001) only under= 0.55). Volume

progressed slowly and over 60% of the sea urchinsinvariant photoperiod. Nutritive phagocyte lengths

did not reach maturity even by early September.

increased under both photoperiod treatments, but

The feeding experiment also revealed that feeding

the volume fraction containing nutrients was higher

with live

could increase theunder invariant photoperiod. Volume fractions ofgonadal volume efficiently under low-temperature.

gonial/gametogenic cells increased significantly (These results demonstrate that low-temperature< 0.001) only under ambient photoperiod from 20.4%

rearing, supplemented with feeding, is effective± 5.5 to 37.8% ± 1.8 in males and 0% to 22.6% ±in suppressing gametogenesis to allow for the

3.6 in females. The volume of fraction of residual

harvesting of high-quality sea urchins during theoocytes from last yearʼs oogenesis increased undersummer tourist season.

invariant photoperiod while that of both residual andnew oocytes increased under ambient photoperiod.

Maturation Control of the Short-Spined Sea Urchin
Residual oocyte diameters increase from 56.2 µm ±2.2 initially to 93.5 µm ± 3.7 under invariant andthose of residual and new oocytes to 126.0 µm ± 7.3
under ambient photoperiod. Invariant photoperiod
yields gonads in both sexes ofthat

do not initiate fall gametogenesis but attain large
size as their nutritive phagocytes grow substantiallyin size. A Canadian study of wild-collectedindicated that gonads taste best when
they contain pre-dominantly nutritive phagocytes
and not copious gametes, however gonad taste inour study was unsatisfactory suggesting that the
only commercially available sea urchin diet requires
modiﬁcation to support commercial development ofland-based aquaculture.
Nabata S., Hoshikawa H., Sakai Y., Funaoka T.,
Oohori T., and Imamura T., 1999: Food valueof several algae for growth of the sea urchin,(in Japanese withEnglish abstract).
., 54, 33‑40.
Rearing experiments of the sea urchin,, were carried out during
June to July, 1995 and June to August, 1996, using
as food marine algae which settled and grew on
the coralline ﬂats after the removal of sea urchins.
Feeding rate and growth rate at 17℃ of the sea
urchin fed on, andwere high and those for, andwere low in the single

food item experiment. Among the algae supplied
as food, the daily amount of food eaten was highin large-sized groups of the sea urchins, while thesmall-sized groups show the highest feeding rate.
To examine any effect on gonad growth, we fed
3 algae,andto sea urchins. Two month later, the gonad index
was found to be the highest in thefedgroup. Among the algae fed, based on the gonad
growth, the highest feeding rate and the highest
growth rate, we estimated thearethe most nutritionally valuable algae for growth of.

165

水研センター研報，第40号，167−172，平成27年
Bull. Fish. Res. Agen. No. 40，167−172，2015

167

Long-term Outcomes in the Tech Transfer of Scallop Spat Collection
Techniques, from Aomori Prefecture, Japan to Maine, USA
Dana L. MORSE＊

Abstract: Improved management, aquaculture and stock enhancement of the sea scallop() in Maine are examples of long-term beneﬁts from a technology transfer

project. In 1999, a delegation from Maine traveled to their sister-state of Aomori Prefecture in
Japan to gain a firsthand view of the world-famous scallop industry of Mutsu Bay. While thetrip was informative from many perspectives, the detail with most immediate application was
the use of so-called ʻspat bagsʼ to capture competent larvae for stock enhancement and intensive
aquaculture, and the way in which rotational management could be used to maximize production.
In subsequent years, over 100 ﬁshermen in Maine used spat bags to capture juveniles, and thecurrent progress made in aquaculture of sea scallops is largely due to the excellent success incollecting spat as a reliable seed supply. Maineʼs ﬁshery is currently managed in rotation and area
closures - sometimes at the request of the ﬁshermen. Fishermen are also involved in discussions ofspat collection for the purposes of stock enhancement. In addition, spat collectors have provided aninvaluable window for ﬁshermen to observe details of this early life stage, observations that theywould not otherwise make. Overall, spat bags have been useful both directly and indirectly forwell over a decade.
Many of these outcomes were not foreseen during the visit of 1999. Technology transfer often
aims for immediate impact, but it seems likely that the normal case is that impacts and beneﬁts
may take many years to be observed. It is important to keep this in mind both in reportingto funders of various tech transfer efforts, but to appropriately gauge the long-term value ofcontemporary eﬀorts.
Keywords: aquaculture, enhancement,

Maineʼs fishery for the sea scallop () has traditionally been an important, technology transfer

limited, with no limit on the number of licenses, anda 3.5” (88.9mm) minimum shell height. Prompted by

option for the stateʼs fishermen; it was an open-

low landings and stimulated by examples of better

access fishery until recently, and provided much-management - such as discussed below - the Maine

needed wintertime income. Direct on-the-water

Dept. of Marine Resources engaged fishermen andemployment in the 2012/2013 ﬁshing year amounted

scientists in a process to improve the landings andto 547 mobile-gear licensees (draggers), and 84 divers

value of the fishery. Since then, several measures(Trish DeGraaf, ME Dept. of Marine Resources, pers.

have been adopted, such as a larger minimum shellcomm.). Landings have varied widely, from a historicheight of 4” (101.6mm), limited ﬁshing days, a daily

high of 3.8 million pounds in 1981, to a low of 33,000

landing limit, rotational management, and closed

pounds in 2005 (Fig. 1). Only the adductor muscle,

areas, and to date, landings have improved (Fig. 1).

or meat, is landed.
Prior to 2007, regulation of the ﬁshery was fairly

The process of industry engagement and inclusion
into the management process was not always

2015年 1 月30日受理（Received on January 30, 2015）＊
Maine Sea Grant College Program and UMaine Cooperative Extension, 193 Clarkʼs Cove, Darling Marine Center, Walpole, ME 04573 USA
E-mail: dana.morse@maine.edu

168

Dana L. MORSE

Fig. 1. Maine annual landings of scallop adductor muscles (meat).
smooth: the issues were highly contentious, andat one point of the year or other; and competition

the bringing-together of industry with science andin the marketplace. A few small trials have yielded

management meant that there was a good deal ofgenerally positive results though with several

learning to be done, toward and from one another.

important caveats: work by Pottle and Hastings

Conversely, it appears that these interactions have(2001) and Kuenstner (1996) - Maine polyculture

begun to pay dividends for all sides: management isstudy demonstrated that scallops would growmore aware and responsive to industry perspectives,

relatively quickly. The Pottle study also showedindustry is more fluent in the science of scallopthat whole scallops were a marketable item, but

management and ecology, and science is being

Kuenstner and Pottle both demonstrated the hurdles

conducted on issues important both to fishermen

posed by algal toxins (saxitoxin, okadaic acid andand regulators.

domoic acid, principally) in selling products other

Before the period reported in this document - 1999

than just adductor muscles.

a n d o n w a r d - en g ag emen t b y fish erme n and

Attitudes toward aquaculture have changed

scientists in enhancement and aquaculture

somewhat for many fishermen, however, with aproduction was extremely limited and variable,

major reason being the limited opportunities oﬀered

with little in the way of planning or broader-scaleon the water in recent decades: limited access tothinking. Fishermen traditionally have experimented

licenses or resources have generally put ﬁshermen ʻinwith enhancement through such actions as moving

a boxʼ where moving through several ﬁsheries in theseed - meaning sublegal scallops, usually larger

course of the year has been supplanted by accessthan 2” (5cm) - to a location they felt was protected

to only one or two fisheries, and therefore highlyor productive, or removing predators like crabs

dependent upon them. The second reason concernsand starfish when they came up in the drag.

the subject of this paper: technology and information

Enhancement with seed smaller than 2” was verytransfer becomes integrated in the knowledge,

limited, and none performed in a structured fashion.

perspectives and ideas of the people who participate,

Scallop aquaculture has historically been opposedand can support an adaptive stance toward current

by many fishermen for two principle reasons:occupation of space that would normally be fishedand future challenges.

Technology Transfer of Scallop Spat Collection from Japan to USA

169

Materials and Methods
Maine enjoys a sister-state relationship with thePrefecture of Aomori, Japan; the northernmost
region of the island of Honshu. Delegations havepassed with some regularity between Maine andAomori, addressing such interests as energy,
affordable housing, and fisheries. One suchdelegation traveled to Aomori in May of 1999, withthe focus of learning about the highly-productive
scallop industry there, one which has itsʼ basis inaquaculture techniques, and one that has become aworldwide model of financial and social success. It
was hoped that the group could identify aspects of
the Japanese industry that would transfer well to

Fig. 2. Citizen environmental group aboard the F/V
Lindsay Marie (Stonington, ME) to assist and learn
about scallop spat collection and stock enhancement.

the scallop ﬁshery in Maine.
Between the 14th and 21st of May, ten representatives
from Maine toured scientific facilities, processing
plants, waterfront infrastructure, seafood markets,a fishermenʼs cooperative, and sea farms. Not
unexpectedly, the delegation was impressed at thediversity, volume and vigor of the farming industry
there. A brief list of conditions, opportunities andapproaches that could potentially be beneficial toMaine ran as follows:‑ A diverse suite of products from scallops would
be beneficial for Maine, rather than just deriving
income from the adductor muscle.
‑ Spat collection equipment and practices could
be useful to stock enhancement and aquaculture

Fig. 3. Spat collections in 1999‑2000 (left) and 2000‑
2001 in coastal Maine. Note that in the second year,
most attempts were made further oﬀshore becauseof prior success there: ﬁshermen adapted rapidly to
new knowledge.

in Maine. Techniques and equipment are directlytransferrable.

collection areas, transferring captured seed to the‑ Collaboration with and support by scientists

wild, and educational connections with citizens

is beneficial to all involved; good monitoring forand environmental groups (Fig. 2). As expected,

biotoxins is criticalfishermen quickly proved their expertise, and‑ There is no diﬀerence between ﬁsherman and sea-

discovered that best spat collections were notfarmer in Japan: sea farmers are ﬁshermen.

done in the immediate coastal waters, but further

A full report was delivered to the Maineoﬀshore, near the limits of state jurisdiction (Fig 3).

Aquaculture Innovation Center, detailing all aspects

In these areas, counts were easily above 1,000 seedof the trip (Bealper collector, and frequently as high as 3,000. Many., 1999).

In the years following the visit, several activities

areas were found to result in collectors catchingand projects were conducted in Maine, in an eﬀort

nearly exclusively scallops and with low foulingto build upon the knowledge gained.

rates, whereas others yielded low scallop catch,

Spat Collection: From 1999 to 2002 several meetingshigh bycatch of other bivalves, and heavy fouling.

were held around the state, to familiarize ﬁshermen

It is estimated that between 1999 and 2002, overwith spat collection equipment and methodology.

100 ﬁshermen had participated in some level of spat

The emphasis was on searching for good spatcollection.

170

Dana L. MORSE

Perhaps more importantly, fishermen found that

oceanography, predation, conservation, water quality

spat collectors are most successful when they are

and other topics, all of which entered the ensuingobserved regularly: the coast of Maine is heavily

discussions about management of the fishery andfished for lobster, and entanglements with lobster

the scallop resource. Similarly, spat collectors also

trap lines were common, unless the collector lines

spurred fishermenʼs interest in aquaculture: seeing

were tended. This was an important observation for2000 small scallops in a spat collector immediately

the co-existence of spat collection with the lobster

led them to consider what it would be like to raiseand other coastal ﬁsheries. It was also during thesethem in cages. Both of these phenomena haveyears that the inclusion of spat collection, stock

played a large role over time in the evolution ofenhancement and aquaculture became better known

scallop management in Maine, and the beginnings ofto the state regulatory agency - the Maine Dept. of

an aquaculture sector.

Marine Resources (DMR); this familiarity has proven

Aquaculture: The investigation of sea scallops as a

to be very beneﬁcial in the long run.

candidate for aquaculture in Maine has been a story

Stock Enhancement: Stock enhancement efforts inof stops and starts. From 1999 to 2000, a trial ofMaine can be generally characterized as haphazard,

cage culture for scallops was undertaken by Tom

with a few exceptions. Eﬀorts by the ME DMR and

Pottle, who was one of the delegates from the 1999

the Northwest Atlantic Marine Alliance (NAMA)

trip, and a ﬁsherman from the Cobscook Bay region.

were well coordinated, but of limited scope. PrimaryHis project successfully demonstrated reasonablelessons from those efforts - which were both done

growth rates, useful equipment and husbandrywith excellent collaboration between science andpractices, and positive feedback from test sales into

industry - were to reinforce the highly mobile nature

the marketplace (Pottle, 2001). It also underscoredof juvenile scallops, and the supposed high predation

the importance of thorough testing for toxic algae,

rates on seed less than 20mm or so (Schick andand presence of algal toxins in scallop tissues. Mr.

Feindel, 2005; Deese-Riordan, 2005). Unfortunately,

Pottle did not continue his project past the fundedthese were general observations, and not thoroughly

time frame, though has remained optimistic about

described in the detail desirable, due to limited

the chances for scallop aquaculture in Maine.

ability to dive on the reseeded sites with thefrequency necessary for such detail.

From 2000 through approximately 2010, scallop
aquaculture was at a virtual standstill in Maine; theOther smaller eﬀorts in stock enhancement wererisks associated with sales of live or roe-on productundertaken between 1999 and 2004; often amounting

were prohibitive for state regulators. In recent years

to a fishermen taking the spat from his collectors

however, conversations between industry and theand deploying it in a favorite spot. The benefits

regulatory community have increased, with bothof these activities have remained anecdotal andsides joining in an effort to explore the possibility

uncertain. The uncertainty over the success of stock

for sales of roe-on or live/whole product. A current

enhancement led to the gradual decline of ﬁshermen

project involves five pilot scallop farms, withparticipating in spat collection. From roughly 2004

excellent growth observed thus far (up to 0.19mm/

and onward, only a handful of fishermen per yearday), and low mortality (Fig. 4). Conversations

participated; this area of study remains to this day

continue regarding the protocols necessary to

one where industry interest would align well withguarantee product and public safety - but they are

scientiﬁc interest.

occurring and all parties have felt optimistic for

However, one area that has been of obvious

future development.

benefit is the way in which spat collectors have

To date, ﬁrst sales of live scallops are anticipatedallowed fishermen to observe and understand thein 2014, accompanied by sales of seed from spat

earlier life phases of scallops; a subject that hascollectors to new growers. Project details located at:been lightly discussed or considered previously.

http://www.seagrant.umaine.edu/research/projects/

Spat collection led to conversations amongst

dv/scallop-trials

fishermen and with scientists or managers about

Technology Transfer of Scallop Spat Collection from Japan to USA

171

items come to mind. The Aomori visit supported
an improved understanding of biology and ecology
of scallops, compared to the level of understanding
prior, particularly by industry. By focusing on the
deployment of spat collectors and observing thecatches, fishermen started to better understandimportant factors such as proximity of spawners
to one another, the influences of oceanographic
conditions (currents, tides, temperatures, etc) on
larval dispersal and settlement, and in particular,
the challenges faced by very young post-set
scallops. In fact, the simple identification of young
scallops improved a good deal: ﬁshermen have often
mistaken so-called ʻjingle shellsʼ ()

as young scallops; spat collectors have allowed
fishermen access to the very smallest sizes, suchthat identiﬁcation is now much more accurate - andquestions have naturally followed those observations.
The concepts of rotational management have been
reinforced; many had focused on the managementof oﬀshore scallops on Georgeʼs Bank as the primaryFig. 4. Scallops growing in a cage during the pilot
project; Blue Hill, ME

example, but the Japanese example has figured
into management discussion. Today, Maineʼs
inshore scallop fishery is managed in rotation, in

Results and Discussion

some measure due to a changed way of thinking
by fishermen, scientists and managers resulting

Technology transfer clearly yields both short-from the Aomori visit (Fig. 5). Other issues suchterm and long-term results. While it may be possible

as mandatory stock enhancement and creation of

to anticipate or predict the near-term beneﬁts andspawner sanctuaries have been included in theactivities that stem from a technology transfer

management discussion and although not included

activity, real predictions over the longer term arein regulations, have been a noteworthy step aboutmuch less achievable, other than to say that they

how fishermen and managers have discussed thewill exist. Documenting such developments is a

responsibilities and possibilities in supporting aworthwhile endeavor however, because of the local

healthy resource and ﬁshery.

relevance but also because of the larger implications

Production remains an area where the Aomori

for future efforts: funding for travel is usually

visit has had a profound inﬂuence, all starting with

diﬃcult, and when good documentation is available

the technology of spat collection. Stock enhancementon the value of such travel, it improves the rationale

continues to gather support from fishermen whenfor proposals involving travel to support technology

the subject arises, and itʼs likely that more worktransfer. Therefore, while itʼs very difficult towill be done in this area, and with greater scientiﬁc

anticipate long-range outcomes, it is of value to note

scrutiny than before, which would permit a better

them as they do become apparent.

evaluation of eﬀectiveness. An aquaculture industry

In the case of the 1999 trip to Aomori, threehas begun to develop in the state as well withprincipal areas of impact can be easily identified.

small sales of the ﬁrst crop (and scallop seed from

These topics - which are closely linked - are: biology

collectors) expected later in 2014. Moreover, theand ecology, management, and production.

tenuousness of the lobster, shrimp and groundfish

In the category of Biology and Ecology, several

industries have made commercial fishermen more

172

Dana L. MORSE

Fig. 5. Ten-year rotational management plan for the Maine scallop ﬁshery;in this case the eastern zone.
open to the possibility of aquaculture as a way toReferences

generate income, and thus the integration of theﬁshing and aquaculture industries has a pathway for
progress, through the culture of sea scallops.

Brian F. B., (ed. and principal writer). 1999: The
culture of Japanese Scallops (The quantification of impacts is more difficult,), Report of the Maine Delegation tothough not impossible. Landings for scallops have

Aomori Prefecture, Japan, May 14‑21, 1999. The

increased in recent years, and surveys of theMaine Aquaculture Innovation Center, Orono,

areas closed as part of the rotational management

Maine, USA.

approach have provided strong indications that

Deese-Riordan H., 2005: Saco Bay Scallop Stockmanagement plays a large role in the improvements.

Enhancement Project. Final Report to the

Therefore, landings and licenses are two metrics

Northeast Consortium. Project Developmentthat can be easily identified. As the aquaculture

Grant #03‑194. 31p.

industry continues to advance, it is straightforward

Kuenstner S., 1996: Polyculture of sea scallops

to record economic activity and the number ofsuspended from salmon pens. New England

farmers as measures of success. These data are

Fisheries Development Association, ﬁnal reportimportant in the sense of tracking the inﬂuence ofto the NOAA Saltonstall-Kennedy Program.

the original information.

Project No. 93-NER-009, Grant No. NA46FD0327.

The natural complement to the numbers

91p.

themselves (sales, licenses, leases, etc) is the use of

Pottle T., and Hastings M., 2001: Sea Scallop

anecdotes to tell the story of what happened over

Demonstration Project. Final Programmatictime. When taken together, the quantiﬁable data andReport, National Fish and Wildlife Foundation.

the qualitative assessments, the stories can create

10p

an understandable and informative landscape of

Schick D. F., and Feindel S. C., 2005: Maine Scallop

the value of technology transfer in both the shortFishery: Monitoring and Enhancement. Final

and long terms. In fact, both are necessary to fully

Report to the Northeast Consortium, September,

understand the impacts. In the case of the Aomori

1, 2005. Award No. 013‑138‑2051‑30. 73p.

trip of 1999, the payoffs became apparent rapidly,
and it appears that beneﬁts will continue well into
the future.

水研センター研報，第40号，173−177，平成27年
Bull. Fish. Res. Agen. No. 40，173−177，2015

173

Oyster Culture in Hokkaido, Japan
Natsuki HASEGAWA＊1, Toshihiro ONITSUKA＊2, Satoru TAKEYAMA＊3,and Kimihiko MAEKAWA＊4
Abstract: The Pacific oyster,is the one of the most commercially importantaquaculture species in the Japanese ﬁsheries industry and is cultured in various Japanese coastal
areas including Hokkaido. The Saroma Lake and the Akkeshi Bay with estuary, which face
Ohotsuku Sea and Paciﬁc Ocean, respectively, are major oyster production areas in Hokkaido withtotal annual production of about 700 tons a year. The seedling spat supplied from Miyagi known as
Miyagi seedlings are widely used in Japanese oyster culturing. Therefore, when the catastrophic
tsunami on March 11, 2011 damaged the Miyagi fisheries, many oyster culturing areas wereheavily affected. Moreover, introduction of seedlings from geographically separated population
have risks of invasion of diseases and alien organisms as hitchhiking species. Using seedlings thatoriginated from local populations in each area is one of the approaches for decreasing some risks.
For example, in the Akkeshi area, the artiﬁcial seedling spats collected from the locally protected
adults are also used for aquaculture, which are marketed as the value-added oysters with shell“Kaki-Emon” and popular among consumers as local special products.
Key words: Paciﬁc oyster (The Pacific oyster,), Miyagi seedlings, Saroma Lake, Akkeshi Bay

is the one

catastrophic tsunami on 2011 damaged the Miyagiof the most commercially important aquacultureﬁsheries, many oyster farmers were heavily aﬀected

species in the Japanese fisheries industry withbecause of uncertainty of seedling supply (Tanabe,

harvest of around 200, 000 metric tons a year, the2012)same as the Japanese scallop

Hokkaido, a northern island of Japan is one of the(Ministry of Agriculture, Forestry and Fisheries,

premier oyster culturing areas with production of

Ministerʼs Secretariat, 2012). Although a lot of

700 metric tons a year (Marinenet Hokkaido; http://

scallops were exported to other countries includingwww.fishexp.hro.or.jp/marineinfo/internetdb/index.

the United States, most oysters are consumedhtm), using primarily Miyagi seedlings. Hokkaido

domestically. Oyster culturing in Japan is reviewed

has two main production areas, Saroma Lake andby Inui (2013). The Oyster is cultured using raft or

Akkeshi Bay and estuary (Fig. 1). Saroma Lake andlong line hanging method. Hiroshima and Miyagi

Akkeshi Bay face towards the Ohotsuku Sea and theprefectures are the main production areas in Japan,

Paciﬁc Ocean, respectively. These areas have similar

and there are also many other production areas

production scales and Oyster industry history.

along Japanese coastal area. Most of these areas

the early days in Hokkaido natural oyster bedsIn

use oyster seedlings which are naturally collected

were very productive in these areas and the oysters

in Miyagi using scallop shell collectors and are

were harvested from there (Inukai and Nishio, 1937).

known as Miyagi seedlings. Therefore, when the

However, natural oyster resources were lost around2015年 1 月30日受理（Received on January 30, 2015）＊1
National Research Institute of Aquaculture, Fisheries Research Agency, 422‑1, Nakatsuhamaura, Minamiise, Watarai, Mie, 516‑0193, Japan
E-mail: hasena@fra.aﬀrc.go.jp＊2
Hokkaido National Fisheries Research Institute, Fisheries Research Agency (Kushiro Laboratory), 116 Katsurakoi, Kushiro, Hokkaido, 085‑
0802, Japan＊3
Akkeshi Oyster Hatchery, 1‑1, Wakatake, Akkeshi, Hokkaido, 088‑1118, Japan＊4
Aquaculture Cooperative of Saroma Lake, Sakaeura, Tokoro, Hokkaido, 093‑0156, Japan

174

Natsuki HASEGAWA, Toshihiro ONITSUKA, Satoru TAKEYAMA, and Kimihiko MAEKAWA

Ohotsuku Sea

Oyster culturing
Scallop culturing

Saroma Lake

Ohotsuku
Sea

HokkaidoPaciĮcOcean

Miyagi

Akkeshi-ko
Estuary
Hiroshima
3

2

1

4
Akkeshi BayPacŝĮcOcean
5 km

Fig. 1. Main Oyster culturing areas in Hokkaido, Japan.
the 1930s and since then the oyster industry has

Saroma Lake in Hokkaido where production reaches

relied on Miyagi seedlings.

150,000 metric tons a year. Most areas of Saroma
Lake are utilized for scallop culture and unsuitable

Features of oyster and scallop culturing inareas for scallop culture due to shallow depth

Saroma Lakeand high water temperatures during summer are

Ground and suspended culture of scallops are theculture is a major fishery (> 77% of total catch),main ﬁsheries in the Abashiri Sub prefecture withhowever, most culture activities must be stopped

used for oyster culture (Fig. 1). In the lake, scallop

175

Oyster Culture in Hokkaido, Japan
during winter due to lake freezing. Oyster ﬁsheries

Culture experiments using single-seedling oysters

contribute a little (< 1%) to increase the diversity ofshowed variations of growth rate and nutritionalﬁsheries and reduce dependency on scallop culture,

condition index between oysters from the Bay andbut oyster culturing brings jobs to farmers in winterthe Estuary. The ratio of ﬂesh wet weight to wholesuch as shucking because oysterʼs in this area arebody weight of oyster, which is one of the indices ofmainly composed of age 1+ and so small oysters

oyster nutritional condition was lower in the estuary

are shipped as a shucked product. Moreover, older

than the bay throughout the experiments (Fig. 3a

fishermen have special feeling for oyster culturing

and b). The speciﬁc growth rate of shell height was

because oyster fishing had been prospered in thehigher in the Estuary than the Bay in early summer

lake until 1928 when the continuous channel was(Fig. 3c and d). The early increase in temperature

opened between Lake and cold Ohotsuku Sea andfrom spring to summer enhances oyster growth

the oyster could not reproduce in stable (Nishihama,and maturation and early spawning occurs in the1994).

Estuary. These features of both culture areas arerecognized by farmers and efficiently utilized. The

Features of oyster culturing in Akkeshi Bay

ﬁshermen use the Estuary as the warmer area forand Estuary

enhancing oyster growth and maturation with early
spawning. Otherwise, the Bay has a greater surface

The most important feature of Akkeshi is thearea, water volume, and food availability than

topography (Fig. 1), Akkeshi has two different

the Estuary, which contributes to improved body

ecosystem types; Akkeshi Bay and the Akkeshi-

conditions of oysters before shipping seasons.

ko estuary. Akkeshi Bay faces the Pacific Ocean

Therefore, ﬁshermen use these two areas properly,

where a cold subarctic ocean “Oyashio” current

and Akkeshi oysters have some advantages to otherﬂows, while the Estuary is shallow (average depth:

culturing areas. But carrying capacity is limited by

1.5 m). The continuous measurement of surface

the topography of the shallow and small estuary. To

water temperature shows that the temperature

avoid this problem, single-seed oysters are cultured

in this Estuary increases earlier than in the Bay

in Akkeshi, which are suitable for farming in shallow

from spring to summer, reaching temperaturesareas. In the hatchery, larvae are collected from local
adult spawns and settled on small oyster shell pieces

in the Bay is around 20 ℃ in late summer (Fig. 2).

individually as single-seedling oysters. The juvenile

Water temperature（℃）

higher than 25 ℃. However, water temperature

J

J

A

S

O

N

Fig. 2. Fluctuation of water temperature at Akkeshi Bay and Estuary, Japan during 2012.

176

Natsuki HASEGAWA, Toshihiro ONITSUKA, Satoru TAKEYAMA, and Kimihiko MAEKAWA

40
Flesh body weight/ Total weight

35

（a）

（b）

30

Bay center25

Bay harbor

20

Estuary center

15

Estuary recesses

Specific growth rate（% d -1）

10
4.0
3.0

（c）

（d）

2.0
1.0
0.0

J A S O N D
2011

J
A
2012

S

O

Fig. 3. Temporal and spatial variation in ratio of fresh wet weight to total weight to whole
weight (a and b) and speciﬁc growth rate of shell height (c and d) ofin Akkeshi
Bay and Estuary, Japan.
oysters are cultured in the bay and the estuaryusing baskets. By using basket rearing, more oysters
can be cultured in shallow areas such as Akkeshi-ko
estuary. And basket hanging keeps oysters in thewater column away from pollution on the estuary
bottom. Moreover, through basket hanging, singleseed oysters have deeper shell breadth than oysters
on scallop shells. These oysters are marketed asvalue-added oysters with shell “Kaki-Emon” and are
popular among consumers as local special products(Fig. 4). Akkeshi town is aiming to be known as“Oyster town” using single-seed oysters to promote
tourism and sightseeing, and to develop brandstrength of other fisheries products. However, theyield of single-seed oysters was lower than thatof Miyagi-seedling oysters at the beginning of thisculturing, because the standard culturing methods
for Miyagi seedlings were not suitable for single-seedoysters. Therefore, the culture of single-seedlingoysters does not increase as much as the researchand promotional organizations expected, even today.

Fig. 4. PR poster of local premium oyster in
Akkeshi “Kaki-Emon”.

177

Oyster Culture in Hokkaido, Japan
Problems facing oyster culturing in Hokkaido

Research “Analysis and future prediction of coastal
ecosystem” provided by the Hokkaido National

Miyagi-seedling has been widely used in Japanese
local production areas including Hokkaido because

Fisheries Research Institute, Fisheries Research
Agency of Japan.

most of the seeding are naturally collected using
scallop shells with low costs and a stable source

References

provided by the specialized farmers in Miyagi
prefecture. These Miyagi oysters have faster

Inui M., 2013: Enclopedia・Oyster culturing in Japan.

growth potential, and thus are preferred by many

Suisanshinkou, 544, Tokyo Fisheries Promotion

oyster farmers. Even though there is wide-scale use

Foundation, Tokyo, 120pp. (In Japanese)of Miyagi-seedlings throughout the area, genetic

Inukai T., and Nishio S., 1937: A limnological study

differences can be detected among some local/of Akkeshi Lake with special reference to thewild populations in Japanese local production areas

propagation of the oyster.

(Usuki., 2002). The tsunami in 2011 exposed therisks of excessive dependence on Miyagi seedling,40, 1‑33. (In Japanese)

supplies. Moreover, introduction of seedlings from

Itoh N., Oda T., Ogawa K., and Wakabayashi H.,

geographically separated populations have risks

2002: Identification and development of aof introducing diseases and alien organisms asparamyxean ovarian parasite in the Pacifichitchhiking species. For example, invasive ascidians

oysterwere also recorded in some bivalve culturing areasin Japan. In particular,, 37, 23‑

28.

strongly

Matsuyama Y., Nishitani G., and Nagai S., 2010:

aﬀect the scallop culturing in Funka Bay, Hokkaido

Direct detection of harmful algae from the(Nishikawa., 2014). The paramyxean parasite,

oyster spat and live fish transporting trailer, has negative impacts

Proceedings of 13th International Conference onon the oyster industry in some Japanese production
areas with heavily infected oysterʼs showing
abnormal tissue (Ito., 2002). Moreover, theHarmful Algae, 185‑189.
Ministry of Agriculture, Forestry and Fisheries,
Ministerʼs Secretariat 2012: Annual report ofpotential transport of harmful algae together withcatch ststistics on ﬁsheries and aquaculture (Inbivalves has also been reported (Matsuyama

Japanese).,

2010). Using local seedlings is one of the approaches

Nishihama Y., 1994: Scallop fisheries in Ohotsuku

to decreasing these risks. After the Miyagi seedlingSea. Hokkaido University Press, Sapporo 218p.

crisis, the use of local populations is beginning inNishikawa T., Oohara I., Saitoh K., Shigenobu Y.,

some production areas. However, similar to the

Hasegawa N., Kanamori M., Baba K., Turonexperience in Akkeshi, it is necessary to improveX., and Bishop D. D. J., 2014: Molecular andculturing techniques and management for culture

morphological discrimination between anof local seedlings because they have different

invasive ascidian,characteristics. Moreover, sales strategies of “local

congeneroyster” with premium price are also important topromoting use of local seedlings to compensate for
additional cost and eﬀort.

, and its
(Urochordata: Ascidiacea).

., 31, 180‑185.
Tanabe T., 2012: The Natural Seed Collection of
Paciﬁc Oyster,, After the Great

East Japan Earthquake on 3.11
Acknowledgements., 12, 47‑59. (In Japanese)
Usuki H., 2002: Evaluation of characteristics andThis research was partly supported by annual
research grants “Study of coastal shellﬁsh ﬁsheries
to support the recovery from tsunami damage ineastern Hokkaido” and grants-in-aid for Scientificpreservation of Pacific oyster,, in view of the genetic resources.
, 40, 40‑104 (InJapanese with English abstracts).