Genetics in Aquaculture

Proceedings of the 42nd
U.S.-Japan Aquaculture
Panel Symposium
NOAA Southwest Fisheries Science Center
8901 La Jolla Shores Drive
La Jolla, CA
October 1, 2014

U.S. DEPARTMENT OF COMMERCE
National Oceanic and Atmospheric Administration

National Marine Fisheries Service

NOAA Technical Memorandum NMFS-F/SPO-168

Genetics in Aquaculture

Proceedings of the 42nd U.S.-Japan
Aquaculture Panel Symposium
NOAA Southwest Fisheries Science Center
8901 La Jolla Shores Drive
La Jolla, CA
October 1, 2014

Kristen Gruenthal1, Mike Rust2, Paul Olin3, and Emily Trentacoste1 (editors)
1

Contractor with Earth Resources Technology, Inc.
NOAA Fisheries Office of Aquaculture1315 East West Hwy, Room 12615
Silver Spring, MD 20910

2

NOAA National Marine Fisheries Service
Office of Aquaculture1315 East-West Highway
Silver Spring, MD 20910

3

California Sea Grant
UCSD / Scripps Institution of Oceanography
133 Aviation Blvd., Suite 109
Santa Rosa CA 95403

NOAA Technical Memorandum NMFS-F/SPO-168
March 2017
U.S. Department of Commerce
Wilbur Ross, Secretary of Commerce
National Oceanic and Atmospheric Administration
Benjamin Friedman (Acting), NOAA Administrator
National Marine Fisheries Service
Samuel Rauch III (Acting), Assistant Administrator for Fisheries

SUGGESTED CITATION:

Kristen Gruenthal, Paul Olin, Mike Rust, and Emily Trentacoste (editors). 2017.
Genetics in Aquaculture: Proceedings of the 42nd U.S.-Japan Aquaculture Panel
Symposium, La Jolla, CA, October 1, 2014. U.S. Dept. Commerce, NOAA Tech.
Memo. NMFS-F/SPO-168, 87 p.

A COPY OF THIS REPORT MAY BE OBTAINED FROM:

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OR ONLINE AT:

http://spo.nmfs.noaa.gov/tm/
Reference throughout this document to trade names does not imply endorsementby the National Marine Fisheries Service, NOAA.

Cover photo of juvenile rainbow trout raised in a tank at the ARS National Centerfor Cool and Cold Water Aquaculture in Leetown, West Virginia. Scientists there
have been working to breed resistance to bacterial cold-water disease into rainbow
trout. Courtesy of Stephen Ausmus, USDA Agricultural Research Service. Image
Number D1946-4

Table of Contents
Introduction

1

Plans, Challenges and Opportunities for Warm Water Aquaculture
and the Role of the Southwest Fisheries Science Center

2

The Strategy for Breeding Study of Fisheries Organisms
by Fisheries Research Agency (FRA)

3

Selective Breeding Research at USDA ARS

8Assessment of Global Population Genetics of Seriola lalandi:
Implications for Genetic Management in Aquaculture

9Michael B. Rust

Russ Vetter, John R Hyde, Cisco Werner

Hiroyuki Okamoto, Hiroyuki Nagoya, Hironori Usuki
Jeffrey T. Silverstein

John R. Hyde, Catherine M. Purcell

The De Novo Draft Assembly of the Yellowtail,
Seriola dorsalis, Genome

Catherine Purcell, Andrew Severin, Mark Drawbridge, Kevin Stuart, John HydeIdentification of Quantitative Trait Loci and Marker-Assisted
Selection by Using Genomics Information in Yellowtail(Seriola quinquediata)
Akiyuki Ozaki, Jun-ya Aoki, Kazuo Araki, Kazuki Akita, Satoshi Kubota,
Takashi Koyama, Takashi Sakamoto, Takurou Hotta, Tsutomu Noda,
Hirotaka Mizuochi, Yasuhiro Shima, Kazunori Yoshida

Examining Host-Pathogen Interactions at Mucosal Surfaces Reveals
Novel Molecular Targets for Columnaris Disease Intervention
Benjamin H. Beck, Eric Peatman

Genetics and Breeding of Highly Fecund Marine Species
Dennis Hedgecock

Assessment of Fertilization Ability of Cryopreserved Sperm in
Fish Using Interspecific Hybridization
Yukinori Shimada, Hiroyuki Okamoto, Hiroyuki Nagoya, Toshiya Yamaguchi

iii

16

23

32
37

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57

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69

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iv

Introduction
Exciting new developments in genetics and genomics contribute significantly toadvances in aquaculture production today and will be of even greater importancein the future. Genetic improvements through selective breeding, genetics andhealth management, understanding genetic interactions of wild and cultured
stocks and genetics and climate change are all research priorities of the JapaneseFisheries Research Agency, the National Oceanic and Atmospheric Administrationand the United States Department of Agriculture. The primary focus of this
symposium will be on solving production problems faced by the aquacultureindustries of the two nations using genetic and genomic approaches. This will
facilitate development of more competitive new and existing aquacultureindustries. Selective breeding has a long history of improving production traits inmany livestock species and is having progressively greater impacts in aquaculture;enhancing genetic improvement through application of genomics can significantly
accelerate this process. In health management genetic approaches to improve
resistance, enhance immune response, better understand pathogens and improve
vaccines are all tools to strengthen the aquaculture industry. Climate change will
present many challenges to aquacultured species and genetics and genomics will
prove to be valuable tools in addressing these. This Symposium will include aworkshop component for broad ranging discussions of some of the new
approaches being developed and how they might be applied in collaborative
research efforts to resolve key bottlenecks and facilitate aquaculture industry
expansion in Japan and the United States.

Michael Rust,
NOAA Fisheries Office of Aquaculture
United States Panel Chair

1Plans, Challenges and Opportunities for Warm Water Aquaculture and the Roleof the Southwest Fisheries Science Center
improvement. The California Cooperative Fisheries
Investigations (CalCOFI) has maintained an oceanobserving program that has routinely sampled waterconditions in the California Current since 1949 and isproviding baseline information and predictivemodeling data for site selection and evaluation of
impacts. Likewise the Southern California Ocean
Observing System (SCOOS) is providing real-timeobservations for impacts evaluation. Along with site
impacts, successful aquaculture must document andmaintain natural genetic structure and genetic
diversity while addressing issues associated withdisease, parasitism and artificial diet in culture.
Genomic approaches are being developed for abalone
species and Seriola species to provide a baseline recordof natural genetic variance and a means of monitoring
genetic changes in brood stocks. Economic studies
focus on individual economics but more importantevaluate systemic impacts and also explorations of the“transfer effect”, the ecological and protectedresources consequences of our reliance on wild
capture seafood imports. Protected resources surveys
provide information on migratory pathways andareas of high occupancy and can be used to guide site
selection and monitor interactions via passive andactive acoustics.

Russ Vetter, John R Hyde, Cisco Werner
NOAA NMFS Southwest Fisheries Science Center,
8901 La Jolla Shores Dr, 92037-1508 La Jolla, CA
Corresponding author: russ.vetter at noaa.gov

ABSTRACT

Each region of the US EEZ provides uniquechallenges and opportunities for marine aquaculture.
Access to markets, regulatory frameworks, competing
ocean uses, water quality and prevalence of extreme
weather events are all part of considerations for thedevelopment of a successful industry. The southwest
portion of the western US coastline and adjoining
waters in Mexico are characterized by: a. minimal
continental shelf habitat, b. low terrestrial runoff andnutrient loading, c. reliable water exchange via
currents, upwelling and tidal mixing, and d. low
prevalence of extreme weather events. Naturalfeatures of the southwest US coupled with a diverse
and educated urban population provides economic
opportunities for the marketing of high value
aquaculture products. It also requires strict adherence
to the highest standards of seafood quality and aminimum of adverse environmental impacts. Siting of
aquaculture facilities in federal waters (beyond 3
miles) provides access to high quality habitat but islimited by the availability of locations suitable formoorings.
The Southwest Fisheries Science Center (SWFSC) isinterested in partnering with private entrepreneurs,
academic researchers, and State and international
regulatory partners to explore opportunities toincrease seafood supply, economic activity and job
creation while minimizing the impacts to the marine
ecosystem. The SWFSC and academic partners are
particularly strong in Fisheries Oceanography,
Protected Resources Monitoring, Biotechnology andEconomics.
In this presentation we will discuss current andplanned research opportunities and how they relate tosite selection, impacts evaluation and brood stock2

The Strategy for Breeding Study of Fisheries Organisms by Fisheries Research
Agency (FRA)
Hiroyuki Okamoto, Hiroyuki Nagoya, Hironori Usuki

INTRODUCTION AND DISCUSSION

Fisheries Research Agency (FRA) made the strategy
for breeding study of fisheries organism in 2013 (Fig.
1). This strategy is expected with dissolving many
problem of aquaculture, by proceeding breeding
study for improving economic condition of
aquaculture farm and activating the industry. Mainly,
the scheme of the strategy is consisted of two parts,“Present situation of research and development ofaquatic breeding study” and “Direction and policy forpromotion of aquatic breeding study” (Fig. 2). At first,
we introduce the past and current aquaculture in
Japan, and the outline of the strategy for breeding
study of FRA.

National Research Institute of Aquaculture,
Fisheries Research Agency, Kamiura, Saiki, Oita
879-2602, Japan
Corresponding author: hiokamot at affrc.go.jp
Keywords: selective breeding, Fisheries Research
Agency of Japan, marker assisted selection, assisted
reproductive technology, genetic resources, pedigree
construction, genetic improvement

ABSTRACT

Recent progresses of seed production techniques andof molecular biological techniques are increasing
expectations to genetic improvement of cultured
species, that is facing high price of feeds, high cost for
disease prevention, low farm gate price and manyother problems. For effective and efficient promotionof aquatic breeding studies which lead to accurate
measures against problems in actual culture sites, theFisheries Research Agency of Japan (FRA) announced
the “Strategy for breeding study of fisheriesorganisms” on March 2013, through several
discussions and cooperation with prefectural
institutes, universities, governmental officials andprivate corporations. The strategy consists of twoparts, namely, “Present situation of research anddevelopment of aquatic breeding study” and“Direction and policy for promotion of aquatic
breeding study”. We introduce the latter parts of thestrategy in this paper. As a fundamental concept, weshould decide on priority in industrially large-scale
and widely spread species, while strains adapting toregional environments are expected to be made byeach prefectural institute. Development offundamental technology, evaluation methods of traits
and preservation methods of genetic resources arethought to be necessary as schemes for promotion of
aquatic breeding. In addition, improving cooperative
system of R&D, provisions for protection of
intellectual property and consideration to naturalenvironment are thought to be necessary to enhance
improved strains in actual culture sites. We introduced
the outline of the strategy for breeding study.

Figure 1. Publication of“The strategy forbreeding study offisheries organisms”.

Figure 2. The scheme and contents of “The strategy for breeding study offisheries organisms”.

3Past and current aquaculture in Japan
History of fish breeding in Japan
Beginning of fish breeding in Japan could bestarted, at least from Edo era or before. It has
started at introduction of goldfish, and ornamental
carp as Nishiki-goi, in Japanese. These aretransferred in Japan from China. Selective andcross breeding of these fish started at least in
mid-18th century. Goldfish was getting in popular
as a pet during 19th century. For edible fish
breeding, it has started by rainbow trout (RT)derived from California, USA, in 1877, after “Meiji
Restoration” when it had started to modernize
Japan’s social system. Selective, cross and polyploidy
breeding of RT has been operated to acquire disease
resistance, fast growth and several other traits. Aboutsalt water fish, in 1960’s, almost a hundred years laterfrom the introduction of RT, selective breeding of red
seabream (Pagrus major) has begun for growth, colorand body shape by Kindai University. After over 40
years of selection, the culture period to gain 1kg bodyweight had reduced to one and half year from three
years. Now, this selective strain is the most popularpedigree in Japanese marine aquaculture. Later, many
marine and fresh water fish had been started toresearch for breeding study.

Figure 4. Ratio of production amount of aquaculture and fishery in Japan.

The ratio of production amount of aquaculture andfishery in 2011 was different depending of fish species(Fig. 4). Seabream aquaculture are operated in afull-life cycle. On the contrary, yellowtail and bluefin
tuna seeds are almost originated from natural
resources. The production amounts of these fish arerather high compare to that of fishery. In 2013,
yellowtail (Seriola quinqueradiata) occupy 44%, redseabream 23% and greater amberjack (Seriola dumerii)
16% in total production amount of aquaculture (Fig.
5). These three species are very important in Japan,
because they occupy over 80% of 240 thousand tons of
total aquaculture production.

Present situation of aquaculture in JapanThe amount of total aquacultural production was
having a peak as 1.43 million ton in 1988, and going
down to 1.07 million ton until 2012 (Fig. 3). The
reason of this decline was estimated to high price offeeds, high cost for prevention of disease, low farm
gate price and severe competition with foreign
products. However, the ratio of production amount ofaquaculture to total fisheries production is going up
to 22 % in 2012, twice as that of 1988 (Fig. 3). Thus, theimportance of aquaculture in Japan is getting higher
in these years.

Figure 5. Production amounts of major aquaculture fish in Japan.

Figure 3. Transition for production amount of aquaculture in Japan, andthe ratio of production amount of aquaculture against to total fisheries
production including in aquaculture and fishery.

However some fish species are still underconstruction of full-life cycle aquaculture system, e.g.
yellowtail, greater amberjack, longtooth grouper(Epinephelus bruneus), bluefin tuna (Thunnus orientalis)and Japanese eel (Anguilla japonica). Some of themalready established the “Systematic study of seedproduction on fish farming and aquaculture”, e.g. red
seabream, Japanese flounder (Paralichthys olivaceus)and pufferfish (Takifugu rubripes), which achieved
41. R&D of fundamental breeding technology
Marker-assisted selection (MAS) is one of the mostpowerful and practical methods to promote
breeding. Meanwhile, new breeding techniques isbeen trying to apply vigorously all over the world.

some “Progress of molecular biological technique”.
They are expected to fulfill genetic improvement tocontribute for aquaculture industry.
Direction and policy for promotion of aquaculture
breeding study in Japan
Fundamental concept
About fundamental nationwide concept, we will
focus on the priority based on the size of industry or
the size of influence, widely or not. In this sense,
important objective fish are yellowtails, Japaneseflounder, groupers, Japanese eel, bluefin tuna andsalmonids in finfish; for seaweeds, Laver as Nori inJapanese name; for shellfish, shortneck clam, pacificoyster and other bivalves; and for shrimp, Kuruma
prawn are important species in Japan aquacultureindustries. For regional or prefectural breeding, theyneed to develop strains adapting to regional
environment by prefectural institute. In both cases fornationwide and regional or prefectural breeding, theyshould develop pedigrees quickly in consideration forpractical use.

i) R&D of DNA marker and MAS• Development of DNA markers for eachspecies using next generation sequencer(NGS)• Making of genetic linkage map, QTL
analysis of quantitative traits, analysis ofcorrelation between phenotype and DNA
polymorphism, etc.
• Acceleration of linkage analysisii) R&D of new breeding techniques
Genome editing using artificial nuclease as
TALEN or CRISPR/Cas (defined below) isgetting widely and rapidly spread to use every
fields of life science. It will be a powerful tool
not only to analyze the function of interested
gene, but also to make a basic pedigree having amodified gene function for making an applied
strain for commercial use by crossing.
• Development of artificial mutagenesis
techniques, e.g. random mutagenesis known
as TILLING (Targeting Induced Local
Lesions In Genomes), genome editing as
TALEN (Transcription activator-like effector
nucleases) or CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeat)/Cas9(CRISPR associated protein 9), etc.
• GMO (Genetically modified organism) is
still powerful method to give a new genefunction as long as the purpose is forbenefits

Objectives of breeding
Making a breeding plan for which species and what
trait should be improved are very important to start
breeding. However, in Japan’s aquaculture, there aremany species which product amount is small, except
for yellowtails or red seabream, there are a few
common trait for breeding for all species. Therefore, abreeding plan should be determined in each species
depending on the case.
In this part, we categorized fundamental four
objectives for breeding.
1. Improvement of administrative character: fast
growth, high food efficiency, easy to handle, etc.
2. Improvement of environmental adaptability:
tolerance to high temperature, low oxygen
concentration, harmful algae, etc.
3. Improvement of economical character: highproductivity of flesh, high concentration offunctional ingredient, fine appearances, etc.
4. Improvement of disease resistance: strength offundamental immunity, resistance to several
pathogens, etc.

iii) R&D of assisting technology for breeding• Development of new sterility technique of
cultured fish: it could be useful to reduce therisk of breeding strain escaping into naturalenvironment• Promotion of surrogate propagation ofcultured fish: it will be a useful tool forestablishing strains in future

Scheme for promotion of breeding
To efficiently promote aquaculture breeding, we needto research and development of many breeding
related methods and technologies, and to prepare
rearing and testing facilities. We summarized theimportant R&D study to promote breeding at the
point of a long-term viewing.

2. Development of evaluation methods for traits
To make a useful pedigree having a commercial
trait, it is important to evaluate a useful trait
correctly and ideally.
i) Evaluation method of fast growthii) Evaluation method of disease resistanceiii) Evaluation of other trait5

freshwater. It is favorable to use FRA researchfacilities in all Japan for preservation of genetic
resources.

3. Development of pedigrees
Genetic resources for development of new
pedigrees in prefectural institute or privatecompany should be bred for their aim bythemselves. Especially, marine and fresh water fish
will be produced pedigrees with fast growth ordisease resistance.

3. Improving cooperative system of R&D
To reinforcement of cooperation with prefectural
institute, universities and private company, it isimportant to make a system to exchange theinformation of each organization through thedepartment for social cooperation, about how toutilize the products of bred strains, from at the
beginning of breeding program to at the stage oftransferring the technology to industry.

4. Development of preservation methods of resources
Keeping many genetic resources and evaluating of
pedigrees are essential to promote breeding. We
focused on the genetic resource species from ourmain aquaculture species. We will keep rearing
such resources, if possible, and develop a method topreserve for the species that cannot be kept as
pedigreed lines.
• Keeping strains for long time is the job of the
Public Preservation Center (described below)• Developing the method of cryopreservation andrestoration techniques of the germ cells

4. Protection of intellectual property and consideration
to environment
We don’t have a law to protect the aquaculture
breeder’s right except for seaweed. To protect theirright, the only way is to make the pedigree sterile,and to register a record to the information ofbreeders in a public database for breeding
information. We need to establish the technology
and the database system.

Propagate promotion for improved pedigrees
We just pointed out several essences of propagate
promotion for improved pedigrees.
• Adjust role-sharing of each institute or company
from the start of development• Confirm prospective policies of pedigrees asintellectual property• Patent law, trademark law, measures against
outflow, traceability, etc.

Also regarding established pedigrees, we need to
manage them not to escape to nature, because of
their genetic profile should be biased during
breeding.
• Protection of right of breeder• Preventive measures against genetic
disturbance in natural area

Preparation of promoting system for aquaculture
breeding
1. Organization of “R&D Center for Aquatic
Breeding”
In FRA, “Research Center for Aquatic Breeding”
was established in 2014. To proceed producing
useful pedigrees or strains requested from various
customer and manufacture needs, we need topromote breeding by widely combining outcomesfrom genetics, molecular biology, physiology,
nutrition, systems engineering and etc. For that
purpose, we need an organization and a system
including research facility and researchers, like as
R&D Center for Aquatic Breeding, to promote sucha modern breeding.
2. Organization of “Preservation Center of Genetic
Resources”
To disperse of risks of loss of genetics resources andpedigrees, we need Preservation Center of Genetic
Resources, which are settled in multiple places inJapan, because aquaculture species are originatedfrom various environments as in northern (cold)and southern (warm) area, or in marine andFinal notes
Japan has 130 years of history of aquaculture about
seeds production and raising techniques. FRA willreinforce the cooperation with prefectural institute,
universities and company in accordance with “thestrategy for breeding study of fisheries organisms”.
FRA will promote to develop DNA marker assisted
selection, reproduction techniques and new breeding
technologies, vigorously.

6

Wada, K. T. (2012). Evaluation of parental breedingvalues in aquaculture – reviews. Fish Genetics
and Breeding Science, 41, Issue 2, 87-96. (inJapanese)

REFERENCES

Fisheries Research Agency, Japan (2013). Strategy forbreeding study of fisheries organisms –A planof proceedings of fisheries breeding in Japan.
1-20.
Wada, K. T. (2012). Evaluation of Parental Breeding
Values in Aquaculture-Reviews. Fish Genetics
and Breeding Science, 41, Issue 2, 87-96.
Ozaki, A., W. Kai, J. Aoki, K. Nomura, K. Araki, K.
Yoshida, T. Tsuzaki, T. Sugaya, M. Sano, K.
Kobayashi-Fuji, T. Sakamoto and N. Okamoto(2012). Application to the genetic breeding
using by genomics information in
Aquaculture. Fish Genetics and Breeding
Science, 41, Issue 2, 173-178.

The strategies in evaluation of parents and in mating
design are important in aquaculture breeding
programs. This review deals with topics of evaluating
the breeding values of parents in fish and shellfish
species of aquaculture. (1) In order to evaluate
offspring reared at a communal environment, various
chemical or physical tags and molecular markers
have been developed for assigning the parentage inmating experiments or in selection programs. (2)Accuracy of selection is a main target in genetic
evaluation of parental animals and the breeding value
has been estimated in many traits of many species.
Methodology like BLUP developed in breedingsciences of livestock can be applied to aquatic animals
with some modification. (3) Increased cost andinbreeding rate and decreased genetic diversity areconcerns in selective breeding since the number ofoffspring is much larger in aquaculture breeding
program than in those of livestock.

ANNOTATED BIBLIOGRAPHY

Fisheries Research Agency, Japan (2013). Strategy forbreeding study of fisheries organisms – a planof proceedings of fisheries breeding in Japan.
1-20. (in Japanese)
A sea area of around Japan is one of the fertile areas toproduce marine natural resources in the world, theproduct amount of Japanese fisheries and aquaculture
is only covered 60 % of the domestic demand itself.
Japanese Fisheries Agency made a plan to raise thefood self-sufficiency ratio up to 70% for fin-fish, and
73% for seaweeds in 2022. The domestic aquaculture
production is mostly consumed in the domestic
consumption, and therefore needs to develop itself toachieve the goal of marine food self-sufficient ratio.
However, the business condition of Japaneseaquaculture is rather serious, because of hardness tokeep fish-meal for feeding from overseas, to save thecost for preventing disease, and to compete against
cheaper import marine products. To overcome these
serious problems of aquaculture farmer in Japan,
Fisheries Research Agency (FRA) made strategy forbreeding study of fisheries organisms to achieve
genetically improvement, for example, about theadaptation in aquaculture environment, or moresuitable products for consumer demand. The
establishment of breeding in aquaculture is expected
to maintain the amount of domestic production, toenhance international competitiveness and to activate
related industries, by reduction of production costand enhancement of domestic consumption. The
strategy is comprised of two parts, one is “Present
situation of research and development of aquatic
breeding study”, the other is “Direction and policy forpromotion of aquatic breeding study”.

Ozaki, A., W. Kai, J. Aoki, K. Nomura, K. Araki, K.
Yoshida, T. Tsuzaki, T. Sugaya, M. Sano, K.
Kobayashi-Fuji, T. Sakamoto and N. Okamoto(2012). Application to the genetic breeding
using by genomics information in aquaculture.
Fish Genetics and Breeding Science, 41, Issue2, 173-178. (in Japanese)
Ten years ago, genetic breeding study of fisheriesorganisms was mainly about salmonids because it
was the main production of world aquaculture. But
now, the aquaculture of marine fish is getting
important, the center of breeding study is moving inmarine fish. As developing technique of fish seedling,
not only the main aquaculture fish species, forexample, flounder, puffer fish and yellowtail, but also
the hard seedling species like eel and tuna are
expected to be applied with breeding study based on
genomic information after establishment of complete
raising technique in aquaculture farm. We are
introducing about studies of Quantitative Trait Loci
(QTL) and Marker-Assisted Selection (MAS) inaquaculture fish, and discussing about necessity andpossibility of breeding study in aquaculture.

7

Selective Breeding Research at USDA ARS

cross-breeding-different species. I will highlight
aspects of the development of genetic improvement
programs for catfish, rainbow trout, Atlantic salmon,
yellow perch, striped bass and oysters. While growth
performance is a trait of primary concern in mostprograms, other traits have gained importance,
too. With catfish, the recent development of methods
for mass production of the hybrid catfish (blue catfish
male x channel catfish female) has raised theimportance of reproductive performance. Atlantic
salmon selective breeding programs are some of themost advanced in the world (Norway, Chile, Iceland,
Scotland), however, due to their listing as endangered
species in North America, continent of origin has
become a fundamental criterion for breeding. Disease
resistance, the ability to withstand specific pathogens
and have higher survival, has been a critical focus forrainbow trout and other species as well. Productaspects such as greater fillet yield are being targeted;and the possibility to selectively breed for greater
ability to convert and deposit long chain n-3 fatty acids
is an intriguing target for selective breeding, too. In
addition to reviewing progress in these projects, I will
discuss some of the research and breeding strategies
that have been adopted recently to maximize industry
relevance and to incorporate genomic information into
our genetic improvement programs.

Jeffrey T. Silverstein
USDA Agricultural Research Service,
PO Box 225, Stoneville, MS 38776
Corresponding author: jeff.silverstein at ars.usda.gov

ABSTRACT

There is great potential for intensifying aquaculture
production. Selective breeding to improve production
efficiency is an important tool and the potential tomodify performance traits through selective breeding
is considerable. The development of successful
selective breeding programs for aquaculture is along-term commitment. While the gains fromselective breeding are permanent and cumulative,
initiation of breeding programs often require
government support in the early stages, with regular
input from industry. The aquaculture program within
the US Department of Agriculture, Agricultural
Research Service has supported development of
numerous selective breeding programs for food fishand shellfish species using a variety of methods
including mass selection, family selection andcrossbreeding both intra-specific crossbreedingdifferent strains, same species; and interspecific8Assessment of Global Population Genetics of Seriola lalandi: Implications for
Genetic Management in Aquaculture

Corresponding author: john.hyde at noaa.gov

complex of three regional species (S. aureovittata, S.
dorsalis, S. lalandi). By creating the framework for a
global genetic diversity monitoring program,substantial progress can be made towards establishingyellowtail as a commercially viable aquaculture
species on the west coast.

Key words: Seriola lalandi, cryptic speciation, genetic
differentiation, yellowtail, microsatellites

INTRODUCTIONJohn R. Hyde, Catherine M. Purcell
National Marine Fisheries Service,8901 La Jolla Shores Dr, 92037-1508 La Jolla, CA

Seriola species comprise an important component of
global aquaculture production, with an estimatedannual value of $1.3 billion (FAO 2012). Althoughaquaculture is seen as a way to reduce fishingpressure on natural populations, culture can also havea significant impact on the health and stability of wild
fish assemblages. Transport and production ofcultured fish into different geographic locations orinto non-local populations creates a threat to wild fish
through introduction of non-native genes and/or
maladaptive traits into native populations (Hutchings
and Fraser 2008; Dann et al. 2010).

ABSTRACTOn the west coast of the United States, the California
yellowtail, Seriola lalandi, is considered a greatcandidate for mariculture due to its high market
demand and value. While most yellowtail production
relies on capture of wild juveniles and fattening in
offshore pens, successful spawning and rearing
techniques for yellowtail have been developed at the
Hubbs-SeaWorld Research Institute (HSWRI), with
the intent to translocate reared individuals to offshore
pens to raise to market size. As aquaculture foryellowtail grows here and around the world, theimpact of unintentional releases on wild populations
has become an increasingly important issue. As such,
most future aquaculture projects will likely require a
genetic analysis component for the permitting
process. This study aims to examine the genetic
diversity of yellowtail over both a global scale andmore regionally in the California-Mexico region todevelop a baseline of the genetic variability in wild
populations and to evaluate locally adapted traits forbroodstock selection. A total of 260 specimens
collected around the Pacific and in the Atlantic and755 specimens collected in the northeast Pacific were
evaluated using 16 nuclear microsatellite markers.
Overall, genetic population structure was highly
significant at both the global and regional scale (FST =0.0858 and 0.0091, respectively). Pairwise comparisonsindicated four distinct groups at the global scale:
northeast Pacific, northwest Pacific, south Pacific, andSouth Africa. However, the pairwise results at theregional scale were not as clear but may indicate
differences between yellowtail found nearshore
versus offshore. Combined phylogenetic analyses
using two mitochondrial and four nuclear genes
strongly support at least three evolutionarily distinctgroups among the global samples, supporting
previous taxonomic hypotheses that S. lalandi is a

Spatial genetic structure has previously been detectedamong yellowtail sampled from different regions(Nugroho et al. 2001; Miller et al. 2011). In addition to
genetic differentiation among populations of S.
lalandi, factors such as size at reproductive maturity
and temporal separation of spawning seasons haverecently reignited the question of whether
populations of S. lalandi may actually be distinct
species. Initially, several nominal species names
existed in the literature; Valenciennes 1833 described
S. lalandi (type locality Brazil), and Castelnau 1872
described S. grandis from Australia, New Zealand andChile. Seriola aureovittata (Temminck and Schlegal,1845) was described in Japan, and Gill 1863 described
Seriola dorsalis from California. Smith-Vaniz (1986,
1990) placed the latter three names (i.e., S. grandis, S.
aureovittata, and S. dorsalis) into synonymy with S.
lalandi based on a lack of morphometric variation
throughout their range and concluded that previous
descriptions of distinct species were due solely to the
disjunct nature of the populations. Currently, S.
lalandi Val. 1833 is considered a single species broadly
distributed throughout the Indian, Atlantic andPacific Oceans.
Levels of genetic diversity, migration, and genetic
differentiation among the different regions still need
9For phylogenetic analyses, portions of two
mitochondrial genes (cytochrome oxidase I (COI) andcontrol region (CR)) as well as four nuclear genes(enoyl-CoA, hydratase/3-hydroxyacyl CoA
dehydrogenase (EHHADH), ubiquitin protein ligase
E3A (UBE3A), mixed-lineage leukemia-like protein(MLL), recombination activating gene 2 (RAG2)) were
amplified by polymerase chain reaction (PCR). Direct
sequencing of these regions was conducted using
either an Applied Biosystems 3130XL Genetic
Analyzer or an Applied Biosystems 3730XL Genetic
Analyzer and further aligned and edited using
GENEIOUS v4.8.5 (Drummond et al., 2010),
SEQUENCHER v4.5 (Gene Codes), and CLUSTALW(Thompson et al., 1994). Phylogenetic trees for S.
lalandi were built using maximum likelihood (ML)and Bayesian posterior probability analyses using 42
individuals of S. lalandi, with Seriola dumerili as an
outgroup. Evolutionary model selection was doneusing JModeltest (Posada, 2008) and phylogenetic
analyses were done using MEGA 6.0 (Tamura et al.,2013) and MrBayes v3.2.3 (Ronquist et al. 2012).

to be resolved for yellowtail. The impact ofunintentional releases on wild populations has
become an increasingly important issue asaquaculture grows for this species, particularly inlight of international trade of hatchery seed. The goalsof the present study were to: 1.) conduct aphylogenetic analysis using multiple mitochondrialand nuclear genes to investigate the phylogenetic
structure of S. lalandi sensu lato, 2.) determine thevalidity for the current treatment of the yellowtail jackas a single, globally distributed species, and 3.)
establish a baseline of existing genetic divergenceamong populations prior to potential introgression
from translocated fish.

METHODS

Specimens of yellowtail were collected from sevenlocations throughout their range between May 2007
and July 2010. The locations included Japan (JP),California (CA), Baja California Mexico (MX-P or
MX-Pacific), the Gulf of California, Mexico (MX-G or
MX-Gulf), Chile (CH), New Zealand (NZ), and South
Africa (SA) (Fig. 1). For the microsatellite analyses, a
total of 288 tissue samples were analyzed: 20 from JP,
58 from CA, 50 from MX-P, 44 from the MX-G, 32
from CH, 34 from NZ, and 50 from SA. A subset of
specimens from this sample was sequenced: 10 fromJP, 4 from CA, 10 from MX-P, 8 from MX-G, 4 fromCH, 2 from NZ, and 4 from SA. Genomic DNA wasextracted from tissues using the DNeasy Blood andTissue Kit (QIAGEN) following the manufacturer’s
protocol.

Fifteen microsatellite loci developed for Seriola (Portaet al. 2009; Renshaw et al. 2007, 2006; Ohara et al.
2005, 2003; Nugroho and Taniguchi 1999) were used
to examine population genetic structure in this study.
Samples were analyzed on an ABI 3730XL Genetic
Analyzer (Applied Biosystems) and fragment data
were analyzed using GENEMAPPER 4.0 (Applied
Biosystems) and scored visually. Genetic
differentiation was calculated using Weir andCockerham’s (1984) FST estimates in GENETIX 4.05
(Belkir et al. 2004). To assess hierarchical structuringof microsatellite variation among groups, withingroups, and within
populations, AMOVA
analyses (implemented
in ARLEQUIN),
including bothmitochondrial control
region sequence data andmicrosatellite genotypes,
were performed. The
model-based Bayesianclustering program
STRUCTURE v2.3.4(Pritchard et al. 2000;
Hubisz et al. 2009) wasused to examine
population structureusing a maximum
likelihood approach andFigure 1. Sample locations of Seriola lalandi, taken from (Martinez-Takeshita et al., in press).
correlation between
10Gene flow among N.E. Pacific locations was highrelative to the other locations, and pairwise estimates
did not detect significant differentiation among theselocations. Gene flow between the N.W. and N.E.
Pacific appears limited, with significant genetic
structure detected between these regions (FST = 0.0592,
0.0547, 0.0664 with P < 0.001 for CA, MX-P, and
MX-G, respectively). In the S. Pacific, gene flow
between NZ and CH prevents significant spatial
structure from being detected. Strong spatial
subdivision was observed between SA and the other
Pacific locations (FST between 0.0775 and 0.1200, P <0.001 for all comparisons).

geographic distances (average distance among
sampling locations in kilometers) and population
structure (FST) was tested with IBDWS (Jensen et al.
2005).

RESULTS AND DISCUSSION

Spatial genetic structure of yellowtail
Overall genetic differentiation among locations, as
revealed by microsatellite data, was highly significant
and large (FST = 0.085; P < 0.001). STRUCTURE results,
and pairwise estimates of divergence derived from
mtDNA and microsatellite data, support the presenceof four significantly differentiated populations in thesampled range corresponding to the N.E. Pacific,
N.W. Pacific, S. Pacific, and South Atlantic (Fig. 2).

Genetic differences among lineages
Phylogenetic and morphological data on the present
study of the cosmopolitan yellowtail jack strongly
support three cladescorresponding to theNortheast Pacific, theNorthwest Pacific, and thesouthern hemisphere (Fig. 3).
These findings are consistent
with and support thefindings from the population
genetic analyses based on the
mitochondrial sequence dataand nuclear microsatellite
markers (Purcell et al., inreview). Mitochondrial
divergence among the three
Seriola clades ranged
between 4.24% and 4.63%,
Figure 2. Graphical representations of Bayesian cluster analyses forwhich corresponds to 2.1 toyellowtail without location as a prior, based on data from 15 microsatellite
2.3 million years of separation among these lineages
loci. The three plots show population assignment results for different valuesof K: (a) K = 2; (b) K = 3; (c) K = 4. Each vertical line represents an individual using the mitochondrial divergence estimate of 2%
and its assignment to a particular cluster; solid lines separate the 7 sampled per million years (Brown et al. 1979; Bowen et al.
locations. This figure was taken from Purcell et al. (in review).
2001). In the southern hemisphere, mitochondrial
divergence was much lower between the S. Pacific
Genetic structure was especially strong acrossand South Africa (1.19%); by the same calculation, thisequatorial regions. For both marker classes, thecorresponds to a separation of only 595,000 years.
largest pairwise estimate occurred between the N.E.
and S. Pacific. STRUCTURE results showed nearly
Nuclear markers were also used to examinecomplete assignment between the northern anddivergence among the clades. Estimates of genetic
southern hemispheres when K = 2. Hemispheric
divergence were much lower, 0.15% to 0.31%,
genetic structure may largely be due to thermal
however Bayesian posterior and Maximum
restrictions and distinct spawning seasons in each
Likelihood bootstrap values showed strong (93 – 95%)
hemisphere. Yellowtail are a temperate andsupport of the three lineages (Fig. 3). In contrast to
subtropical species found in regions where
the microsatellite data, the nuclear sequences did nottemperatures range between 18° and 24° C (Fielder
reveal additional separation of the South Pacific fromand Heasman 2011); equatorial temperatures exceed
the South Atlantic, thus this divergence in thethe thermal range for this species, thereby limiting
southern hemisphere needs to be investigated incross-hemispheric movements (Martinez-Takeshita etgreater depth. There were no fixed sequenceal., in press).
differences between specimens collected in the S.
11

Pacific and South Africa using the nuclear
genes, and while the pattern varies slightly
between the mitochondrial and nuclear
genes, given the low divergence (0.06%) andlack of fixed differences in the nuclear
markers, there is inadequate evidence tosupport these regions as separate species.
Based on the strong phylogenetic structure,it has been demonstrated that three distinct
genetic lineages exist within the currently
recognized Seriola lalandi Val. 1833 species
occurring in the NW Pacific, the NE Pacific,
and the South Hemisphere (Fig. 4)Pacificand the South Atlantic. We conclude thatthree cryptic species currently bear the name
Seriola lalandi Val. 1833. Therefore, we
propose the resurrection of the currently
available names for these species in eachregion based on nomenclatural priority:
Seriola lalandi Valenciennes 1833 in Brazil,
South Africa, Australia, New Zealand andChile, Seriola aureovittata Temminck andSchlegel, 1845 in Japan, and Seriola dorsalis(Gill, 1863) in California, Pacific Baja, and
the Gulf of California, Mexico.

Figure 3. Bayesian derived consensus tree of combined mitochondrial andnuclear DNA sequence data (CR, COI, EHHADH, UBE3A, MLL, RAG2) for
Seriola lalandi and rooted with S. dumerili. Specimen designations are JP =Japan; CA = California; MP = Mexico Pacific; MG = Mexico Gulf (Gulf of CA);CH = Chile NZ = New Zealand; SA = South Africa. Values above nodes reflect
Bayesian posterior probabilities while values below nodes indicate Maximum
Likelihood bootstrap support values, values <80 are not shown. This figure
was taken from Martinez-Takeshita et al. (in press).

Figure 4. Distributions of proposed species of Seriola formerly referred to as S. lalandi.

12Jensen J.L., A.J. Bohonak, and S.T. Kelley. 2005.
Isolation by distance, web service. BMC
Genetics 6, 13. v.3.23 http://ibdws.sdsu.edu/
Martinez-Takeshita N., C.M. Purcell, C.L. Chabot,
M.T. Craig, C.N. Paterson, J.R. Hyde, and L.G.
Allen. In press. A tale of three tails: Cryptic
speciation in a globally distributed marine fishof the genus Seriola. Copeia
Miller, P. A., A. J. Fitch, M. Gardner, K. S. Hutson, and
G. Mair. 2011. Genetic population structure of
Yellowtail Kingfish (Seriola lalandi) in
temperate Australasian waters inferred frommicrosatellite markers and mitochondrialDNA. Aquaculture 319:328–336.
Nugroho E., and N. Taniguchi. 1999. Isolation ofgreater amberjack microsatellite DNA andtheir application as genetic marker to speciesof genus Seriola from Japan. Fish Sci 65:353–
357.
Nugroho, E., D. J. Ferrell, P. Smith, and N. Taniguchi.
2001. Genetic divergence of kingfish fromJapan, Australia and New Zealand inferred by
microsatellite DNA and mitochondrial DNA
control region markers. Fisheries Science
67:843-850.
Ohara E., T. Nishimura, Y. Nagakura, T. Sakamoto, K.
Mushiake, and N. Okamoto. 2005. Genetic
linkage maps of two yellowtails (Seriola
quinqueradiata and Seriolalalandi). Aquaculture 244:41-48.
Ohara E., T. Nishimura, T. Sakamoto, Y. Nagakura, K.
Mushiake, and N. Okamoto. 2003. Isolationand characterization of microsatellite loci fromyellowtail Seriola quinqueradiata and crossspecies amplification within the genus Seriola.
Mol Ecol Notes 3:390– 391.
Porta J.M., P. Novel, G. Martinez-Rodriguez, M.C.
Alvarez, and J. Porta. 2009. Isolation andcharacterization of microsatellites from Seriola
dumerili (Risso 1810). Aqua Res 40:249-251.
Posada, D. 2008. jModelTest: Phylogenetic Model
Averaging. Molecular Biology and Evolution
25:1253-1256.
Pritchard J.K., M. Stephens, and P. Donnelly. 2000.
Inference of population structure using
multilocus genotype data. Genetics 155:945–
959.
Renshaw M.A., J.C. Patton, C.E. Rexroad, and J.R.
Gold. 2006. PCR primers for trinucleotide andtetranucleotide microsatellites in greateramberjack, Seriola dumerili. Mol Ecol Notes 6:1162–1164.

REFERENCES

Belkhir K., P. Borsa, L. Chikhi, N. Raufaste, and F.
Bonhomme. 1996-2004. GENETIX 4.05,
Logiciel Sous Windows TM Pour la Génétiquedes Populations. Laboratoire Génome,
Populations, Interactions, CNRS UMR 5000,
Université de Montpellier II, Montpellier.
Bowen, B. W., A. L. Bass, L. A. Rocha, W. S. Grant,
and D. R. Robertson. 2001. Phlogeography of
the trumpetfishes (Aulostomus): ring species
complex on a global scale. Evolution 55:10291039.
Brown, W. M., M. George Jr., and A. C. Wilson. 1979.
Rapid evolution of animal mitochondrialDNA. Proceedings of the National Academyof Sciences, USA 76:1967-1971.
Castelnau, F. L. 1872. Contribution to the ichthyologyof Australia. No. 1.--The Melbourne fish
market (pp. 29-242). No. II.--Note on someSouth Australian fishes (pp. 243-247).
Proceedings of the Zoological andAcclimatisation. Society of Victoria 1:29-247.
Dann T.H., W.W. Smoker, J.J.Hard, and A.J. Gharrett.
2010. Outbreeding depression after two
generations of hybridizing southeast Alaska
coho salmon populations? T Am Fish
Soc 139:1292-1305.
Drummond, A. J., B. Ashton, S. Buxton, M. Cheung,
A. Cooper, C. Duran, M. Field, J. Heled, M.
Kearse, S. Markowitz, R. Moir, S. StonesHavas, S. Sturrock, T. Thierer, and A. Wilson.
2010. Geneious v5.5 http://www.geneious.
com
FAO. 2012. The state of world fisheries andaquaculture. Rome:209 pp.
Fielder, D. S., and M. P. Heasman, 2011. Hatcherymanual for the production of Australian bass,
mulloway and yellowtail kingfish. New South
Wales Department of Industry andInvestment, Fisheries and Research
Development Corportation (Australia):170pp.
Gill, T. N. 1863. Catalogue of the fishes of Lower
California, in the Smithsonian Institution,collected by Mr. J. Xantus. Part IV. Proceedingsof the Academy of Natural Sciences ofPhiladelphia v. 15:80-88.
Hubisz M.J., D. Falush, M. Stephens, and J.K.
Pritchard 2009. Inferring weak population
structure with the assistance of sample group
information. Mol Ecol Res 9:1322–1332.
Hutchings J.A., and D.J. Fraser. 2008. The nature offisheries- and farming-induced evolution. Mol
Ecol 17:294-313.
13

Renshaw M.A., J.C. Patton, C.E. Rexroad, and J.R.
Gold. 2007. Isolation and characterization of
dinucleotide microsatellites in greateramberjack, Seriola dumerili. Conserv Genet
8:1009–1011.
Ronquist F, Teslenko M, van der Mark P, et al.
MrBayes 3.2. 2012. Efficient Bayesian
Phylogenetic Inference and Model Choice
Across a Large Model Space. Syst Biol 61:539542. doi:10.1093/sysbio/sys029.
Smith-Vaniz, W. F. 1986. Carangidae. In: Smiths’ sea
fishes (eds. MM Smith, P Heemstra), SpringerVerlag, Berlin:638- 661.
Smith-Vaniz, W. F., J.-C. Quéro, and M. Desoutter.
1990. Carangidae. In J. C. Quero, J. C. Hureau,
C. Karrer, A. Post and L. Saldanha (eds.)
Check-list of the fishes of the eastern tropicalAtlantic (CLOFETA). JNICT, Lisbon; SEI, Paris;
and UNESCO, Paris. Vol. 2:729-755.
Tamura, K., et al. 2013. MEGA6: molecular
evolutionary genetics analysis version 6.0.
Molecular Biology and Evolution 30:27252729.
Temminck, C. J. and H. Schlegel. 1845. In: Temminck
and Schlegel 1843. Fauna Japonica, sive
description animalium quae in itinere per
Japoniam. Parts 7-9:113-172.
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994.
CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment
through sequence weighting, position-specific
gap penalties and weight matrix choice.
Nucleic Acids Research 22:4673-4680.
Valenciennes, A. 1833. In: Cuvier, G. and A.
Valenciennes 1833. Histoire naturelle despoissons. Tome neuvième. Suite du livre
neuvième. Des Scombéroïdes. Histoire
naturelle des poissons v. 9: i-xxix + 3 pp. +1-512, Pls. 246-279. [Cuvier authored pp. 1-198,
330-359, 372-427; Valenciennes the balance).
i-xxiv + 1-379 in Strasbourg edition. Pp.
429-512 contains additions and corrections forvols. 2 through 5.]
Weir B.S., and C.C. Cockerham. 1984. Estimating
F-statistics for the analysis of population
structure. Evolution 38:1358-1370.

ANNOTATED BIBLIOGRAPHY

Miller, P.A., A.J. Fitch, M. Gardner, K.S. Hutson, and
G. Mair. 2011. Genetic population structure ofyellowtail kingfish (Seriola lalandi) in
temperate Australasian waters inferred frommicrosatellite markers and mitochondrialDNA. Aquaculture, 319: 328-336.
The authors aimed to determine if there was spatial
genetic structure among populations of S. lalandi intemperate Australasia. This study was building on
research conducted by Nugroho et al. (2001) who
found significant variation between the northwest
and southwest Pacific, but not within the southwest.
A total of 272 specimens were collected from 17 sites
in five regions: New South Wales, New Zealand,South Australia, Victoria, and Western Australia.
Samples were analyzed using nine nuclear
microsatellite markers and a subset of the samples
was sequenced at the mitochondrial ND4 region. Boththe microsatellite and mitochondrial data indicatedthat Western Australia was significantly distinct fromthe other locations. However, no significant genetic
structure was detected among the other locations. The
authors state that population structure is an importantconsideration in aquaculture development and should
be used to help guide decisions regardingtranslocation of aquaculture fish among regions.
Nielsen, E.E., J. Hemmer-Hansen, N.A. Poulsen, V.
Loeschcke, T. Moen, T. Johansen, C.
Mittelholzer, G.-L. Taranger, R. Ogden, andG.R. Carvalho. 2009. Genomic signatures of
local directional selection in a high gene flow
marine organism; the Atlantic cod (Gadus
morhua). BMC Evolutionary Biology, 9: 276.
In this study, the authors tested the hypothesis that
populations of Atlantic cod show signals of local
adaptation to environmental conditions, and therefore
show patterns of divergent selection. A genome scan
examined variation in 98 gene-associated SNPs toidentify outlier loci potentially associated withadaptive population divergence over various
geographic scales. A landscape genetic analysis was
also used to investigate environmental factors
believed to be involved in local adaptation. Resultsfrom this study identified eight outlier gene loci thatare likely being acted upon by directional selection
over a global scale, but regional analyses also revealed
seven loci displaying strongly elevated levels of14

genetic differentiation. This study is important inconsidering broodstock selection for aquaculture
purposes. Given the very low level of neutral genetic
variation (what is measured in most population
genetic studies), the number of loci with strong
statistical support for selection is surprising. As theauthors state, despite being connected by variablelevels of gene flow, populations can follow semiindependent adaptive trajectories shaped by theirlocal environments. This is an importantconsideration in utilizing locally adapted species orpopulations in aquaculture.

15

The De Novo Draft Assembly of the Yellowtail, Seriola dorsalis, Genome

approximately 1.2 billion raw reads, for estimated180X coverage of the 685Mb genome. Two programs
were used to assemble the sequencing reads,
ALLPATHS-LG (v1.1) and MaSuRCA. MaSuRCA
resulted in a greater number of scaffolds (n = 86,357)
than ALLPATHS-LG (n = 2,460) and, similarly, more
contigs (n = 102,628 and n = 40,683, respectively).
However, the longest scaffold size was nearly threetimes larger using MaSuRCA; 14,669,447 bp versus
5,745,343 bp. The N50 scaffold length was also greaterwith MaSuRCA (N50 = 2,145,274) than with
ALLPATHS-LG (N50 = 900,636), as was the N50
contig length (N50 = 171,508 and 36,582, respectively).
Genes were annotated using Maker2. A web portal(Genome Browser) to directly view the genome, geneannotations, gene models, and markers was also
created to provide a publically available resource.
This study represents the first draft genome for Seriola
dorsalis. When correctly applied, this genomic
approach has the potential to improve broodstock
selection through identification of genes underlying
complex and/or economically important traits,characterization of variation (both beneficial anddetrimental), and marker-assisted selection for thisand other Seriola species. Investment in the genetic
resources for Seriola will contribute to making
aquaculture practices more economically viable andwill help improve domestic seafood production forthese valuable finfish.

Catherine Purcell1, Andrew Severin2, Mark
Drawbridge3, Kevin Stuart3, John Hyde1
1

National Marine Fisheries Service,8901 La Jolla Shores Dr, 92037-1508 La Jolla, CA

2

Iowa State Genome Informatics Facility,
Ames, IA 50011

3

Hubbs Sea World Research Institute,2595 Ingraham Street, San Diego, CA 92109

Corresponding author: catherine.purcell at noaa.gov
Keywords: aquaculture, genomics, broodstock
selection, marker assisted selection

ABSTRACT

Seriola species, collectively known as amberjacks, arefish of particular interest to the growing aquacultureindustry due to their high value, forming a billion
dollar plus component of the sashimi industry. Of
these species, the native California Seriola dorsalis, isconsidered a prime candidate for aquaculture
development in southern California. In developing
aquaculture for this species, methods to improve
culture efficiency and effectiveness are of greatinterest. Genetic resources have been developed andused extensively in agriculture and livestock to
improve product quality and quantity, and only more
recently have these approaches been applied to selectaquaculture species (e.g. rainbow trout, tilapia,
catfish, flounder, and Atlantic cod). Previously,
technology costs were prohibitive for many species;
however accessibility has improved throughdecreasing sequencing costs and enhanced
bioinformatics analyses. With the improved
accessibility to this technology, we are working todevelop genetic resources for S. dorsalis to help
improve aquaculture techniques.

INTRODUCTION

In the United States, the yellowtail, Seriola dorsalis, is astrong candidate for future development of offshore
commercial aquaculture in southern California.
Although artificial propagation and production frombroodstock populations have been successful inyellowtail, production may be further improved by
employing the genomic tools and breeding program
designs that have already proven successful in otheraquaculture species (Dong et al. 2015). In order todevelop environmentally friendly and economicallysustainable aquaculture, an understanding of thegenetic basis of traits that currently limit/enhancedevelopment and progress of domestic aquaculture isdesirable.

In a collaborative effort between the Southwest
Fisheries Science Center (SWFSC) and Genome
Informatics Facility at Iowa State University, we havecreated a de novo draft assembly of the genome for
Seriola dorsalis. DNA sequencing was conducted on
juvenile S. dorsalis using mate-paired and paired-end
reads with the Illumina HiSeq 2500, resulting in
16

Genomic selection and various genetic techniques are
powerful tools for the improvement of plant,
livestock, and aquaculture breeding practices (Taylor
2014); they have led to large increases in the rate ofgenetic improvement in a short amount of time
(Goddard and Hayes 2009). In particular, marker
assisted selection has the potential to greatly increase
the efficiency and effectiveness of culture practices.
Compared with traditional breeding practices, marker
assisted selection is faster, more reliable, and could
help avoid inbreeding depression by not requiring
highly inbred family lines to target complex traits(Ozaki et al. 2013). The genomic approaches would
also make it easier to identify genes underlying thosecomplex traits, which would not only improve theunderstanding of these traits but would also be used
to benefit culture practices (Goddard and Hayes
2009). Next generation sequencing (NGS) has helped
to revolutionize this area of research throughdecreasing costs and increasing number of research
applications (Dunham et al. 2014); this has enabled
the development of genetic resources for breeding
and culture purposes in a much wider range of
species (Huete-Pérez and Quezada 2013).

dorsalis. The genomic tools and resources created inthis project will provide a much-needed framework
for researchers, hatcheries and producers to begin
using marker-assisted selection in Seriola culture
practices. This framework is indispensable in working
to overcome the limitations that currently hinder
rapid growth of hatchery and offshore cage
production for Seriola culture in the continental U.S.
and Hawaii. Developing these genetic resources andtools are necessary steps for making broodstock-based
aquaculture of Seriola sustainable and economicallyfeasible.

METHODS

DNA was extracted from a single juvenile yellowtail(S. dorsalis) (approximately 50 days post hatch) rearedat Hubbs-SeaWorld Research Institute (HSWRI). The
purified DNA from the single fish was sent to Iowa
State for sequencing on the Illumina HiSeq 2500. Fourlanes were used to sequence the S. dorsalis genome: a150bp paired end lane, a 2,000bp mate pair lane, an
8,000bp mate pair lane, and a 12,000bp mate pair lane.
This sequencing generated 1.2 billion raw sequencing
reads providing 180X coverage for the genome.
Sequence quality was assessed using FastQC (v0.10.1). Since the quality scores were satisfactory notrimming was performed. To assemble the raw reads
for S. dorsalis, two assembly programs were used,
ALLPATHS-LG (1.1) (Gnerre et al. 2011) and
MaSuRCA (2.1) (Zimin et al. 2013; Neale et al. 2014).
ALLPATHS-LG (1.1) is an older program but is still
considered one of the better programs that specializes
in assembling shorter sequencing reads (such as theones generated by the Illumina HiSeq). MaSuRCA(2.1) is a newer assembly program that specializes ingenerating longer super-reads from the shorter
sequences. The genome was assembled using bothprograms to determine which assembly method
generated a better and more complete de novo draft
genome. MaSuRCA assembly pipeline uses
QuORUM kmer based error correction to correct raw
reads. From these error corrected reads, super reads
were generated by extending short reads on either
ends. For the average reads size of 106bp, kmer size of
74 was used for graph construction. The super reads
along with the filtered long linking reads were thenused with the CABOG assembler (Miller et al. 2008) togenerate the final assembly. CABOG assembler was
configured to use default parameters except for
scaffolder error rate (cgwErrorRate) of 0.15 andoverlap kmer size of 30. Maker2 (Holt and Yandell2011) was used for gene annotation. Maker was
initially run with just the evidences (using protein/

Only more recently have these genomic approaches
been applied to select aquaculture species (e.g.
rainbow trout, Atlantic salmon, tilapia, catfish,
flounder, Atlantic cod, and the Japanese eel) (Henkel
et al. 2012; Ozaki et al. 2012; Terova et al. 2013;
Dunham et al. 2014). Genotyping By Sequencing(GBS), Genome-Wide Association Studies (GWAS),
and Quantitative Trait Loci (QTL) mapping
approaches can be used in aquaculture to identify
genetic variation affecting phenotypic traits of interest
or other economically relevant qualities; identificationof that variation can in turn facilitate efficient
implementation in breeding schemes by use of
marker assisted selection (Sodeland et al. 2013). These
analyses have been used to improve various aspectsof aquaculture production and life history traits, suchas disease resistance, growth rate, tolerance ofenvironmental stressors, diet/nutrition, reproduction,
general health, and other key traits in aquaculture
species (Quinn et al. 2012; Cerdá, and Manchado
2013; Huete-Pérez and Quezada 2013; Dunham et al.
2014; Fuji et al. 2014).
We are developing genetic resources for S. dorsalis tohelp improve aquaculture techniques. In acollaborative effort between the SWFSC, the Genome
Informatics Facility at Iowa State University and the
Hubbs-SeaWorld Research Institute, we have createda de novo draft assembly of the genome for Seriola
17gene sequences from closely related species). With theinitial crude predictions, SNAP (v2013-11-29)(Korf
2004) hmm were generated. GeneMark (v2.3) (selftrained) (Borodovsky and Lomsadze 2011) hmm’s
were also generated using only the scaffolds. Revised
gene predictions were carried out using hmm’s from
GeneMark and SNAP. These revised gene models
were then used to train AUGUSTUS (v2.5.5) (Stanke
et al. 2006) and final gene predictions were calledusing AUGSUTUS, SNAP, GeneMark and FGENESH(v20150830) (Softberry Inc.,) (using Cynoglossus
matrix). The genome and the gene models were thenloaded as tracks on local, and GBrowse (v2.0) (Stein2013) was used for visualization.

Figure 2: Longest scaffold in the S. dorsalis draft genome using
two assembly programs, MaSuRCA (2.1) and ALLPATHS-LG(1.1).

RESULTS AND DISCUSSION

In the genome assembly, the longest contiguous
sequence (a.k.a. contig) MaSuRCA generated was
almost three times the length of the longest
ALLPATHS contig (Fig 1), and the scaffold was morethan twice as large as the ALLPATHS longest scaffold(Fig. 2). For the total number of scaffolds, the
MaSuRCA assembly ended up with 700 fewer
scaffolds than the ALLPATHS assembly did (Fig. 3).
The most widely used measure to describe genome
assemblies is the N50 length of scaffolds or contigs,
which is essentially a weighted mean, favoring thelonger (or more informative) contigs or scaffolds(Baker 2012; Adams 2003).

Figure 3: Number of scaffolds in the S. dorsalis draft genome
using two assembly programs, MaSuRCA (2.1) and ALLPATHSLG (1.1).

Figure 1: Longest contiguous sequence in the S. dorsalis draft
genome using two assembly programs, MaSuRCA (2.1) and
ALLPATHS-LG (1.1).

The N50 contig length using MaSuRCA was
141,285bp and for ALLPATHS-LG it was 36,582bp(Fig. 4). Similarly the N50 scaffold length was longer
using MaSuRCA than the ALLPATHS-LG assembly
program, with N50 scaffold lengths of 1,514,642 and
900,636, respectively (Fig. 5). However, both programs

Figure 4: The N50 contig length of the S. dorsalis draft genome
using two assembly programs, MaSuRCA (2.1) and ALLPATHSLG (1.1).

18

performed similarly in the placing the assembly into
scaffolded contigs, with 98.8% of the assembly placedusing MaSuRCA and 99.2% placed using ALLPATHSLG (Fig. 6).
Another measure of genome assembly quality isdetermining the CEGMA (Core Eukaryotic Gene
Mapping Approach) number for the genome (Parra etal. 2007). This approach determines how many ofthese ‘core genes’ can be found at full-length (or nearlyfull-length) within a single scaffold. For the MaSuRCA
assembly, 243/248 genes were found (or 97.98%), andfor the ALLPATHS-LG assembly, 240/248 (or 96.77%)of the genes were found. Both are considered highvalues among draft genome assemblies (http:www.
acgt.me/blog/2014/4/8/which-genome-assemblergives-you-the-best-genome-assembly).
This de novo draft genome assembly resulted in 180x
coverage of the small-to-moderately sized 685 Mb
genome of S. dorsalis. Both the MaSuRCA and
ALLPATHS-LG programs worked well for theassembly; it was estimated that 97% and 93% of the
genome was captured by these two programs,
respectively. After filtering for low quality scaffolds,
the current version of this genome has 1,439 scaffolds,
with an N50 value of 2,560,911bp. Ninety percent of
the total assembled bases are represented by only thelargest 410 scaffolds. Overall, the S. dorsalis genome
assembled remarkably well using the Illumina
sequencing data.

Figure 5: The N50 scaffold length of the S. dorsalis draft
genome using two assembly programs, MaSuRCA (2.1) and
ALLPATHS-LG (1.1).

To be able to utilize the sequencing data, we created a
web-based tool to visualize the genomic,transcriptomic, and genetic data. Sequences areorganized by scaffold (Fig. 7) and then can be zoomed
on to view individual transcripts, with clickable links
to BLAST results and the transcript sequences (Fig. 8).
With the additional transcriptomic data (Buonaccorsi

Figure 6: The percent of the S. dorsalis draft genome assembly
contained within scaffolded contigs using two assembly
programs, MaSuRCA (2.1) and ALLPATHS-LG (1.1).

Figure 7: Genome browser for S. dorsalis showing an entire scaffold.

19

Figure 8: Genome browser for S. dorsalis showing BLAST search results
for a transcript and the accompanying sequence.

et al. in press) incorporated into the genome browser,
we can also look at gene variants and splicing models
in the data.
Utilizing this draft genome assembly, we may
be able to use comparative analyses to aid ourunderstanding of S. dorsalis genome. In
particular, exploring other genomes that havebeen characterized for traits of interest may
greatly accelerate research for those traits in
Seriola. These analyses will continue toimprove as the genomes for additional species
are sequenced(Fig. 9).
In aquaculture, genomic data have been used
to identify disease resistance, genes associatedwith growth rates and resilience to different
types of stressors, fillet quality, and genes
related to reproduction and general health(Whatmore et al. 2013). Creating a de novo
draft assembly for S. dorsalis is an important
step to looking at these economicallyimportant traits. Analyses are ongoing in thisproject, and while much work is still needed
to be able to best utilize these genomic resources, thedevelopment of a draft genome for Seriola is anenormous first step to helping improve broodstock
selection and aquaculture practices for these species.

Figure 9: NCBI common tree of teleost genome sequences with S. lalandi(now S. dorsalis) placed within the tree.

20

REFERENCES

Adams. M., Sutton, G.G., Smith, H.O., Myers, E., andJ.C. Venter. 2003. The independence of ourgenome assemblies. PNAS, 100:3025-3026.
Baker, M. 2012. De novo genome assembly: what
every biologist should know. Nature Methods,
9:333-337.
Borodovsky, M. and A. Lomsadze. 2011. “Eukaryotic
gene prediction using GeneMark.hmm-E andGeneMark-ES.” Curr Protoc Bioinformatics
Chapter 4: Unit 4 6 1-10.
Bolger, A. M., M. Lohse, and B. Usadel. 2014.
Trimmomatic: a flexible trimmer for Illumina
sequence data. Bioinformatics, btu170.
Cerdá, J. and M. Manchado. 2013. Advances ingenomics for flatfish aquaculture. Genes andNutrition, 8: 5-17.
Dong, Z., Nguyen, N.H., and W. Zhu. 2015. Genetic
evaluation of a selective breeding program forcommon carp Cyprinus carpio conducted from2004 to 2014. BMC Genetics. 16:94.
Dunham, R., et al. 2014. Development of strategies for
integrated breeding, genetics and applied
genomics for genetic improvement of aquaticorganisms. Aquaculture 420-421:S121-S123.
Fuji, K., et al. 2014. Construction of a high-coveragebacterial artificial chromosome library andcomprehensive genetic linkage map ofyellowtail Seriola quinqueradiata, BMC Res
Notes 7:200.
Gnerre, S. et al. 2011. High-quality draft assemblies ofmammalian genomes from massively parallel
sequence data. Proc Nat Acad Sci 108:1513–
1518.
Goddard, M., and B. Hayes. 2009. Mapping genes forcomplex traits in domestic animals and theiruse in breeding programmes. Nat Rev
Genet 10:381-391.
Henkel, C.V., R.P. Dirks, D.L. de Wijze, Y. Minegishi, J.
Aoyama, H. Jansen, B. Turner, B. Knudsen, M.
Bundgaard, K. Lyneborg Hvam, M. Boetzer,
W. Pirovano, F.-A. Weltzien, S. Dufour, K.
Tsukamoto, H. Spaink, G. and van den
Thillart. 2012. First draft genome sequence of
the Japanese eel, Anguilla japonica. Gene, 511:
195-201.
Holt, C., and M. Yandell. 2011. MAKER2: anannotation pipeline and genome-databasemanagement tool for second-generation
genome projects. BMC bioinformatics, 12(1),491.
Korf, I. 2004. “Gene finding in novel genomes.” BMC
Bioinformatics 5: 59.
21

Huete-Pérez, J. A., and F. Quezada. 2013. Genomic
approaches in marine biodiversity andaquaculture. Biol Res 46:353-361.
Ozaki, A., K. Araki, H. Okamoto, M. Okauchi, K.
Mushiake, K. Yoshida, T. Tsuzaki, K. Fuji, T.
Sakamoto, and N. Okamoto. 2012. Progress of
DNA marker-assisted breeding inmaricultured finfish. Bulletin of Fisheries
Research Agency, 35, 31-37.
Ozaki, A., et al. 2013. Quantitative Trait Loci (QTL)
associated with resistance to a monogenean
parasite (Benedenia seriolae) in yellowtail(Seriola quinqueradiata) through genome wide
analysis. PLOS One 8:e64987.
Miller, J. R., A. L. Delcher, S. Koren, E. Venter, B. P.
Walenz, A. Brownley, J. Johnson, K. Li, C.
Mobarry and G. Sutton. 2008. “Aggressive
assembly of pyrosequencing reads withmates.” Bioinformatics 24(24): 2818-2824.
Neale, D.B. et al. 2014. Decoding the massive genomeof loblolly pine using haploid DNA and novel
assembly strategies. Genome Biol 15:R59.
Parra, G., K. Bradnam, and I. Korf. 2007. CEGMA: apipeline to accurately annotate core genes ineukaryotic genomes. Bioinformatics, 23(9),1061-1067.
Quinn, N., et al. 2012. Genomics and genome
sequencing: benefits for finfish aquaculture. In
Aquaculture.
Sodeland, M., et al. 2013. Genome-wide association
testing reveals quantitative trait loci for fillet
texture and fat content in Atlantic
salmon. Aquaculture 408:169-174.
Stanke, M., O. Keller, I. Gunduz, A. Hayes, S. Waack
and B. Morgenstern. 2006. “AUGUSTUS: abinitio prediction of alternative transcripts.”
Nucleic Acids Res 34(Web Server issue):W435-439.
Stein, L. D. 2013. “Using GBrowse 2.0 to visualize andshare next-generation sequence data.” Brief
Bioinform 14(2): 162-171.
Taylor, J. 2014. Implementation and accuracy ofgenomic selection. Aquaculture 420-421:S8S14.
Terova, G., et al. 2013. Molecular cloning and gene
expression analysis in aquaculture science: areview focusing on respiration and immune
responses in European sea bass (Dicentrarchus
labrax). Rev Fish Biol Fisheries 23:175-194.
Whatmore, P., N.H. Nguyen, A. Miller, R. Lamont, D.
Powell, T. D’Antignana, E. Bubner, A. Elizur,
and W. Knibb. 2013. Genetic parameters for
economically important traits in yellowtail
kingfish, Seriola lalandi. Aquaculture, Vol
400-401: 77 – 84.

200 bp and a maximum scaffold size of 1.14 Mbp. The
authors state that development of this genome is an
important component to the development of genetic
resources for fresh water eels and key in selective
breeding of this species for aquaculture purposes.
There are many similarities between the Japanese eel
and the California yellowtail in terms of aquaculture
importance, currently existing genetic resources, andthe motives in generating a draft genome.

Zimin A.V., Marcais, G., Puiu, D., Roberts, M.,
Salzberg, S.L., et al. 2013. The MaSuRCA
genome assembler. Bioinformatics 29:2669–
2677. doi: 10.1093/bioinformatics/btt476

ANNOTATED BIBLIOGRAPHY

Cerdá, J. and M. Manchado. 2013. Advances ingenomics for flatfish aquaculture. Genes andNutrition, 8: 5-17.

Whatmore, P., N.H. Nguyen, A. Miller, R. Lamont, D.
Powell, T. D’Antignana, E. Bubner, A. Elizur,
and W. Knibb. 2013. Genetic parameters for
economically important traits in yellowtail
kingfish, Seriola lalandi. Aquaculture, Vol
400-401: 77 – 84.

In this review, the authors describe the various ‘omics’
approaches that have been used to investigate
different aspects of flatfish biology, and that havebeen used to develop genomic resources available forflatfish research and aquaculture improvement. The
authors go over the next-generation sequencing
approaches that have been used in de-novo genomic
sequencing and transcriptome sequencing for various
flatfish species, along with the advantages anddisadvantages of these approaches. In this review, theauthors also discuss how the technology is being
applied to specifically address aquaculture questions
such as diet (e.g., nutrigenomics) and changes in theculturing techniques during the different life-stages(e.g., changes in transcriptome over larval
development). This review was of interest as an
overview of how these genomic and transcriptomic
approaches have been applied toward improving
aquaculture for these species, and how we may
similarly improve yellowtail culture.

The authors report on the genetic properties of muscle
fat content, condition score and deformity and their
genetic associations with body and carcass traits inyellowtail kingfish (Seriola lalandi). A total of 10
microsatellites were used to construct pedigrees forthe progeny of 22 full- and half-sib families (eightsires and six dams). Heritability was estimated forbody and carcass traits (fillet weight, fillet yield), fat
content, condition score and deformity, and genetic
relationships among these traits. The estimates ofheritability for body and carcass traits were moderate(h2 = 0.15 to 0.30), and fillet fat content showed
moderate heritability (0.41 ± 0.26).The estimate forcondition score was 0.15 ± 0.11, but the heritability for
deformity was close to zero (h2 = 0.02). The genetic
correlations between body and carcass (fillet weight
and fillet yield) traits were high and positive (0.57 to0.94). The results suggest that selection for improving
body weight, fillet weight and fat content could be
effective as evidenced by their moderate heritability.
Genomic data may reveal QTL-associated markers forthese traits, which could then be used in broodstock
selection.

Henkel, C.V., R.P. Dirks, D.L. de Wijze, Y. Minegishi, J.
Aoyama, H. Jansen, B. Turner, B. Knudsen, M.
Bundgaard, K. Lyneborg Hvam, M. Boetzer,
W. Pirovano, F.-A. Weltzien, S. Dufour, K.
Tsukamoto, H. Spaink, G. and van den
Thillart. 2012. First draft genome sequence of
the Japanese eel, Anguilla japonica. Gene, 511:
195-201.
In this study, the authors used a de novo assembly
protocol to create the first draft genome of thecommercially important Japanese eel, Anguillajaponica. Although considerable research has been
conducted on this species, including the developmentof some genetic resources such as a recently
developed linkage map, the tools are still far frombeing able to understand the genomic mechanisms ofthis organism’s biology. The genome of this nonmodel species was assembled using a whole-genome
shotgun sequencing strategy utilizing the Illumina
GAIIx or HiSeq 2000. The assembled genome size is
1.15 Gbp, which is contained in 323,776 scaffolds thathave an N50 of 52,849 bp, a minimum scaffold size of22

Identification of Quantitative Trait Loci and Marker-Assisted Selection by
Using Genomics Information in Yellowtail (Seriola quinqueradiata)
Two major QTL regions (BDR-1, BDR-2) were
employed for marker-assisted selection in F1 siblings
from QTL selection candidates to produce F2 full-sib
families. F1 siblings were placed in two groups which
are according to whether or not they have inherited
the QTL significant LD about Benedenia disease
resistance. Two crossbreed types of F2 full- sib families
were established by one-on-one crossing from F1
siblings. The families were putative resistant families(R-families) whose parents were selected for theirinheritance of QTL significant LD block, and the otherfamilies were putative susceptive families (S-families)
whose parents were selected for not having inherited
the QTL significant LD block.

Akiyuki Ozaki1†, Jun-ya Aoki1, Kazuo Araki1, Kazuki
Akita2, Satoshi Kubota2, Takashi Koyama2, Takashi
Sakamoto2†, Takurou Hotta3, Tsutomu Noda3,
Hirotaka Mizuochi3, Yasuhiro Shima3, Kazunori
Yoshida3†
1

National Research Institute of Aquaculture,
Fisheries Research Agency, 422-1, Nakatsuhamaura,
Minamiise-cho, Watarai-gun, Mie, 516-0193, Japan

2

Faculty of Marine Science,
Tokyo University of Marine Science and Technology,
4-5-7, Konan, Minato-ku, Tokyo, 108-8477, Japan

3

Goto Branch of Seikai National Fisheries ResearchInstitute, Fisheries Research Agency, 122-7,
Nunoura, Tamanoura-machi, Goto-shi, Nagasaki,
853-0508, Japan†

In artificially induced infection, we had compared theF2 R-families (total, n=1200) and F2 S-families (total,
n=1200) in three month old stage. The two different
type of QTL significant LD block were reared together
and challenged by exposure to B. seriolae. F2 R-family,
F2 S-family and about 120,000 B. seriolae hatchlings per
fish were placed in each tank. After 10 days, wemeasured the number of parasites in each fish. We
had performed infection experiment six times. All
infection experiment showed the same result, the F2
R-family fish had significantly fewer parasites thanthe F2 S-family fish.

These authors contributed equally to this work

Corresponding author: aozaki at affrc.go.jp
Keywords: yellowtail, Seriola quinqueradiata, Benedenia
seriolae, disease resistance, quantitative trait loci
(QTL), marker-assisted selection (MAS)ABSTRACTIn natural induced infection, we had compared thesusceptibility of B. seriolae with F2 R family (n=67), F2
S family (n=40) and F1 progeny from the wildparental fish (n=50). One hundred fifty-seven of the
one year old fish were pit tagged in total, and thenumber of B. seriolae was counted eight rounds for
one year in each fish. All eight rounds of naturalinfection were showed the significant results. The F2
R-family fish had significantly fewer the number ofparasites than the F2 S-family fish.

Benedenia infections caused by the monogenean fluke
ectoparasite Benedenia seriolae seriously impact marine
finfish aquaculture. We have discovered the evidence
that contributes to detailing the phenotypic resistanceto Benedenia disease in yellowtail (Seriola
quinqueradiata). Two putative quantitative trait loci
(QTL) associations, of medium to large effect of with
Benedenia disease resistance, were localized tolinkage groups Squ2 and Squ20. Finding the QTL
region strongly supports the potential for success ofmarker-assisted selection (MAS) for resistance to
Benedenia disease. The aim of this study was toconfirm the QTL significant region and compare thesusceptibility, depending on the difference of linkage
disequilibrium (LD) block for Benedenia disease
resistance in yellowtail, for use in marker-assisted
selection to increase the rate of genetic improvement
for this trait.

We employed LD blocks based on QTL significant
region for MAS, and confirmed their effects insubsequent F2 full- sib families. Furthermore, it means
QTL significant regions are identified from these wild
ancestors to the breeding population. It would bepossible to introgress certain properties of the wild
population in the breeding population. These results
indicate that it is possible to rapidly develop
domesticated strains having commercially important
traits by MAS in aquaculture.
23

et al. 2007), and therefore have had little time to adapt
to the new disease pressures.

INTRODUCTION

Construction of genetic linkage map andidentification of quantitative trait loci (QTL) are
beginning to make an impact on animal breeding, by
providing DNA markers linked to QTL affecting
phenotypic traits. Such molecular markers can beused in marker-assisted selection (MAS), selection
based partly or fully on DNA marker genotypes.
Among the traits relevant for MAS, disease resistancetraits are of particular interest, since the phenotypicmeasurement of these traits is often difficult.

The yellowtail amberjack (Seriola quinqueradiata) is, as
its relative the gold-striped amberjack (S. lalandi) andthe greater amberjack (S. dumerili), an important
species in marine finfish aquaculture in Japan.
Production of cultured species of yellowtail in Japan
was approximately 152,800 tons in 2009, which
accounts for 59% of marine finfish aquaculture in
Japan (MAFF Japan 2009). Yellowtail has been
cultured in southern areas of Japan using juveniles
caught from natural stock. Although research on
disease, nutrition and pond management has
supported the development of the yellowtail
aquaculture industry, genetic improvement programs
leading to improve yellowtail lines are only at the
beginning.

MAS involves selection of animals carrying genomic
regions that are involved in the expression of traits ofinterest through molecular markers. With thedevelopment and availability of an array of molecularmarkers and dense genetic linkage map in animals,
MAS has become possible for traits both governed bymajor genes as well as QTL.

Benedenia disease caused by infection by Benedenia
seriolae is a serious parasitic disease for yellowtail inaquaculture, leading to secondary infection due toviral or bacterial disease. This is because fish rub theirbodies against the fish cage to remove the parasite. In
certain conditions, the mortality is quite highespecially in juvenile fishes. Although the way ofremoving the parasite is generally to soak the fish in a
freshwater bath, this method requires a high cost andis labor intensive. Thus, Benedenia disease is difficult
to prevent in marine aquaculture systems. Besides
from the point of view of wildlife conservation,
yellowtail aquaculture is considered as a hotbed forenhancement of parasite transmission (Ogawa 1996;
Hutson et al. 2007). Risk management is an importantconsideration for the long-term sustainability of theaquaculture industry.

The advantages of MAS are obvious as comparedwith the conventional selective breeding. MAS areespecially useful for traits that are difficult tomeasure, or are expressed the phenotype late indevelopment. Implementation of MAS requires DNA
markers that are tightly linked to QTL for traits ofinterest based on QTL mapping or association studies(Lande and Thompson 1990). Ideally, the DNA
markers should be the linkage disequilibrium (LD)block underlying the phenotypic variation. QTL
studies in aquaculture species covered a wide rangeof traits including disease resistance, growth, stressresistance, reproduction and other traits. The resultsof these studies provide a good starting point tosearch for QTL within breeding populations. Of the
QTL from experimental crosses, only a small numberof them have been followed up by confirmation andfine-mapping. The responsible genes have not been
identified for detected QTL. However, there are
already a few applications of MAS in commercial
breeding programs in aquaculture species (Fuji et al.
2007; Moen et al. 2009; Ozaki et al. 2012).

In previous study, we performed QTL analyses using
wild F1 strains of S. quinqueradiata to elucidate thegenetic evidence of resistance to Benedenia disease.
By using the high-density linkage map withmicrosatellite and SNP markers, we identified two
chromosomal regions containing QTL (BDR-1, BDR-2)
that were associated with Benedenia disease
resistance. Two putative QTL associations, of medium
to large effect of with Benedenia disease resistance,
were localized to linkage groups Squ2 and Squ20.
These two loci were responsible for ranging from 32.9
to 35.5% of the total phenotypic variation (Ozaki et al.
2013). Finding the QTL region strongly supports thepotential for success of MAS for this disease.

In aquaculture, disease resistance traits are ofparticular importance. In intensive culture systems,
opportunities for avoidance or escape are minimal.
Furthermore, interactions between fish and viral,
microbial, parasite pathogens that may be harmless
under natural conditions often result in diseaseproblems in aquaculture systems because of theadded stress from biological, physical and chemicalfactors (Wedemeyer 1996). In contrast to farmanimals, the animal strains used in aquaculture areusually very recent derivatives of wild strains (Duarte

Here, we report on results from a project that used
MAS and confirmed their effects in subsequent F2
24

In both confidence intervals of QTL regions, we had
confirmed the genotypes within each locus in F1
selection candidates. In the QTL (BDR-1) region at
Squ2F linkage group, we confirmed that a linkage
disequilibrium (LD) block was conserved without
reorganization of chromosome in meiosis between
marker loci Sequ0672TUF and Sequ1067TUF (Fig. 1a).
And also in the other QTL (BDR-2) region of Squ20F,
we confirmed that an LD block was conserved
without recombination between Sequ1071TUF and
Sequ0288TUF (Fig. 1b). And we had known the LD
block associated with Benedenia disease resistancefrom wild ancestor within QTL reference families.

full-sib families. These results indicate that it ispossible to rapidly develop domesticated strains
having commercially important traits by MAS, where
investigation of a QTL at the level of an entire
breeding population yielded results that are directly
applicable for MAS within that population.

MATERIALS AND METHODS

Confidence intervals of QTL region about
Benedenia disease resistance
We show the confidence intervals of QTL regions for
Benedenia disease resistance from previous study in
Figs. 1a and 1b. Two regions of the linkage groups
were identified to be significantly associated with
Benedenia disease. The QTL (BDR-1) at Squ2F
identified by simple interval mapping (SIM) was
observed as a high single peak as genome-wide
logarithm of the odd score (LOD) significance level(LOD = 4.71). And the other QTL (BDR-2) region of
Squ20F linkage group had slightly exceeded the
genome-wide LOD significance level (LOD=2.98).
Each of the LOD peaks, at locus Sequ1295BAC in
Squ2F, and at Sequ0808TUF in Squ20F, can explain thephenotypic variance 21.4 and 14.1% by SIM [10].
When LOD peaks were combined, the two loci could
explain the phenotypic variance ranging up to 35.5%.
QTL confidence intervals were estimated by 1.8-LOD
support interval with 95% confidence interval
probability coverage (Manichaikul et al. 2006).

Simple sequence repeat analysis to confirm theLD block in QTL regions
Simple sequence repeat (SSR) genotyping was
performed in a 10 μl reaction volume containing 0.5
pmol/μl of unlabeled primer, 0.05 pmol/μl of
fluorescence-end-labeled primer with [5›-TET], plus
1× buffer, 2.0 mM MgCl2, 0.2 mM dNTP, 1% BSA,
0.025 U of Ex Taq DNA polymerase (TaKaRa Bio, Inc.,
Shiga, Japan) and 25 ng template DNA. Suitableannealing temperatures for each microsatellite marker
were used. PCR was performed on a MJ PTC-100(Bio-Rad, CA, USA), and the program conditions
were 95 °C for 2 min for initial denaturation, followed
by 35 cycles of 30 s at 95 °C, 1 min at the annealing
temperature (56–58 °C), 1 min at 72 °C and 3 min at 72°C for final extension. Amplification products weremixed with an equal volume of loading buffer [98%formamide, 10 mM EDTA (pH 8.0), 0.05%
bromophenol blue], heated for 5 min at 95 °C andthen immediately cooled on ice. The mixture was
loaded onto 6% PAGE-PLUS gel (Amresco, OH, USA)
containing 7 M urea and 0.5× TBE buffer.
Electrophoresis was performed in 0.5× TBE buffer at
1800 V constant voltage for 1.5 h. Afterelectrophoresis, the gel was scanned and imaged
using an FMBIO III Multi-View fluorescence image
analyzer (Hitachi-soft, Tokyo, Japan).
Wild ancestor, F1 selection candidates and LDMAS in F2 full-sib families
The wild ancestor fish of S. quinqueradiata were
captured in the coastal waters of Goto Fukue Island(Tsushima Strait, Nagasaki Prefecture). One thousand
juveniles were kept in a growing fish pen for two
years. Two hundred of the three year old fish were pit
tagged, and the number of B. seriolae counted six
times to select the ideal parents. The selected fish
Figure 1. Localization of significant markers for Benedenia disease
resistance in linkage group Squ2F and Squ20F. Squ(linkage group)F
marker distance in female map: (left) Squ2F, (right) Squ20F. Marker
absolute map distances are given in (cM), with 95% confidence probability
LOD support interval indicated as a bold line.

25

were reared until maturity. We prepared F1 families
for QTL analysis (Fig. 2). And we had found two QTL
medium to large effects for Benedenia disease
resistance in F1 families (Ozaki et al. 2013).

established by one-on-one crossing from F1 selection
candidates. Six families were putative resistantfamilies (R-families) whose parents were selected fortheir inheritance of Benedenia disease resistance LD
block, and six families were putative susceptive
families (S-families) whose parents were selected for
not having inherited the Benedenia disease resistanceLD block (Figs. 2, 3).

Two major QTL regions were employed for markerassisted selection in F1 selection candidates to produce
F2 full-sib families. F1 selection candidates wereselected in two groups according to whether or notthey inherited the LD block for Benedenia disease
resistance. Then, two types of F2 families were

Phenotypic measurement and comparison dependon the LD block for Benedenia disease resistancein artificially induced infection
About parasite collection for artificial infection
experiment, B. seriolae used for artificial infection werecollected from adult fish stock in growing pens. Mesh
nets were hung in fish pens in which adult fish wereparasitized by B. seriolae. Eggs of B. seriolae stuck to
the net and a method to collect only B. seriolae wasestablished and confirmed under experimental
conditions several times for reproducibility(Nagakura et al. 2010). Collected B. seriolae eggs could
be induced to hatch by a 15 minute exposure to
fluorescent light. Hatched larvae were kept in ashaded tank before infection.
In artificially induced infection, we had compared theF2 R-families (total, n=1200) and F2 S-families (total,
n=1200) in three month old stage. In preliminary
experiments, it was confirmed that the parasite did
not exist on the test fish bodies, which were soaked ina freshwater bath for five minutes. And the fish wereallowed to recover from freshwater stress for twodays before artificial infection. Hatched B. seriolae
larvae were introduced to experimental fish tanks,and individual fish were exposed to one hundred
larvae of B. seriolae. One F2 R family (n=200), one F2 S
family (n=200) and about 40,000 B. seriolae hatchlings
were mixed up in same tanks (Fig. 4). Water
temperature was kept at 25±1 centigrade degrees
during the infection experiment for 10 days until thelarvae grew to the countable adult stage. Ten days
after exposure to B. seriolae, each fish was individuallydipped in a tank of freshwater to remove theparasites. Thus, in the context of this study werecorded the total number of B. seriolae per fish. We
prepared set of test water tanks in six times.

Figure 2. Schema of fish strains. Two major QTL regions were employed
for marker-assisted selection in F1 selection candidates to produce F2
full-sib families. F1 selection candidates were selected in two groups
according to whether or not they inherited the LD block for Benedenia
disease resistance.

Figure 3. Schema of crossbreeding in MAS. Two types of F2 families were
established by one-on-one crossing from F1 selection candidates. Six
families were putative resistant families (R-families) whose parents wereselected for their inheritance of Benedenia disease resistance LD block,and six families were putative susceptive families (S-families) whoseparents were selected for not having inherited the Benedenia disease
resistance LD block.

Phenotypic measurement and comparison dependon the LD block for Benedenia disease resistancein natural induced infection
We had prepared the families in the same of artificial
infection experiment, which have the difference of LD
block associated with Benedenia disease resistance.
Those families are F2 R family (n=67) and F2 S family(n=40). In addition for the control group, we prepared26

40cm to 70cm.The fish raising methods are same
condition as practical mariculture of yellowtail.
When we started the natural infection experiment,each fish was injected the pit tag in their body for the
tracking of the susceptibility of Benedenia disease inindividual. Subsequently, fish are bathed in fresh
water to eliminate B. seriolae on the body surface.
Then natural induced infection had started, all fish
were kept in the same fish cage during theexperimental period. The first investigation isconducted at 4-8 weeks after starting the naturalinfection experiment. And we counted the number ofB. seriolae in each fish, which were stick on the surfaceof yellowtail skin as susceptibility of Benedenia
disease.
The investigation is conducted using the procedure
that: (a) put the yellowtail in the bag made of Tetoron
Russell Weave Net individually; (b) put the bag into
the fresh water tank for 5 minutes to remove theparasites; (c) identify the fish by the pit tag and countthe parasites that have appeared on the body surface
in each fish; (d) calculate the sum of the number ofparasites, which was on the fish the body surface, andparasites in the bag, which were peel off the fish thebag surface; and (e) after the investigation, return thefish to the fish cage to continue maintained. In total
number of times, we performed the investigation foreight rounds.

Figure 4. Schema of Artificially induced infection. We compared the F2
R-families (total n=1200) and F2 S-families (total n=1200) at the threemonth old stage.

F1 progeny (n=50) from wild parental fish. The F1progeny were selected random sampling from mixed
three families, and mixed three families were
sampling same number of progeny from each family
crossbred by one on one mating. Total number of 157
fish were prepared with beginning of natural infection
experiment. Fish were cultured from July to June forone year in fish cage at the coastal waters of Goto
Fukue Island. During the year, fish were grown from

Table 1. Evaluation of phenotypic data, in the tank experiment: artificially induced infection.

Total number of parasite
Tank1

F2-R

F2-S

Tank2

F2-R

Tank3

F2-R

Tank4

F2-R

Tank5

F2-R

Tank6

F2-R

F2-S
F2-S
F2-S
F2-S

F2-S

Mean(SD)

53.8 ± 17.8

The number of parasites/body surface

p.valuens

56.7 ± 18.2

0.16660

55.0 ± 21.4

1.22E-1160.0 ± 16.7

4.27E-07

43.0 ± 13.9

8.11E-08

68.1 ± 18.8

1.69E-1236.8 ± 16.1

1.26E-05

40.0 ± 18.8
51.0 ± 17.8
35.3 ± 14.4
55.7 ± 14.8
30.3 ± 13.8

***
***
***
***
***

Mean

1.01

p.value***

1.12

0.00088

1.09

3.91E-16

0.96

6.19E-11

0.66

3.01E-06

0.81

3.15E-07

0.45

0.04311

0.74
0.78
0.56
0.71
0.41

***
***
***
***

ns: not significant
p.value< 0.05*
p.value< 0.01**
p.value< 0.001***
The pairwise t test compared the two type of cross using F2 R-families vs. F2 S-families in each tank. And the graphs are indicatedtest water tank in six times.

27*

RESULTS

Evaluation of phenotypic data, in the field
experiment: natural induced infection
ANOVA and Dunnett test compared the three type ofcross in F2 -families using F2 R-families vs. F2
S-families, and F2 R-families vs. F1 progeny from wildparental fish (Table 2). In all of investigation results,
the number of parasite in F2 R-families are significant
difference than F2 S-families. And the investigation
days except July 20, Feb. 5, and June 3 in 2012, theEvaluation of phenotypic data, in the tankexperiment: artificially induced infection.
The pairwise t test compared the two type of crossusing F2 R-families vs. F2 S-families in each tank. And
the graphs are indicated test water tank in six times(Table 1). In infection experiment, the total number ofparasite in F2 R-families are significantly fewer than F2
S-families except Tank 1.

Table 2. Evaluation of phenotypic data, in the field experiment: naturally induced infection.

Total number of parasite
July. 20, 2012

Aug. 10, 2012

Nov. 2, 2012

Nov. 28, 2012

Dec. 27, 2012

Feb. 5, 2013

May.2, 2013

June. 3, 2013

Sum

F2-R

Mean(SD)19.5±11.6

F2-S

30.2±15.6

F2-R

31.1±18.5

F1-W

21.4±9.1

F2-S

49.4±24.8

F2-R

22.2±13.8

F1-W

58.3±23.9

F2-S

28.8±14.7

F2-R

25.7±19.4

F1-W

31.6±10.6

F2-S53.1±26.4

F2-R

36.6±22.5

F1-W
F2-S

36.8±20.4
88±44.5

F1-W

58.4±29.2

F2-S

30.3±11.7

F2-R

6.5±3.8

F2-R

F1-W

17.1±8.919.7±7.3

F2-S

13.4±4.4

F2-R

18.1±8.6

F1-W
F2-S

9.6±4.130.7±11.7

F1-W

21.8±11.3

F2-S

325.5±121.2

F2-R

F1-W

ANOVA p.value

177.1±88257.3±84.7

0.002 **

6.23E-09 ***5.01E-04 ***1.03E-05 ***4.46E-08 ***5.90E-06 ***1.01E-09 ***1.09E-05 ***3.79E-08 ***Dunnett p.value

F2-R vs. F2-S

F2-R vs. F1-W
F2-R vs. F2-S

P-

4.42E-05 ***0.645 ns-

1.72E-04 ***F2-R vs. F1-W

1.41E-09 ***F2-R vs. F2-S

0.045 *-

F2-R vs. F1-W

3.93E-04 ***F2-R vs. F2-S

1.10E-07 ***F2-R vs. F1-W
F2-R vs. F2-S-

0.013 *-

1.03E-11 ***F2-R vs. F1-W

4.86E-04 ***F2-R vs. F2-S

2.69E-09 ***F2-R vs. F1-W
F2-R vs. F2-S-

0.251 ns-

5.05E-12 ***F2-R vs. F1-W

1.16E-04 ***F2-R vs. F2-S

3.49E-07 ***F2-R vs. F1-W
F2-R vs. F2-S

F2-R vs. F1-W-

0.107 ns-

2.77E-10 ***3.08E-05 ***ns: not significant, p.value< 0.05*, p.value< 0.01**, p.value< 0.001***
ANOVA and Dunnett test compared the three type of cross in F2 -families using F2 R-families vs. F2 S-families, F2 R-families vs. F1 progeny
from wild parental fish.

28

total number of parasite in F2 R-families are fewer
than F1 progeny from wild parental fish. Even in anatural induced infection, it is presume that the
number of parasite in F2 R-families are significant
difference than F2 S-families and F1 progeny from wildparental fish.

REFERENCES

Duarte CM, Marba N, Holmer M (2007) Rapid
domestication of marine species. Science
316:383-383.
Fuji K, Hasegawa O, Honda K, Kumasaka K,
Sakamoto T, Okamoto N (2007) Markerassisted breeding of a lymphocystis diseaseresistant Japanese flounder (Paralichthys
olivaceus). Aquaculture 272:291-295.
Hutson K, Ernst I, Whittington ID (2007) Risk
assessment for metazoan parasites ofyellowtail kingfish (Seriola lalandi)(Perciformes: Carangidae) in South Australiansea-cage aquaculture. Aquaculture 271:85-99.
Lande R, Thompson R (1990) Efficiency of markerassisted selection in the improvement of
quantitative traits. Genetics 124:743-756.
MAFF Japan (2009) Ministry of Agriculture. Forestry
and Fisheries. Annual Statistics of Fishery and
Aquaculture Production. Statistics
Department. (in Japanese)
Manichaikul A, Dupuis J, Sen S, Broman KW (2006)
Poor performance of bootstrap confidence
intervals for the location of a quantitative trait
locus. Genetics 174:481-489.
Moen T, Baranski M, Sonesson AK, Kjoglum S (2009)
Confirmation and fine-mapping of a major
QTL for resistance to infectious pancreatic
necrosis in Atlantic salmon (Salmo salar):population-level associations between markers
and trait. BMC Genomics 10:368.
Nagakura Y, Yoshinaga T, Sakamoto T, Hattori K,
Okamoto N (2010) Susceptibility of fourfamilies derived from two Seriola species to themonogenean parasite (Benedenia seriolae) using
a new challenge method. Journal of Fisheries
Technology 3:21-26. (in Japanese)
Ogawa K (1996) Marine parasitology with specialreference to Japanese fisheries andmariculture. Veterinary Parasitology 64:95-105.
Ozaki A, Araki K, Okamoto H, et al. (2012) Progress of
DNA marker-assisted breeding inmaricultured finfish. Bulletin of Fisheries
Research Agency 35:31-37.
Ozaki A, Yoshida K, Fuji K, Kubota S, Kai W, Aoki JY,
Kawabata Y, Suzuki J, Akita K, Koyama T,
Nakagawa M, Hotta T, Tsuzaki T, Okamoto N,
Araki K, Sakamoto T (2013) Quantitative traitloci (QTL) associated with resistance to amonogenean parasite (Benedenia seriolae) inyellowtail (Seriola quinqueradiata) throughgenome wide analysis. PLoS One 8(6):e64987.
Wedemeyer G (1996) Physiology of fish in intensive
culture systems. London: Chapman & Hall.

DISCUSSION

We employed LD blocks based on QTL significant
region for MAS, and confirmed their effects insubsequent F2 full-sib families. Furthermore, it means
QTL significant regions are identified from these wild
ancestor to the breeding population. It would bepossible to introgress certain properties of the wild
population in the breeding population. These results
indicate that it is possible to rapidly develop
domesticated strains having commercially important
traits by MAS in aquaculture.
MAS will greatly increase the efficiency andeffectiveness of breeding compared to traditional
breeding programs. The fundamental advantage ofMAS compared to conventional phenotypic selectionis that it is faster since the early selection could bemade without collecting the phenotypic data, and fish
will be selected with high reliability, and successive
generations will enable avoidance of inbreeding
depression. And it is more accurate based on genetic
selection instead of phenotypic selection.
In addition, the potential of MAS and marker-assisted
introgression (MAI) use for disease risk managementof marine aquaculture finfish is considered. Wild
aquatic species are not selected and still maintain highgenetic diversities. Individuals have high potential for
genetic breeding regarding phenotypic variation.
Natural populations will be more appropriate tocontribute to those genetic resources to find large QTL
effects than strain populations, as breeding materials.
Therefore, aquatic species have a real possibility thatmarker-assisted selection could be a success.

29

authors constructed a high-coverage bacterial
artificial chromosome (BAC) library, obtained
sequences from the BAC-end, and first comprehensive
female and male linkage maps of yellowtail using
Simple Sequence Repeat (SSR) markers developed
from the BAC-end sequences and a yellowtail
genomic library. The total insert length of the BAC
library the authors constructed here was estimated tobe approximately 11 Gb and hence 16-times larger
than the yellowtail genome. Sequencing of the BACends showed a low fraction of repetitive sequencescomparable to that in Tetraodon and fugu. A total of837 SSR markers developed here were distributed
among 24 linkage groups spanning 1,026.70 and
1,057.83 cM with an average interval of 4.96 and 4.32
cM in female and male map respectively without any
segregation distortion. Oxford grids suggested
conserved synteny between yellowtail andstickleback. In addition to characteristics of yellowtail
genome such as low repetitive sequences andconserved synteny with stickleback, the genomic andgenetic resources constructed and revealed here will
be powerful tools for the yellowtail breeding programand also for studies regarding the genetic basis oftraits.

ANNOTATED BIBLIOGRAPHY

Ozaki A, Yoshida K, Fuji K, Kubota S, Kai W, Aoki JY,
Kawabata Y, Suzuki J, Akita K, Koyama T,
Nakagawa M, Hotta T, Tsuzaki T, Okamoto N,
Araki K, Sakamoto T (2013) Quantitative traitloci (QTL) associated with resistance to amonogenean parasite (Benedenia seriolae) inyellowtail (Seriola quinqueradiata) throughgenome wide analysis. PLoS One 8(6):e64987.
Benedenia infections caused by the monogenean fluke
ectoparasite Benedenia seriolae seriously impact marine
finfish aquaculture. Genetic variation in host has beeninferred to play a significant role in determining thesusceptibility to this parasitic disease. To evaluate thegenetic basis of Benedenia disease resistance inyellowtail (Seriola quinqueradiata), a genome-wide andchromosome-wide linkage analyses were initiatedusing F1 yellowtail families (n = 90 per family) basedon a high density linkage map with 860 microsatelliteand 142 single nucleotide polymorphism (SNP)
markers. Two major quantitative trait loci (QTL)
regions on linkage groups Squ2 (BDR-1) and Squ20(BDR-2) were identified. These QTL regions explained
32.9–35.5% of the phenotypic variance. On the other
hand, the relationship between QTL for susceptibility
to B. seriolae and QTL for fish body size were
investigated. The QTL related to growth was foundon another linkage group (Squ7). As a result, theauthors present first genetic evidence that contributes
to detailing phenotypic resistance to Benedenia
disease, and the results will help resolve themechanism of resistance to this important parasitic
infection of yellowtail.

Aoki J-y, Kai W, Kawabata Y, Ozaki A, Yoshida K,
Tsuzaki T, Fuji K, Koyama T, Sakamoto T,
Araki K (2014) Construction of a radiation
hybrid panel and the first yellowtail (Seriola
quinqueradiata) radiation hybrid map using ananofluidic dynamic array. BMC Genomics
15:165.
Yellowtail (Seriola quinqueradiata) are an economicallyimportant species in Japan. However, there are
currently no methods for captive breeding and earlyrearing for yellowtail. Thus, the commercial
cultivation of this species is reliant upon the captureof wild immature fish. Given this, there is a need todevelop captive breeding techniques to reducepressure on wild stocks and facilitate the sustainable
development of yellowtail aquaculture. the authors
constructed a whole genome radiation hybrid (RH)
panel for yellowtail gene mapping and developed aframework physical map using a nanofluidic dynamicarray to use SNPs (single nucleotide polymorphisms)
in ESTs (expressed sequence tags) for the DNAassisted breeding of yellowtail. Clonal RH cell lines
were obtained after ionizing radiation; specifically, 78,
64, 129, 55, 42, and 53 clones were isolated after
treatment with 3,000, 4,000, 5,000, 6,000, 8,000, or
10,000 rads, respectively. A total of 421 hybrid celllines were obtained by fusion with mouse B78 cells.

Fuji K, Koyama T, Kai W, Kubota S, Yoshida K, Ozaki
A, Aoki J-y, Kawabata Y, Araki K, Tsuzaki T,
Okamoto N, Sakamoto T (2014) Constructionof a high-coverage bacterial artificial
chromosome library and comprehensive
genetic linkage map of yellowtail Seriola
quinqueradiata. BMC Research Notes 7:200.
Japanese amberjack/yellowtail (Seriola quinqueradiata)
is a commonly cultured marine fish in Japan. For costeffective fish production, a breeding program that
increases commercially important traits is one of the
major solutions. In selective breeding, information ofgenetic markers is useful and sufficient to identify
individuals carrying advantageous traits but if theaim is to determine the genetic basis of the trait, large
insert genomic DNA libraries are essential. In thisstudy, toward prospective understanding of genetic
basis of several economically important traits, the30

Ninety-four microsatellite markers used in the genetic
linkage map were genotyped using the 421 hybrid celllines. Based upon marker retention and genome
coverage, the authors selected 93 hybrid cell lines toform an RH panel. Importantly, the authors
performed the first genotyping of yellowtail markers
in an RH panel using a nanofluidic dynamic array(Fluidigm, CA, USA). Using this, 580 markers
containing ESTs and SNPs were mapped in theyellowtail RH map. The authors successfully
developed the first yellowtail RH panel to facilitate
the localization of markers. This high-density physicalmap will serve as a useful tool for the identification ofgenes related to important breeding traits using
genetic structural information, such as conserved
synteny. Moreover, in a comparison of 30 sequences in
the RH group 1 (SQ1), yellowtail appeared to be
evolutionarily closer to medaka and the green-spotted
pufferfish than to zebrafish. The authors suggest thatsynteny analysis may be potentially useful as a tool toinvestigate chromosomal evolution by comparison
with model fish.

31

Examining Host-Pathogen Interactions at Mucosal Surfaces Reveals Novel
Molecular Targets for Columnaris Disease Intervention
Benjamin H. Beck1, Eric Peatman2
1

United States Department of Agriculture,
Agricultural Research Service, Stuttgart National
Aquaculture Research Center, Stuttgart, AR USA

2

Auburn University School of Fisheries, Aquaculture,
and Aquatic Sciences, Auburn, AL USA

INTRODUCTION

In contrast to their terrestrial agriculturalcounterparts, farmed fish species rely more heavily
on mucosal barriers as they are continuouslyinteracting with an aquatic milieu teeming withmicrobes. Unlike classical immune centers, such as
the spleen or anterior kidney, the accessibility of
mucosal surfaces through immersion/dip treatments
or dietary changes allows for tailored chemical andnutritional strategies to maximize mucosal and,
therefore, organismal health (Beck and Peatman2015). Indeed, many areas of intense research inaquaculture over the last decade have hinged upon abetter understanding of mucosal health.

Corresponding author: benjamin.beck at ars.usda.gov
Keywords: columnaris disease, RNA-seq,transcriptome, mucosal health, rhamnose-binding
lectinHowever, studies on immunity in farmed fish
species, as a whole, have either been comparative innature, seeking to verify the existence of immune
components known in mammals, or oriented aroundartificial disease challenges, with little or no
examination of underlying mechanisms.

ABSTRACT

Columnaris disease, caused by the bacterial pathogen
Flavobacterium columnare, is a major problem globallyand leads to tremendous losses of freshwater fish,particularly in intensively farmed aquaculture species.
Despite its widespread importance, our understandingof F. columnare infectious processes remains limited.
Specifically, little is known regarding the mechanisms
controlling pathogen adhesion and replication on hostmucosal surfaces. Utilizing next-generation
sequencing-based RNA-seq, we profiled thetranscriptome in columnaris disease resistant andsusceptible families of channel catfish to examinetranscriptional differences in mucosal barriers (e.g,skin and gill) before pathogen exposure and at early
timepoints following a columnaris challenge. The
results revealed a consistent pattern of basal immune
polarization between resistant and susceptible fishprior to challenge, including key differences in
expression of genes linked to mucin abundance andcomposition, lysozyme levels, and a rhamnosebinding lectin, a potential receptor for F. columnare.
Next, we profiled the transcriptome of mucosal tissuesof channel catfish under conditions of routine feeding
versus short-term feed deprivation. Surprisingly, we
found numerous shared mucosal gene expression
signatures between resistant/fed fish and susceptible/
fasted fish. The position of these molecular actors on
ectopic mucosal surfaces could make them accessible
for use as prognostic indicators of disease fitness andhighly amenable to modulation through dietary or
topical prophylactic or therapeutant approaches.

Opportunely, an array of new molecular tools has
become available in recent years, which is rapidly
evolving this area of study. The dramatic decline insequencing costs brought about by a shift tomassively parallel sequencing platforms has farreaching consequences for the study of immunity inaquaculture species. Transcriptome sequencing andgene expression profiling (RNA-seq) offer a rapid
approach to characterizing actors in mucosal tissues
and cell types and their changes following diseasechallenge, environmental perturbation, nutritionalchange, or a combination thereof.
In the present series of studies, we sought to better
understand the molecular immune actors that arepresent within mucosal immune compartments, bothat rest in naïve healthy channel catfish and after
experimental infection. We first compared the global
gene expression in the gill and skin isolated from two
different families of channel catfish with known
differential sensitivities (resistant versus susceptible)
to Flavobacterium columnare, the causative agent ofcolumnaris disease. Our analysis revealed earlymechanisms of pathogen adhesion and invasion anduncovered exciting and unanticipated associations
between nutrition and immunity. This led us to nextcompare the transcriptomes of mucosal tissues
32derived from channel catfish that were subjected to aperiod of short-term feed deprivation as compared toa well-fed treatment group. These studies bring tolight new links connecting mucosal immunity, diseasesusceptibility, and nutritional status (Sun et al. 2012;
Beck et al. 2012; Liu et al. 2013; Peatman et al. 2013; Liet al. 2014).

The k-mer size was set from 50 to 96, assemblies fromall k-mers were merged into one assembly by TransABySS. In order to reduce redundancy, the assembly
results from different assemblers were passed to
CD-Hit version 4.5.4 (Li and Godzik, 2006) and CAP3
(Huang and Madan, 1999) for multiple alignments
and consensus building after trimming contigs lessthan 200 bp. The threshold was set as identity equal to
1 in CD-Hit, the minimal overlap length and identity
equal to 100 bp and 99% in CAP3. The high quality
reads from each sample were mapped onto theTransABySS reference assembly using CLC Genomics
Workbench software. During mapping, at least 95% of
the bases were required to align to the reference and amaximum of two mismatches were allowed. The total
mapped reads number for each transcript wasdetermined and then normalized to detect RPKM
(Reads Per Kilobase of exon model per Million
mapped reads). The proportions-based test was used
to identify the differently expressed genes between
resistant and susceptible families with correctedp-value < 0.05 (Kal et al., 1999). After scaling
normalization of the RPKM values, fold changes were
calculated. Analysis was performed using the RNAseq module and the expression analysis module in
CLC Genomics Workbench (Robinson and Oshlack,2010)2010. Transcripts with absolute fold change
values of larger than 1.5 and a false discovery rate ofless than 0.05 were included in downstream analyses
as differentially expressed genes. We utilized qPCR
for validation of RNA-seq results and for examining
putative biomarker expression in additional
individual fish following published protocols (Li et al.
2012).

MATERIALS AND METHODS

Gene expression profiling
We utilized Illumina-based RNA-seq to profile global
transcriptional changes in the gill and skin ofcolumnaris disease resistant and susceptible channel
catfish before and after a columnaris disease challenge
and the gill and skin of channel catfish fed daily
versus fish deprived of feed for 7 days (d).
Columnaris challenges were conducted using the“Ultra Low-Flow System” described by Mitchell andFarmer (2010), which allows for a natural progressionof the disease after challenge in a flow-throughenvironment. Samples were collected at 0 h, 1 h, 2 h,
4 h, 8 h, and 24 h post-challenge. Pooled biologicalreplicates (consisting of equal amounts of tissue from
replicate fish) were collected from each experimentalgroup and stored until RNA extraction. For thefeeding study, juvenile channel catfish were randomly
distributed (30 per tank) among three 600 L tanks.
Fish were then subjected to one of three treatments.
Fish in treatment group 1 were fed to satiation threetimes daily with a standard catfish ration for 7 d. In
treatment 2, fish were withheld feed for 7 d. In
treatment 3, fish were withheld feed for 7 d,subsequently fed to satiation, and at 4 h following the
introduction of food, fish were sacrificed. All fish
were sacrificed on day 7, and portions of the gill andskin were isolated from equivalent locations on eachfish and stored until RNA extraction and sequencing.
Sequencing (Illumina HiSeq 100bp paired-end
sequencing) was performed at the HudsonAlpha
Institute for Biotechnology (Huntsville, Alabama).
RNA-seq library preparation and sequencing was
carried out as previously described (Li et al. 2012; Sunet al. 2012). Before assembly, raw reads were trimmed
by removing adaptor sequences and ambiguous
nucleotides. Reads with quality scores less than 20
and length below 30 bp were all trimmed. The
resulting high-quality sequences were used in thesubsequent assembly (Miller et al., 2010). The de novo
assembly was performed by de Brujin graphassembler ABySS (version 1.3.2) (Simpson et al., 2009).
Briefly, the clean reads were first hashed according toa predefined k-mer length, the ‘k-mers’. After
capturing overlaps of length k-1 between thesek-mers, the short reads were assembled into contigs.

Modulating rhamnose-binding lectin levels in vivo
While numerous changes in gene expression wereobserved, sequencing data revealed that a rhamnosebinding lectin (RBL) was both highly and exclusively
expressed in the susceptible family of catfish and in
fasted fish. To better understand the significance of
this lectin family on columnaris disease pathogenesis,
we blocked RBL with supraphysiological doses ofexogenous ligand. Briefly, a generic population ofchannel catfish fingerlings were stocked at a rate of 30
per tank. In triplicate tanks, the native ligand for
RBL, the sugar L-rhamnose, was added to tanks at
rates of 0 mM, 20 mM, and 40 mM. Fish were
exposed to the sugars for 15 minutes prior to bacterial
challenge. At the same timepoint, all challenged
tanks received the same dose of F. columnare cells fromthe same preparative stock. For each sugar, a single
tank was exposed to the 40 mM concentration but not
challenged with the bacterium as a negative control.
33

status as RBL levels were strongly downregulated
within 4 h of feeding in fasted fish (Fig. 1C).

As an additional negative control, a single tank ofunchallenged fish containing no sugar was used. Fishwere monitored daily and any dead fish were
promptly removed.

RESULTS AND DISCUSSION

Previously, global transcriptome profiling of thechannel catfish gill revealed a rhamnose-binding
lectin (RBL) whose expression was induced greaterthan 100-fold soon after F. columnare experimental
infection (Sun et al. 2012). Here, RBL expression was
found to be highly expressed in susceptible fish
before challenge and strongly upregulated early after
challenge, suggesting RBL expression is positively
correlated with host susceptibility to an F. columnare
infection (Fig. 1A). Saturation of the RBL with theligand L-rhamnose lowered its expression levels (notshown) and decreased F. columnare mortality in adosage-dependent manner in a subsequent challenge
infection (Fig. 1B). Taken together, these results
suggest that host expressed rhamnose-binding lectins
are mediating F. columnare binding to the surface
mucosa. We next asked if changes in feeding,previously tied to F. columnare susceptibility (Klesius
et al. 1999), could impact RBL expression. Indeed, aperiod of 7 d fasting upregulated RBL expression in
the catfish gill greater than 120-fold (Fig. 1C).
Expression of RBL was exquisitely sensitive to feeding

Figure 2. Connecting host susceptibility with nutritional status, evidence
for immune-nutritional co-regulation. Shared patterns of gene expression
are evident in columnaris resistant fish and well-fed fish. Similarly, fishsusceptible to columnaris disease share many transcriptional hallmarks
with fish subjected to short-term feed deprivation. Some example genes
are listed here.

This striking co-regulation of a mucosal lectin by both
infection and nutritional status led us to a broader
examination of the RNA-seq data, which revealed
numerous shared host gene signatures in fasted vs.
fed and F. columnare resistant vs. susceptible catfish.
Our results indicate that, beyond RBL, criticalcomponents of the innate immune response
governing host susceptibility to F. columnare were
perturbed by short-term feed deprivation (Fig. 2; Becket al. 2012; Peatman et al. 2013; Liu et al. 2013; Li et al.
2014a). Both F. columnare susceptible and fasted (for7 days) channel catfish featured altered
arginine metabolism pathways, criticalfor production of inducible nitric oxide
synthase (iNOS) and its effector nitricoxide (NO). iNOS expression, which
we found to be extremely abundant in
the fed catfish gill, was down-regulated
greater than 17-fold following fasting.
We also found evidence of immunonutritional co-regulation of lysozyme
Figure 1. Foe and famine: striking
co-regulation of rhamnose-binding lectin
expression by infection and nutritional
status. A.) Fish from a columnaris
disease susceptible family strongly
upregulate RBL expression after pathogen
encounter. B.) Saturation of RBL
receptors with ligand (L-rhamnose)
protects fish from columnaris disease in adose-dependent manner. C.) RBL
expression in the gill is markedly
upregulated after short-term feed
deprivation in channel catfish.

34

blue catfish (Ictalurus furcatus) Developmental
and Comparative Immunology 44(1):226-234
Li W. and Godzik A., 2006. Cd-hit: a fast program forclustering and comparing large sets of protein
or nucleotide sequences. Bioinformatics 22,
1658-1659.
Liu L, Li C, Su B, Beck BH, Peatman E (2013) Shortterm feed deprivation alters immune status of
surface mucosa in channel catfish (Ictaluruspunctatus). PLoS One 8:e74581.
Miller J. R., Koren S. and Sutton G., 2010. Assembly
algorithms for next-generation sequencing
data. Genomics 95, 315-327.
Mitchell AJ, and Farmer BD. 2010. Evaluation of an
ultra low-flow water delivery system for small
experimental tanks. North American Journalof Aquaculture 72:195-200.
Peatman E, Li C, Peterson BC, Straus DL, Farmer BD,
Beck BH. 2013. Basal polarization of themucosal compartment in Flavobacterium
columnare susceptible and resistant channel
catfish (Ictalurus punctatus). Molecular
Immunology 56:317-327.
Robinson M. D. and Oshlack A., 2010. A scaling
normalization method for differential
expression analysis of RNA-seq data. Genome
biology 11, R25.
Simpson J. T., Wong K., Jackman S. D., Schein J. E.,
Jones S. J. and Birol I., 2009. ABySS: a parallel
assembler for short read sequence data.
Genome research 19, 1117-1123.
Sun F, Peatman E, Li C, Liu S, Jiang Y, Zhou Z, Liu Z.
2012. Transcriptomic signatures ofattachment, NF-ĸB suppression and IFN
stimulation in the catfish gill following
columnaris bacterial infection. Developmental
and Comparative Immunology 38:169-180.

levels, MHC class I/II profiles, mucin secretion, andthe chemokine repertoire (Liu et al. 2013; Li et al.
2014a).
We highlight these studies as an illustration of how
sequencing approaches can reveal key mucosal
players and pathways missed by relying too heavily
on mammalian immune paradigms. To bring theseRNA-seq studies from the level of interestingobservation to application, we are currently studying
the impact of rhamnose on both host susceptibility
and pathogen virulence, formulating diets toexperimentally modulate goblet cell formation andmucin secretion, and pursuing RBL knockout
fish lines.

REFERENCES

Beck BH, Peatman E, Editors. 2015. Mucosal Healthin Aquaculture. Published by Elsevier,
Waltham, Massachusetts.
Beck BH, Farmer BD, Straus DL, Li C, Peatman E.
2012. Putative roles for a rhamnose binding
lectin in Flavobacterium columnare pathogenesis
in channel catfish Ictalurus punctatus. Fish
Shellfish Immunol 33:1008-1015.
Huang X. and Madan A., 1999. CAP3: A DNA
sequence assembly program. Genome research
9, 868-77.
Kal A. J., van Zonneveld A. J., Benes V., van den Berg
M., Koerkamp M. G., Albermann K., Strack N.,
Ruijter J. M., Richter A., Dujon B., Ansorge W.
and Tabak H. F., 1999. Dynamics of gene
expression revealed by comparison of serial
analysis of gene expression transcript profiles
from yeast grown on two different carbon
sources. Molecular biology of the cell 10,
1859-1872.
Klesius P, Lim C, Shoemaker C. 1999. Effect of feed
deprivation on innate resistance and antibody
response to Flavobacterium columnare inchannel catfish, Ictalurus punctatus. Bulletinof the European Association of Fish
Pathologists 19:156-158.
Li, C., Zhang, Y., Wang, R., Lu, J., Nandi, S., Mohanty,
S., Terhune, J., Liu, Z., Peatman, E., 2012. RNAseq analysis of mucosal immune responses
reveals signatures of intestinal barrier
disruption and pathogen entry following
Edwardsiella ictaluri infection in channel
catfish, Ictalurus punctatus. Fish & Shellfish
Immunology 32, 816-827.
Li C, Beck BH, Peatman E. 2014. Nutritional impacts
on gene expression in the surface mucosa of

ANNOTATED BIBLIOGRAPHY

Beck, B.H., Farmer, B.D., Straus, D.L., Li, C., Peatman,
E. 2012. Rhamnose-binding lectins and theirligands: Putative roles in Flavobacterium
columnare pathogenesis in channel catfish
Ictalurus punctatus. Fish and Shellfish
Immunology. 33(4):1008-1015.
The authors examined the role of a family ofcarbohydrate binding molecules termed rhamnosebinding lectins (RBL) in columnaris disease. After alaboratory challenge with Flavobacterium columnare,
the authors monitored the levels of RBL in the gill of
two different families of channel catfish; one family
35was previously shown to be susceptible to columnaris
disease, while the other was completely resistant.
Unexpectedly, the authors found that RBL expression
in the gill was strongly upregulated in thecolumnaris-susceptible family of channel catfish but
not the resistant. To better determine if RBLs were
influencing columnaris pathogenesis, fish werepretreated with the native RBL ligands L-rhamnoseand D-galactose, and investigators found that thesesugars protected channel catfish against columnaris
disease, likely through competition with F. columnare
for binding on host RBL. The research team also
examined the role of nutritional status on RBL
regulation and found that RBL expression was
upregulated in fish fasted for 7 d (as compared to fish
fed to satiation daily), yet expression levels returnedto those of satiated fish within 4 h after re-feeding.
Collectively, these findings position RBL as animportant determinant of columnaris disease
outcomes and reveal new aspects linking innate
immunity to feed availability.

mucosa, with susceptible fish possessing a putativemucosecretory, tolerogenic phenotype which may
predispose them to F. columnare infection.
Liu, L.G., Li, C., Su, B., Beck, B.H., Peatman, E. 2013.
Short-term feed deprivation alters immune
status of surface mucosa in channel catfish(Ictalurus punctatus). PLoS One. 8(9):e74581.
doi:10.1371/journal.pone.0074581.t004.
Short-term feed deprivation is a frequent occurrencein farmed fish species and results from seasonalchanges, production strategies, or as a means tocombat certain disease outbreaks. In channel catfish,periods of fasting have been linked to increases in thesusceptibility of fish to columnaris disease. The
authors examined changes at the transcriptional levelin the gill and skin (the target tissues of columnaris)in fish that were subjected to a 7 day period of fasting.
In comparison to fish fed daily, fasted fish showedchanges in the levels of over 1,500 genes. Strikingly,
the results showed a marked downregulation of
innate immune actors involved in the host defense tobacterial pathogens. These changes are predicted to
impact the delicate recognition/tolerance balance forcommensal and pathogenic bacteria on the skin andgill. The highlighted transcriptional profiles reveal
exciting new interrelationships between nutrition,
mucosal integrity, and immunity in teleost fish.

Peatman, E., Li, C., Peterson, B.C., Straus, D.L.,
Farmer, B.D., Beck, B.H. 2013. Basal
polarization of the mucosal compartment in
Flavobacterium columnare susceptible andresistant channel catfish Ictalurus punctatus.
Molecular Immunology. 56(4):317-327.
Despite recent progress in Flavobacterium columnare
research, little was known about how host molecularand cellular components modulated rates of adhesion,
tissue invasion, and ultimately, mortality. Here, theauthors profiled gene expression differences in gillbetween two channel catfish families (Ictaluruspunctatus) differing in their susceptibility to F.
columnare at both basal (in naïve fish), and at early
timepoints post-infection. Between groupcomparisons revealed over 1,700 unique genes
differentially expressed greater than 1.5-fold at one ormore timepoints. The authors focused on the analysis
of basal differential expression between resistant andsusceptible catfish as these genes could potentiallyreveal molecular actors modulating the differential
susceptibility to infection noted between the two
families. A number of critical innate immune effectors,
including iNOS2b, lysozyme C, IL-8, and TNF-alphawere constitutively higher in resistant catfish gill,while susceptible fish showed high expression levelsof secreted mucin forms, a rhamnose-binding lectin
previously linked to susceptibility, and mucosal
immune factors such as CD103 and IL-17. Taken
together, the immune and mucin profiles obtained byRNA-seq suggest a basal polarization in the gill
36

Genetics and Breeding of Highly Fecund Marine Species(sweepstakes reproductive success) reduce effective
population sizes and increase genetic drift in closed
aquaculture populations, which then have increased
risk of losing genetic diversity and fitness, owing to
inbreeding depression. Crossbreeding programs,
because they are based on controlled, systematic
inbreeding, can prevent inbreeding depression fromreaching farmed populations. Progress incrossbreeding can be accelerated, moreover, byapplication of genomic science to a global,
mechanistic understanding of physiological andmetabolic processes underlying production traits.

Dennis Hedgecock
Department of Biological Sciences,
University of Southern California, 3616 Trousdale
Parkway, Los Angeles CA 90089-0371
Corresponding author: dhedge at usc.edu
Keywords: selection, inbreeding depression,
sweepstakes reproductive success, crossbreeding,heterosis

ABSTRACT

Aquaculture genetics and breeding is dominated by afocus on selection, largely as a result of its
transformative success in improving salmon andrainbow trout. However, application of selection tohighly fecund marine species faces challenges, which
are not found with low fecundity animals, including
freshwater and anadromous fish—higher likelihoodof random genetic drift, owing to sweepstakes
reproductive success, higher genetic loads, and mostimportant, larger non-additive components of genetic
variance in yield-related traits.

INTRODUCTION

The characteristics of farmed plants and animals havetraditionally been improved by two fundamentallydifferent methods, selection and crossbreeding.
Selection is based on additive genetic variance in thetrait or traits to be improved. When a large
proportion of the variance in a trait is caused byadditive genetic variance, the trait has highheritability, and the “breeding values” of individuals
can be assessed by phenotypic information fromprogeny or relatives. Alleles with favorable influenceon the trait in question are passed through thegametes of individuals with high breeding value,
improving that trait in the next generation (Fig. 1A).
Selection has been extraordinarily successful inimproving livestock species and is arguably the single
most important factor responsible for the remarkable
growth of salmonid aquaculture over the last 45 years(Gjedrem 2010, 2012).

Crossbreeding offers an alternative strategy for
improving highly fecund species with thesecharacteristics. For example, the Pacific oyster shows
dramatic heterosis (hybrid vigor) for yield and can be
improved through crossbreeding of inbred lines, as
demonstrated in corn, rice, and other major crops. In
addition to improving yield, crossbreeding provides ameans for improving triploid oysters and thetetraploid lines used to produce triploid seed.
Unfortunately, heterosis has been little investigated inother highly fecund aquatic species, including marine
fishes.

Crossbreeding, on the other hand, is based on nonadditive genetic variation in the traits or traits to be
improved (Fig. 1B). Non-additive genetic variation
results from an interaction between alleles at the same
locus (dominance) or between alleles at different loci(epistasis), such that the performance of a particulargenotype cannot be reliably predicted from theaverage performance of its parents. The classic
example of non-additive variation is hybrid vigor incorn, rice, and other crops (Crow 1998; Birchler et al.
2010). High-yielding corn hybrids are produced bycrossing elite inbred lines that have been selected fortheir special combining ability after testing millions ofcombinations. These inbred lines are typicallyderived from the same species, which is naturally

The Pacific oyster also shows strong inbreeding
depression for yield characteristics, particularly
mortality of early life stages. High early mortality(type-III survivorship) is a consequence of strong
natural selection against deleterious mutations thatare likely generated as a by-product of high fecundity.
Selection for early life-history traits or for survival in
the face of diseases or environmental challenges mustaccount for this strong background selection. Finally,
unequal reproductive contributions by broodstock37

recruitment. At the same time, however, the periodic
life history may impact domestication andimprovement for aquaculture in two ways, (1) bymaking possible large variance in reproductive
success, increasing genetic drift, and (2) by increasing
the number and potentially the initial frequencies of
new mutations, most of which are deleterious,
contributing to a large genetic load and a highpotential for inbreeding depression.
Genetics and breeding of the Pacific oyster
Crassostrea gigas
Selection for disease resistance in different oyster
species has been successful (Haskins and Ford 1979;
Dégremont et al. 2015a, b), largely because the natural
selective pressure on diseases is quite severe. Familybased selection has also been successful in the Pacificoyster for improving resistance (and susceptibility) tosummer mortality (Dégremont et al. 2010) and forincreasing yield (Langdon et al. 2003; Kube et al.
2011). More recently, implementation of family-based
selection programs, utilizing an animal model,
economic weighting of traits, and index selection on
multiple traits (Kube et al. 2011; Small et al. 2016;
Langdon et al. 2016, submitted), has demonstrated thepower of selection to improve the performance and to
reduce the costs of producing farmed oysters. Again,
the demonstrated response to selection in these cases
is compelling evidence of substantial additive genetic
variance in the underlying traits.

Figure 1. Bell-shaped curves represent the relative frequencies of trait
values, say for yield, in large breeding populations. Yield, on thehorizontal axis, is standardized to have a mean of zero and a standard
deviation of 1.0; yields to the right of the zero are above-average, those to
the left, below average. Most individuals are close to the average andvery few families have extremely high or extremely low yields. (A) The
process of selective breeding begins, in the upper left figure, by taking
parents of the next generation from the upper end of the yield-distribution(i.e. to the right of the black vertical line). To the extent that theseselected parents pass genes to their offspring that cause those offspring
to have high yield, we say that the parents have high “breeding value.”
The distribution of yield is shifted to the right in the next generation (lower
left figure), so that the offspring generation has a higher average yield thanits parents. In this way, steady improvement can be achieved. Note alsothat genetic diversity is reduced, as a consequence of having been
propagated by only a select fraction of the population at each generation.
(B) Crossbreeding is indicated when the highest yield is achieved byhybrid offspring of two inbred parent lines. This is the phenomenon of“hybrid vigor,” which has dramatically improved the yields of the major
agricultural crops over the last half-century. Note that hybrid vigor is notstrictly heritable, i.e. the highest performing individuals in the upper end of
the lower right distribution cannot pass on high yield to their offspring
because performance depends on a combination of genes that is
re-shuffled during sexual reproduction. Superior hybrids must be
produced each generation by repeating the same cross of inbred parent
lines. Note also that the distribution of yield in the hybrid population has asmaller standard deviation because the individuals are genetically uniform.

Non-additive genetic variance is also important inoysters, however, as evidenced by yield heterosis(hybrid vigor) in the Pacific oyster that is as dramaticas that in maize (Crow 1998), even in crosses among
inbred lines derived from the same wild population(Hedgecock et al. 1995; Hedgecock and Davis 2007;
Fig. 2). Yield heterosis is associated with equally
dramatic inbreeding depression (Evans et al. 2004),
attributed to a remarkably large load of deleterious
recessive and partially dominant mutations (Bierne etal. 1998; Launey and Hedgecock 2001; Plough andHedgecock 2011; Plough 2012; Plough et al. 2016).
Inbreeding depression (ID) is the proportional
reduction in fitness (w) or traits related to fitness,such as viability or growth, of inbred relative torandom-bred individuals: : ID = 1 - (winbred ⁄ wrandom). Evans et al. (2004) found a 25% reduction in yieldbred
for oysters with an average inbreeding of ~0.2
(slightly less than the expected inbreeding level ofprogeny from a brother-sister mating). Hedgecockand Davis (2007) obtained ID = 0.64 for seed weight in
inbred lines. Most of the inbreeding depression inoysters is expressed as early mortality, which is

outcrossing; thus, crossbreeding is distinguished frominterspecific hybridization. While crossbreeding ismostly applied to crops, it is also used in combination
with selection in cattle, swine, and poultry breeding
programs. Crossbreeding may be an importantmeans for improving production of highly fecundmarine aquaculture species. Here, the Pacific oyster
Crassostrea gigas provides a case study for illustrating
this point of view.
The great fecundity of marine fish and shellfish
distinguishes them from most terrestrial animals andeven from many freshwater and anadromous fishes(Palumbi and Hedgecock 2005). High fecundity is
generally coupled in marine species with high earlymortality (type-III survivorship) and delayed
maturation (adult longevity), forming what
Winemiller (2005) calls the “periodic” life history.
This periodic life history is adaptive to the vagaries ofthe marine environment, especially the oceanographic
variability that impacts early development and38

of individuals and very easy for some individuals tohave greater access to food than others. Suchcompetition can cause an amplification in the varianceof size among individuals within a cage, potentiallyobscuring their inherent “breeding value.” To someextent, this competition can be ameliorated throughvigilant husbandry, in which animals are periodically
redistributed within rearing units. Still, measures ofindividual performance are bound to be much lessreliable indicators of breeding value in oysters than inlivestock, because of close communal rearing andcompetition for food. For this reason, many programs
select among oyster families based on average yield
per production unit, rather than the performance ofindividuals.
Second, individual oysters are worth much, much lessthan prize livestock sires. Prize bulls can sell for
hundreds of thousands of dollars, for example.
Moreover, despite their reputation for fecundity, amale oyster can have a smaller lifetime reproductive
capacity than a prize bull, whose artificiallycryopreserved sperm can be periodically harvested
over his lifetime and used repeatedly to inseminate
thousands of cows over time. The record in thisregard is 400,000 cows inseminated by a single bull.
Since gametes are stripped from mature oysters, asingle oyster will generally be used once, to fertilize
tens of millions of eggs, of which only about 1% willsurvive.

Figure 2. Crossing inbred lines of oysters and of corn produces reciprocal
hybrid oysters that are, in both cases, much higher-yielding than theirinbred parent lines (top). Bottom photograph of hybrid corn reproduced
from original by G.H. Schull in 1908.

substantial, as much as 99% of fertilized eggs (Launeyand Hedgecock 2001; Plough and Hedgecock 2011).
We have estimated that an average wild oyster carries
a minimum of about 15 lethal mutations (compared 3
or 4 in humans or fruit flies).

The value of prized livestock sires comes from theirdemonstrated breeding value, as established throughprogeny testing. Strip spawning precludes progeny
testing. Routine cryopreservation of oyster sperm has
been demonstrated (Dong et al. 2007) and would
make progeny tests possible, but this technology has
not been widely adopted by industry. Breedingvalues can be established from information on theperformance of relatives, and selected populations
propagated by use of related sires and dams.

A large mutational load in oysters was predicted by
G. C. Williams (1975), in the Elm-Oyster model for theadvantages of sexual reproduction in species withhigh fecundity and high early mortality. Since highfecundity and high early mortality are the dominant
life history features among marine fish (Winemiller
2005) and invertebrates (Thorson 1950), considerable
scope for genetic improvement likely lies incrossbreeding of inbred lines.

Second, inbreeding depression is severe in oysters
and likely other highly fecund marine species andmay eliminate or even reverse genetic gains fromselection, if inbreeding is not held in check. Selection
inevitably results in inbreeding, since only a smallpart of the population is used for reproduction, andindividuals in small populations eventually become
highly related. Improvement, then, requires a balance
between the response to selection and the increase ininbreeding depression over time. There are very few
data on long-term selection programs in oysters.
Selection for MSX resistance of eastern oysters,

Challenges for selection of marine species
While selection has clearly proven effective in
improving production traits of oysters, sustaining
these genetic gains over longer time periods poses
several challenges. First, competition for food among
filter-feeding oysters within a rearing unit can bemuch more severe than competition for food among
livestock. It is very difficult to monitor the feed intake
39initiated by Dr. Hal Haskin in Delaware, led
ultimately to such a decline in growth rate that theresistant stocks were not commercially useful. On theother hand, a well-designed family-based selection
program can keep inbreeding at about 1% per
generation (Kube et al. 2011).

Challenges and opportunities for crossbreeding ofmarine species
Like corn, oysters show hybrid vigor in experimental
crosses (Fig. 2; Hedgecock et al. 1995; Hedgecock andDavis 2007). As noted, the life-history and genetic
parallels between oysters and plants were first notedby G. C. Williams (1975) in his Elm-Oyster Model.
Both oysters and corn are naturally outcrossing
species, so the phenomenon of hybrid vigor is notobservable in wild populations but can only be seen,
first, by making inbred parent lines artificially and,then, by making crosses between inbred parents. The
resulting hybrids almost always yield more than theirinbred parents but, on average, may be no different
than the average wild genotype.

Finally, another challenge to the long-term efficacy of
selective breeding is high variance in the reproductive
success of oysters and other highly fecund marine
species (Hedgecock and Pudovkin 2011). Variance inoffspring numbers inevitably causes a reduction in
genetic diversity, by reducing the effective size of the
population, Ne, compared to the actual size of the
population, N. For example, Boudry et al. (2002)
obtained sperm from five male oysters and used it tofertilize eggs from five female oysters, thereby
producing 25 families in a full, factorial cross. In thiscase, N = 5 males + 5 females = 10. In one experiment,
Boudry et al. (2002) made 25, separate fertilizations
and combined equal numbers of embryos from eachfamily at 3 h post-fertilization. After 90 days, theyused genetic markers to determine the proportional
contribution of each family to the mix, which were far
from equal, although most of the 25 families had some
representation. Still, the effective size of the offspring
population was 6.2, substantially lower than 10. In asecond experiment, Boudry and colleagues pooled
equal numbers of eggs and equal amounts of sperm
in a mixed fertilization, mimicking the mass spawns
that are typical of commercial shellfish hatcheries.
Again, after 90 days, the proportional contributions ofeach family to the mix were highly skewed, making
the effective size of the offspring population
only 3.2, instead of 10. Similar results wereobtained by Matson (2010). Mass spawns
can greatly decrease population sizes,
causing a loss of genetic diversity andleading inevitably to inbreeding. This can beameliorated by performing controlled paircrosses and keeping families separate at alllife stages or by use of molecular markers toreconstruct the parentage of individuals in acohort.

There are two reasons why crossbreeding does not
simply restore the average wild type, however. First,
in a crossbreeding program, one is constantly
selecting among lines for those with superiorcombining ability. Our preliminary data suggest thata doubling of oyster yield could be achieved withcurrent “elite” inbred lines (Fig. 3). A doubling ofyield in corn took ~30 years in the double-cross era(from ~1930 to ~1960; see Fig. 1 in Crow 1998).
Second, a hybrid population is genetically uniform,
rather than heterogeneous, like a random-mating wild
population, so that the crop is uniformly highyielding (suggested by the reduced variance in Fig.
1B). Currently, shellfish farmers incur substantial
costs in grading product for the size-dependent, live,half-shell market.

Although risks from these challenges to thelong-term sustainability of selective breeding
can be managed, crossbreeding offers analternative method for improvement thateliminates several of these challenges. Crossbreeding
also allows substantial non-additive variance to be
used.

Figure 3. Hybrids produced by a factorial cross of males and females froma set of superior inbred lines. The best hybrids show twice the yield ofoysters produced from wild parents. Note also that reciprocal hybrids areoften different (e.g. 47×92 > 92×47) and that one inbred line performs
better than the two wild families.

40

Progress in crossbreeding can now be accelerated
through the use of genomic biotechnologies based on
fundamental understanding of the mechanisms ofhybrid vigor (Applebaum et al. 2014). The
publication in 2012 of a genome sequence for the
Pacific oyster (Zhang et al. 2012) provided resources
for fundamental research that are uncommon formarine aquaculture species. When combined withexperimental approaches that provide well-controlled
biological contrasts for physiological analyses, thesegenomic resources allow powerful insights into
biological mechanisms.

(Applebaum et al. 2014). Such fundamental
knowledge could lead to the development ofbiomarkers that could be used to predict which crosswill produce the best hybrids early in the life cycle,saving much effort in progeny testing.
Finally, crossbreeding offers a path to theimprovement of triploid oysters (Piferrer et al. 2009)and ultimately the tetraploid broodstocks that areused to generate triploids for production (Allen 2011).
First, it should be noted that, in plants, heterosis is“progressive” in polyploids, i.e. it increases with thediversity of genomes in polyploids (Birchler et al.
2010); for yield or hardiness of tetraploid plants,
ABCD > AABB > AAAB. Previously, tetraploidoysters were made from triploid females (Guo andAllen 1994; McCombie et al. 2005), which were
derived from chemical inhibition of the second
meiotic division of a fertilized egg. If the female used
for chemical induction of triploidy were a hybrid (AB)and the fertilizing sperm were provided by a malefrom an unrelated but well-combining inbred line(CC), then the triploid progeny would be a mix of
AAC, BBC, and ABC, depending on the distance ofgenes from the centromere on each chromosome. It
would be difficult to optimize the heterosis in such a
mix and to transfer it to the tetraploid level. Recently,
however, a new method for making tetraploids
directly from diploid parents has opened up a
promising new path for inducing heterosis in
tetraploid stocks (Benabdelmouna and Ledu 2015).

One example of an experimental approach, which isgreatly enhanced by having a genome, isQuantitative-Trait Locus (QTL) mapping. A QTL map
for live-weight at 10 months of age in one F2 Pacificoyster family shows five, major, significant peaks,
suggesting that variance in growth is mainly
controlled by five regions of the genome (Fig. 4).
Interestingly, preliminary estimates of genotypicvalues at each of these QTL contradict the two oldest
theories of heterosis, the dominance andoverdominance hypotheses (see Crow 1998; Birchler
et al. 2010), since the heterozygotes are like theinferior inbred genotype at each QTL. With the
genome sequence in hand, we can begin to examine
the several hundred genes in the vicinity of each peak(out of a total of 28,027; Zhang et al. 2012) for theirpotential role in regulating growth physiology

Figure 4. A Quantitative Trait Locus(QTL) map for live weight at 10
months of age in an F2 family of
the Pacific oyster. The x-axis of thelower panel represents the linkage
map of the oyster, in centiMorgans,across the 10 chromosomes; theblack tick marks show the locationsof the 53 genetic markers for which
each of 376 individuals was typed.
The Y-axis gives a measure of thestatistical association between
individual genotypes at eachposition of the genome andindividual live weight; the dottedline is a threshold for significance.
Estimates of genotype live weights
at the five major QTL peaks showsthat the heterozygote is in all cases
like the smaller homozygote, aresult that fits neither thedominance nor the overdominancetheories of hybrid.

41

CONCLUSIONSREFERENCES

1. Marine fish and shellfish can undoubtedly be
improved by selective breeding. Because
broodstock are still being obtained from natural
populations in many cases, traits of importance inproduction are likely to have substantial additive
genetic variance and will respond to selection.
2. The periodic life history of oysters and othermarine animals presents a set of unique challenges
to selection, such as defining the units of selection,
determining breeding values in the face ofcompetition in communal rearing, inbreeding
depression, and high variance in reproductive
success, leading to small effective population
sizes.
3. Oysters and perhaps other highly fecund marine
fish and shellfish can also be improved bycrossbreeding, which can capture substantial,
non-additive, genetic variance in yield and othertraits. Hybrid vigor has been documented in the
Pacific oyster, but there are few studies to date on
inbreeding and crossbreeding of other bivalve
molluscs and marine fish. Crossbreeding alsooffers a path to improvement of polyploidvarieties, which are useful in production and inpreventing reproductive interactions between
farmed and wild populations.
4. There are no major technical impediments todomesticating and improving farmed marine fishand shellfish, but disciplined breeding programs
are needed to conserve natural biodiversity andmake marine aquaculture sustainable in the longrun.
5. Genomic tools are helping to accelerate
improvement, by revealing mechanisms
underlying heterosis and other traits, which may,in turn, lead to the development of biomarkers for
identifying superior combining ability early in thelife cycle.

42

Allen S. K., Jr. 2011. The challenge of domesticating
tetraploid brood stock for the production of
triploid oyster seed. J. Shellfish Res. 30: 481481.
Applebaum S. L., T.-C. F. Pan, D. Hedgecock, and D.
T. Manahan. 2014. Separating the nature andnurture of energy allocation in response to
global change. Integr. Comp. Biol. 54: 284-295.
Benabdelmouna A. and C. Ledu. 2015.
Autotetraploid Pacific oysters (Crassostrea
gigas) obtained using normal diploid eggs:
induction and impact on cytogenetic stability.
Genome 58: 333-348.
Bierne N., S. Launey, Y. Naciri-Graven, and F.
Bonhomme. 1998. Early effect of inbreeding as
revealed by microsatellite analyses on Ostrea
edulis larvae. Genetics 148: 1893-1906.
Birchler J. A., H. Yao, S. Chudalayandi, D. Vaiman,
and R. A. Veitia. 2010. Heterosis. Plant Cell 22:
2105-2112.
Boudry P., B. Collet, F. Cornette, V. Hervouet, and F.
Bonhomme. 2002. High variance inreproductive success of the Pacific oyster(Crassostrea gigas, Thunberg) revealed by
microsatellite-based parentage analysis ofmultifactorial crosses. Aquaculture 204: 283296.
Crow J. F. 1998. 90 years ago: The beginning of hybridmaize. Genetics 148: 923-928.
Dégremont L., E. Bedier, and P. Boudry. 2010. Summer
mortality of hatchery-produced Pacific oyster
spat (Crassostrea gigas). II. Response to
selection for survival and its influence on
growth and yield. Aquaculture 299: 21-29.
Dégremont L., M. Nourry, and E. Maurouard. 2015a.
Mass selection for survival and resistance to
OsHV-1 infection in Crassostrea gigas spat infield conditions: response to selection after
four generations. Aquaculture 446: 111-121.
Dégremont, L., C. Garcia, and S. K. Allen, Jr. 2015b.
Genetic improvement for disease resistance inoysters: A review. J Invert. Pathol. 131: 226-241.
Dong Q., C. Huang, B. Eudeline, and T. R. Tiersch.
2007. Cryoprotectant optimization for spermof diploid Pacific oysters by use of commercial
dairy sperm freezing facilities. Aquaculture
271: 537-545.
Evans F., S. Matson, J. Brake, and C. Langdon. 2004.
The effects of inbreeding on performance traitsof adult Pacific oysters (Crassostrea gigas).
Aquaculture 230: 89-98.
Gjedrem, T. 2010. The first family-based breeding
program in aquaculture. Rev. Aquaculture 2:
2-15.

Gjedrem, T. 2012. Genetic improvement for thedevelopment of efficient global aquaculture: A
personal opinion review. Aquaculture 344:
12-22.
Guo X. and Allen S. K., Jr. 1994. Viable tetraploids in
the Pacific oyster (Crassostrea gigas, Thuneberg)
produced by inhibiting polar body 1 in eggs
from triploids. Mol. Mar. Biol. Biotechnol. 3:42-50.
Haskin, H. H. and S. E. Ford. 1979. Development ofresistance to Minchinia nelsoni (MSX)mortality in laboratory-reared and native
oyster stocks in Delaware Bay. Mar. Fish. Rev.
41: 54-63.
Hedgecock D. and J. P. Davis. 2007. Heterosis for yieldand crossbreeding of the Pacific oyster
Crassostrea gigas. Aquaculture 272S1:S17–S29.
Hedgecock D. and A. I. Pudovkin. 2011. Sweepstakes
reproductive success in highly fecund marine
fish and shellfish: A review and commentary.
Bull. Mar. Sci. 87: 971-1002.
Hedgecock D., D. J. McGoldrick, and B. L. Bayne.
1995. Hybrid vigor in Pacific oysters: An
experimental approach using crosses among
inbred lines. Aquaculture 137: 285-298.
Kube P., M. Cunningham, S. Dominik, S. Parkinson, B.
Finn, J. Henshall, R. Bennett, and M. Hamilton.
2011 Enhancement of the Pacific oyster
selective breeding program. FRDC andSeafood CRC Final Report Project No.
2006/227, CSIRO Marine and AtmosphericResearch, GPO Box 1538, Hobart, TAS 7001,
Australia.
Langdon C., F. Evans, D. Jacobson, and M. Blouin.
2003. Improved family yields of Pacific oysters
Crassostrea gigas Thunberg derived fromselected parents. Aquaculture 220: 227-244.
Langdon C., C. Melo, E. Durland, and B. Schoolfield.
2016. Genetics parameters for performancetraits of the Pacific oyster Crassostrea gigas.
Aquaculture 2016 Abstracts.
Launey S. and D. Hedgecock. 2001. High genetic
load in the Pacific oyster. Genetics 159: 255265.
Matson S. E. 2010. Development, evaluation, andapplication of a mixed-family selective
breeding method for the Pacific Oyster(Crassostrea gigas). PhD dissertation, Oregon
State University.
McCombie H., C. Ledu, P. Phelipot, S. Lapègue, P.
Boudry P., and A. Gérard. 2005. A
complementary method for production oftetraploid Crassostrea gigas using crosses

between diploids and tetraploids withcytochalasin B treatments. Mar. Biotechnol. 7:318-330.
Palumbi S. R. and D. Hedgecock. 2005. The life of thesea: Implications of marine population biology
to conservation policy. In E.A. Norse and L.B.
Crowder (eds), Marine Conservation Biology:
The Science of Maintaining the Sea’s
Biodiversity, Island Press, Washington, D.C.,
Pp. 33-46.
Piferrer F., A. Beaumont, J.-C. Falguière, M. Flajšhans,
P. Haffray, L. Colombo. 2009. Polyploid fishand shellfish: Production, biology andapplications to aquaculture for performance
improvement and genetic containment.
Aquaculture 293: 125-156.
Plough L.V. 2012. Environmental stress increases
selection against and dominance of deleterious
mutations in inbred families of the Pacificoyster, Crassostrea gigas. Molecular Ecology 21:3974-3987.
Plough L. V. and D. Hedgecock. 2011. QTL analysisof stage-specific inbreeding depression in the
Pacific oyster Crassostrea gigas. Genetics 189:
1473-1486.
Plough L.V., G. Shin and D. Hedgecock. 2016. Genetic
inviability is a major driver of type III
survivorship in experimental families of ahighly fecund marine bivalve. Molecular
Ecology 25: 895-910.
Small, J. M., P. Kube, and S. K. Allen, Jr. 2016.
Advances in C. virginica breeding in
Chesapeake Bay, USA: From mass to familybased selection. Aquaculture 2016 Abstracts.
Thorson G. 1950. Reproductive and larval ecology ofmarine bottom invertebrates. Biological
Reviews 25: 1-45.
Zhang, G., X. Fang, X. Guo, L. Li, R. Luo, F. Xu, P.
Yang, L. Zhang, X. Wang, H. Qi, Z. Xiong, H.
Que, Y. Xie, P. W. H. Holland, J. Paps, Y. Zhu,
F. Wu, Y. Chen, J. Wang, C. Peng, J. Meng, L.
Yang, J. Liu, B. Wen, N. Zhang, Z. Huang, Q.
Zhu, Y. Feng, A. Mount, D. Hedgecock, Z. Xu,
Y. Liu, T. Domazet-Lošo, Y. Du, X. Sun, S.
Zhang, B. Liu, P. Cheng, X. Jiang, J. Li, D. Fan,
W. Wang, W. Fu, T. Wang, B. Wang, J. Zhang,
Z. Peng, Y. Li, N. Li, J. Wang, M. Chen, Y. He,
F. Tan, X. Song, Q. Zheng, R. Huang, H. Yang,
X. Du, L. Chen, M. Yang, P. M. Gaffney, S.
Wang, L. Luo, Z. She, Y. Ming, W. Huang, S.
Zhang, B. Huang, Y. Zhang, T. Qu, P. Ni, G.
Miao, J. Wang, Q. Wang, C. E. W. Steinberg, H.
Wang, N. Li, L. Qian, G. Zhang, X. Liu, Y. Li, Y.
Yin, and J. Wang. 2012. The oyster genome43

highly fecund marine metazoan animals. The sameprocess occurs readily in hatchery-propagatedpopulations used for supplementation or aquaculture.
Typical practices in aquatic hatcheries may rapidly
degrade the genetic diversity of hatchery-propagatedpopulations, to the long-term detriment of theseresources.

reveals stress adaptation and complexity ofshell formation. Nature 490:49-52
Williams G. C. 1975. Sex and Evolution. Princeton:
Princeton University Press.
Winemiller K. O. 2005. Life history strategies,
population regulation, and implications forfisheries management. Can. J. Fish. AquaticSci. 62: 872-885.

Plough L. V. and D. Hedgecock. 2011. QTL analysisof stage-specific inbreeding depression in the
Pacific oyster Crassostrea gigas. Genetics 189:
1473-1486.

ANNOTATED BIBLIOGRAPHYApplebaum S. L., T.-C. F. Pan, D. Hedgecock, and D.
T. Manahan. 2014. Separating the nature andnurture of energy allocation in response to
global change. Integr. Comp. Biol. 54: 284-295.

Pacific oysters show inbreeding depression forsurvival and growth. In this study, we show that asubstantial proportion of early mortality, usually
greater than 90%, can be attributed to selection
against deleterious mutations. Each oyster carries
about 12 lethal mutations. Half of the genotypedependent mortality occurs during metamorphosis.
This genetic load on early mortality has implications
for breeding efforts that focus on early life-history
stages.

This paper describes the research necessary forrevealing the physiological and metabolicmechanisms underlying resilience in the face of global
climate and ocean change. The Pacific oyster is amodel system, because it has well developed genetic
and genomic resources for fundamental and appliedresearch into complex phenotypes, such as survival,
growth, sex determination, and disease and stressresistance.

Zhang, G., X. Fang, X. Guo, L. Li, R. Luo, F. Xu, P.
Yang, L. Zhang, X. Wang, H. Qi, Z. Xiong, H.
Que, Y. Xie, P. W. H. Holland, J. Paps, Y. Zhu,
F. Wu, Y. Chen, J. Wang, C. Peng, J. Meng, L.
Yang, J. Liu, B. Wen, N. Zhang, Z. Huang, Q.
Zhu, Y. Feng, A. Mount, D. Hedgecock, Z. Xu,
Y. Liu, T. Domazet-Lošo, Y. Du, X. Sun, S.
Zhang, B. Liu, P. Cheng, X. Jiang, J. Li, D. Fan,
W. Wang, W. Fu, T. Wang, B. Wang, J. Zhang,
Z. Peng, Y. Li, N. Li, J. Wang, M. Chen, Y. He,
F. Tan, X. Song, Q. Zheng, R. Huang, H. Yang,
X. Du, L. Chen, M. Yang, P. M. Gaffney, S.
Wang, L. Luo, Z. She, Y. Ming, W. Huang, S.
Zhang, B. Huang, Y. Zhang, T. Qu, P. Ni, G.
Miao, J. Wang, Q. Wang, C. E. W. Steinberg, H.
Wang, N. Li, L. Qian, G. Zhang, X. Liu, Y. Li, Y.
Yin, and J. Wang. 2012. The oyster genome
reveals stress adaptation and complexity ofshell formation. Nature 490:49-52

Hedgecock D. and J. P. Davis. 2007. Heterosis for yieldand crossbreeding of the Pacific oyster
Crassostrea gigas. Aquaculture 272S1:S17–S29.
This paper provides definitive evidence, from diallelor factorial crosses among inbred lines of the Pacificoyster, that heterosis (hybrid vigor) for yield ispervasive. This same phenomenon underlies genetic
improvement programs for yields of corn, rice, andother major crops. Additive genetic effects on yield,
the basis for selection, exist but generally account forsmaller proportions of yield variance than nonadditive genetic components. Non-additive genetic
effects include special combining abilities andreciprocal effects, which include maternal effects andnon-maternal, nuclear-cytoplasmic interaction effects.
These findings strongly suggest that crossbreeding
should be used to improve the yield of farmed Pacificoysters and, by implication, the yield of many highly
fecund marine fish and shellfish.

This paper reports completion of a draft genomesequence for the Pacific oyster, which has a very highlevel of sequence polymorphism and a highredundancy of genes for resistance to environmental
stress.

Hedgecock D. and A. I. Pudovkin. 2011. Sweepstakes
reproductive success in highly fecund marine
fish and shellfish: A review and commentary.
Bull. Mar. Sci. 87: 971-1002.
Sweepstakes reproductive success, whereby a fractionof the spawning population contributes the vast
majority of recruits, occurs in natural populations of44

Assessment of Fertilization Ability of Cryopreserved Sperm in Fish Using
Interspecific Hybridization
Yukinori Shimada, Hiroyuki Okamoto, Hiroyuki
Nagoya, Toshiya Yamaguchi

INTRODUCTION

Yellowtail Seriola quinqueradiata is one of the mostimportant target species for aquaculture in Japan, andcorresponds to about 60% of the aquaculture
production of marine fish. However, most of theseedling supply of yellowtail for commercial
aquaculture depends on wild fish (Morita et al. 2012).
The development of techniques for artificial seedling
production of yellowtail has recently been initiated at
Fisheries Research Agency (FRA) or a few Japanesefish farms, but the numbers are insufficient for thecontributions to aquaculture. To activate theaquaculture industry of yellowtail, FRA has initiatedthe control of spawning season and the breeding
program to develop yellowtail strains withcommercially desirable traits (e.g. disease resistantand fast growth strains).
Because yellowtail is generally reared in sea cages andrequires several years to sexual maturation, there is arisk of loss of parental fish through infectious disease,
red tide problems or accidents in rearing (Morita et al.
2012). Therefore, it is important to establish thepreservation and restoration method for thedeveloped strain. So far, cryopreservation of sperm(reviewed in Suquet et al. 2000; Cabrita et al. 2010)
and germ cells (Lee et al. 2013) have been more or lesssuccessful although cryopreservation of fish embryos
and mature oocytes have not yet (Mazur et al. 2008),
as result of their large size, high yolk content and low
membrane permeability (Hagedorn et al. 1996, 1998).
Especially, cryopreservation of fish sperm has beendeveloped (Holtz 1993; Babiak et al. 1997; Dreanno etal. 1997; Chen et al. 2004) and widely used to preserve
sperm in many fish species (reviewed in Suquet et al.
2000; Cabrita et al. 2010). However, there is no reporton cryopreservation of yellowtail sperm. We have
tried to develop it, but the viewpoint was limited to
the kinds of extenders or cryoprotectants, rather than
the appropriate concentration of cryoprotectant.

National Research Institute of Aquaculture,
Fisheries Research Agency, Tsuiura, Kamiura, Saiki,
Oita 879-2602, Japan
Corresponding author: yukinoris at affrc.go.jp
Keywords: Seriola quinqueradiata, Epinephelus bruneus,
cryopreserved sperm, fertilization ability, interspecifichybridization

ABSTRACT

Fertilization ability of cryopreserved sperm of theyellowtail Seriola quinqueradiata (YT) in interspecifichybridization was examined to increase opportunities
for assessment of the sperm. At first, we examined theappropriate concentration of dimethyl sulfoxide(DMSO) ranging from 1 to 20% with artificial seminal
plasma. The activity of frozen-thawed sperm wasevaluated after 24 h. A frozen-thawed sperm of 10%
DMSO was shown in the highest sperm activity (73.3 ±
3.3%; mean ± SE) and corresponded to more than 80%of the activity of the pre-freeze sperm (83.3 ± 6.7%). To
assess the fertilization capacity of the frozen-thawed
sperm of YT, which is not easy to collect high quality
eggs frequently, we examined artificial fertilization
using longtooth grouper Epinephelus bruneus (LG)eggs; (1) LG x LG (female x male) and (2) LG x YT. The
fertilized eggs of LG x YT were developed andhatched as similar with those of LG x LG. The rates offertilization, embryogenesis and hatching in LG x YT
were 39.5, 2.5 and 0.8%, whereas those in LG x LG
were 51.5, 20.9 and 19.9%, respectively. Although therates of embryogenesis and hatching in LG x YT waslower rates than those in LG x LG, the rate offertilization in LG x YT was comparatively high andcorresponded to approximately 75% of it in LG x LG.
Relative DNA contents of 29 individuals in LG x YT
were confirmed to be diploid and shown in theintermediate value between both species. That is, thecryopreserved sperm has a fertilization capacity,suggested that it can be assessed its fertilization
capacity by using a rate of fertilization even in the caseof interspecific hybridization.

In the present study, to develop the cryopreservationof yellowtail sperm, we examined the appropriate
concentration of dimethyl sulfoxide, which is used as
an effective cryoprotectant. Then, to evaluate thefertilization capacity of frozen-thawed yellowtail
sperm, we tried to apply an interspecifichybridization using longtooth grouper, as the eggs areeasily collectable, at Kamiura Laboratory in FRA. In
contrast, yellowtail eggs are not easily collected, even45lamp of the flow cytometer PA (Partec GmbH,Germany), after DAPI (4’,6-deamidino-2phenylindole) staining with a preparation kit foranimal cells provided by the manufacturer of the flow
cytometer (Partec GmbH, Germany).

in the spawning season. Indeed, there is a risk of theevaluation for a fertilization capacity using
interspecific hybridization because it sometimes
results in a fatality (Morishima et al. 2002). However,
if our challenge was successful, this kind of method
would be expected as a highly versatile technology
for other fish species.

RESULTS AND DISCUSSIONSTo determine the appropriate concentration of DMSO
in YT sperm, the diluted sperms were cryopreservedin 1-20% DMSO concentrations with artificial seminal
plasma. According to the observation after 24 hours,
the rate of sperm activity was significantly different
among each DMSO concentration (Kruscal-Wallis test;
F2,68=1291.876, P < 0.001, Scheffe’s post hoc test, P <0.05). The sperm frozen with 10% DMSO showed thehighest sperm activity as compared with those withother DMSO concentrations (Fig. 1), and retained
about 80% of the pre-freeze sperm activity. On theother hands, sperms frozen with 1 and 5% DMSO
showed the aggregation of sperm after thawing, andtheir activities were very low (less than 3%, Fig. 1).
The cryopreservation of sperm in some marine fish
species has been available on 10-20% DMSO
concentration (e.g. Wayman et al. 1997; Chen et al.
2004; see also reviewed in Suquet et al. 2000; Cabrita
et al. 2010). Similar concentration was observed in theyellowtail sperm.

MATERIALS AND METHODS

Dilution and freezing of sperm
Each sperm from three males of yellowtail (YT) was
diluted 1:9 in artificial seminal plasma (ASP, Fujinami
et al. 2003) containing 1, 5, 10, 15 and 20% dimethyl
sulfoxide (DMSO, Sigma-Aldrich Inc., St. Louis, MO).
The diluted sperm was loaded into 0.5 ml straw tube
(Fujihira Industry Co., Ltd., Saitama, Japan) and
enclosed by straw powder (Fujihira Industry Co., Ltd.,
Saitama, Japan). The straw tubes filled with diluted
sperm were placed on dry ice plate for 10 min, andthen plunged into liquid nitrogen. After storage for 24
h in liquid nitrogen, the frozen sperm was thawed in a
beaker at 20°C for 30 s for its activity observation.
Evaluation of fertilization capacity
After two months storage in liquid nitrogen, thecryopreserved sperm of YT was thawed in a beaker at
20°C for 30 s for a fertilization test. Fertilization test
was performed using frozen-thawed YT sperm andfresh longtooth grouper (LG) sperm (diluted 1:9 in
ASP) with LG eggs. Five g of eggs (about 10,000 eggs)
was fertilized with each sperm (approximately 1.0 x
109 sperms) and gently mixed before activation with
500 ml seawater. The floating eggs were transferred
into net cages and incubated at 20°C. The rate offertilization, embryogenesis and hatching wasevaluated at 2-4 cells, embryo and hatched larvalstage, respectively.
Sampling and ploidy determination
To determine the ploidy of LG x YT larvae, a total of
71 individuals (30 individuals in LG x LG, 29 in LG x
YT and 12 in YT x YT) were sampled. Note that
hatched larvae in YT x YT were collected at a different
schedule. All individuals were subjected to flow
cytometry for relative DNA content of whole bodycells in order to determine their ploidy status. Wheile
the YT x YT individual adjusts a value of 70 using theflow cytometer PA (Partec GmbH, Germany), the LG
x LG individual exhibits a value of approximately 100
due to the difference of relative DNA content.
Therefore, the LG x YT should be theoretically theirintermediate values of approximately 85. All samples
were measured for relative DNA content by excitationin the near ultraviolet (UV) range from a mercury

Figure 1. Comparisons of the rate of sperm activity in the yellowtail sperm
frozen with different DMSO concentrations. Symbols with the same letterare not significantly different at P < 0.05 (Scheffe’s post hoc test).

The rate of fertilization, embryogenesis and hatchingin LG x LG were 51.5, 20.9 and 19.9%, whereas thosein LG x YT were 39.5, 2.5 and 0.8%, respectively. Notethat those in YT x YT examined at different schedule
were 67.7, 51.0 and 49.9% for the same lot of46

cryopreserved sperm and 83.9, 75.5 and 74.1% forfresh YT sperm, respectively (Y. Shimada,unpublished data). LG x YT showed the lower rates ofembryogenesis and hatching than LG x LG, but therate of fertilization in LG x YT was comparatively
high. In addition, we found a morphologicalcharacteristic of LG x YT was the presence of more
pigment cells like a melanophore than LG x LG (Fig.
2). However, all LG x YT larvae were observed bymalformation (Fig. 2) and died within 12 h after
hatching. This means that LG x YT was a fatality
although it could be fertilized and hatched.

Figure 2. Developmental stages of LG x LG (a-f) and LG x YT (g-l); 2-cell (aand g), 16-cell (b and h), gastrulation (c and i), embryo (d and j), justhatched larva (e and k) and magnified photograph (f and l).

Figure 3. Example of relative DNA content in LG x LG, LG x YT and YT x
YT.

To confirm that LG x YT is a hybrid fish, we
performed the ploidy analysis using flow cytometry.
We showed examples of relative DNA content of YT x
YT, LG x YT and LG x LG (Fig. 3), which were 66.7 ±
0.4 (mean ± SE), 98.2 ± 0.4 and 82.8 ± 0.3, respectively(Fig. 4). The relative DNA content of each fish wassignificantly difference (one-way ANOVA;
F2,68=1291.876, P < 0.0001). We found that relative
DNA content of LG x YT was the intermediate value
between two species (Scheffe’s post hoc test; P <0.0001, Fig. 4). If a haploid individual was observed in
LG x YT, it is thought to be the outcome ofgynogenesis (embryogenesis initiated by only
maternal chromosomes without syngamy),
parthenogenesis (all female reproduction in theabsence of sperm) or androgenesis (embryogenesis
initiated by only paternal chromosomes without
syngamy; Liu et al. 2001; Morishima et al. 2002). No
haploid individuals have been observed in all

sampled LG x YT individuals. Therefore, these results
demonstrate that LG x YT is a hybrid fish. That is, we
could develop the cryopreserved sperm of YT with afertilization capacity. We also showed that it ispossible to assess the fertilization capacity of YT
cryopreserved sperm by rate of fertilization, even ininterspecific hybridization.
In future studies, we aim to develop the large scale ofcryopreserved sperm to apply to yellowtail
hatcheries. In addition, we might be able to evaluate
fertilization capacity of sperm from several fish
species in one female if we found a marine fish
species with more easily collected eggs.

47

REFERENCES

Morita T., N. Kumakura, K. Morishima, T. Mitsuboshi,
M. Ishida, T. Hara, S. Kudo, M. Miwa, C.
Ihara, K. Higuchi, Y. Takeuchi, G. Yoshizaki.
2012. Production of donor-derived offspringby allogeneic transplantation of
spermatogonia in the yellowtail (Seriola
quinqueradiata). Biol. Reprod. 86: 1-11.
Suquet M., C. Dreanno, C. Fauvel, J. Cosson, R.
Billard. 2000. Cryopreservation of sperm inmarine fish. Aquac. Res. 31: 231-243.
Cabrita E., C. Sarasquete, S. Martínez-Páramo, V.
Robles, J. Beirão, S. Pérez-Cerezales, M. P.
Herráez. 2010. Cryopreservation of fish sperm:
applications and perspectives. J. Appl.
Ichthyol. 26: 623-635.
Lee S., Y. Iwasaki, S. Shikina, G. Yoshizaki. 2013.
Generation of functional eggs and sperm fromcryopreserved whole testes. Proc. Nat. Acad.
Sci. 110: 1640-1645.
Mazur P., S. P. Leibo, G. E. Jr. Seidel. 2008.
Cryopreservation of the germplasm of animals
used in biological and medical research:
Importance, impact, status, and future
directions. Biol. Reprod. 78: 2-12.
Hagedorn M., E. W. Hsu, U. Pilatus, D. E. Wildt, W. F.
Rall, S. J. Blackband.1996. Magnetic resonancemicroscopy and spectroscopy reveal kinetics
of cryoprotectant permeation in a
multicompartmental biological system. Proc.
Nat. Acad. Sci. 93: 7454-7459.
Hagedorn M., F. W. Kleinhans, D. Artemov, U. Pilatus.
1998. Characterization of a majpr permeability
barrier in the zebrafish embryo. Biol. Reprod.
59: 1240-1250.
Holtz W. 1993. Cryopreservation of rainbow trout(Oncorhynchus mykiss) sperm: practical
recommendations. Aquaculture 110: 97-100.
Babiak I., J. Glogowski, E. Brzuska, J. Szumiec, L.
Adamek. 1997. Cryopreservation of sperm ofcommon carp, Cyprinus carpio. Aquac. Res.
28: 567-571.
Dreanno C., M. Sequet, L. Quemener, J. Cosson, F.
Fierville, Y. Normant, R. Billard. 1997.
Cryopreservation of turbot (Scophthalmus
maximus) spermatozoa. Theriogenology 48:
589-603.
Chen S-L., X-S. Ji, G-C. Yu, Y-S. Tian, Z-X. Sha. 2004.
Cryopreservation of sperm from turbot(Scophthalmus maximus) and application tolarge-scale fertilization. Aquaculture 236:
547-556.
Morishima K., S. Horie, E. Yamaha, K. Arai. 2002. A
cryptic clonal line of the loach Misgurnus

Figure 4. Mean of relative DNA contents in LG x LG, LG x YT and YT x YT.
Symbols with the same letter are not significantly different at P < 0.0001(Scheffe’s post hoc test). Numbers in bar show sample size.

ACKNOWLEDGMENT

We thank to Ph. D. Hironori Usuki and Ph. D.
Kazuharu Nomura in National Research Institute of
Aquaculture for valuable comments. We thank also to
Mr. Yasuhiro Shima, Mr. Kazunori Yoshida, Mr.
Tsutomu Noda, Mr. Hirotaka Mizuochi and theircolleagues in Seikai National Fisheries ResearchInstitute for their kind donation of yellowtail spermand eggs in part. This work was supported in part by
grants-in-aid for Scientific Research from Fisheries
Research Agency of Japan.

48

with PKH26 fluorescent dye, and transferred into theperitoneal cavities of 8-day-old larvae. Fluorescenceobservation at 28 days post-transplantation revealed
that PKH26-labeled cells were incorporated into
recipients’ gonads. To assess whether donor-derived
spermatogonia could differentiate into functional
gametes in the allogeneic recipient gonads, gametes
collected from nine male and four female adultrecipients were fertilized with wild-type eggs andsperm. Analysis of microsatellite DNA markers
confirmed that some of the first filial (F) offspringwere derived from donor fish, with the averagecontribution of donor-derived F1 offspring being 66%
and the maximum reaching 99%. These findings
confirmed that authors’ method was effective fortransplanting yellowtail spermatogonia into
allogeneic larvae to produce donor-derived offspring.

anguillicaudatus (Teleostei: Cobitidae)
evidenced by induced gynogenesis,
interspecific hybridization, microsatellite
genotyping and multilocus DNA
fingerprinting. Zool. Sci. 19: 565-575.
Fujinami Y., H. Takeuchi, T. Tsuzaki, H. Ohta. 2003.
Sperm motility and short term preservation of
testicular spermatozoa obtained from captured
and dead red tilefish Branchiostegus japonicus.
Nippon Suisan Gakkaishi 69: 162-169.
Wayman W. R., T. R. Tiersch, R. G. Thomas. 1997.
Refrigerated storage and cryopreservation of
black drum (Pogonias cromis) spermatozoa.
Theriogenology 47: 1519-1529.
Liu S., Y. Liu, G. Zhou, X. Zhang, C. Luo, H. Feng, X.
He, G. Zhu, H. Yang. 2001. The formation oftetraploid stocks of red crucian carp x
common carp hybrids as an effect ofinterspecific hybridization. Aquaculture 192:
171-186.

S. Lee, Y. Iwasaki, S. Shikina, and G. Yoshizaki. 2013.
Generation of Functional Eggs and Spermfrom Cryopreserved whole testes. Proc. Nat.
Acad. Sci. 110: 1640-1645.

ANNOTATED BIBLIOGRAPHY

The conservation of endangered fish is of criticalimportance. Cryobanking could provide an effective
backup measure for use in conjunction with theconservation of natural populations; however,methodology for cryopreservation of fish eggs andembryos has not yet been developed. Authors
established a methodology capable of deriving
functional eggs and sperm from frozen type A
spermatogonia (ASGs). Whole testes taken fromrainbow trout were slowly frozen in a cryomedium,and the viability of ASGs within these testes did notdecrease over a 728-d freezing period. Frozen-thawed
ASGs that were intraperitoneally transplanted into
sterile triploid hatchlings migrated toward, and wereincorporated into recipient genital ridges.
Transplantability of ASGs did not decrease after as
much as 939-d of cryopreservation. Approximately
half of triploid recipients produced functional eggs or
sperm derived from the frozen ASGs and displayedhigh fecundity. Fertilization of resultant gametes
resulted in the successful production of normal,
frozen ASG-derived offspring. Feasibility andsimplicity of this methodology will call for an
immediate application for real conservation ofendangered wild salmonids.

M. Suquet, C. Dreanno, C. Fauvel, J. Cosson, and R.
Billard. 2000. Cryopreservation of Sperm in
Marine Fish. Aquac. Res. 31:231-243.
Since 1953, cryopreservation of sperm in fish has been
performed on about 30 marine species. The paper
reviews the techniques used and the results publishedin these species. The quality of frozen-thawed semen
was evaluated using previously standardized biotests(i.e. fertilization capacity). Of the investigatedcryoprotectants, dimethyl sulphoxide (DMSO)
generally leads to the best results. Cooling rates range
from 8 °C to 99 °C min-1; the thawing rate is generally
high. Compared with freshwater species, a highpercentage of sperm survives in cryopreservation.
Therefore, the cryopreservation of marine fish sperm
is suited for application in aquaculture.
T. Morita, N. Kumakura, K. Morishima, T. Mitsuboshi,
M. Ishida, T. Hara, S. Kudo, M. Miwa, S. Ihara,
K. Higuchi, Y. Takeuchi, and G. Yoshizaki.
2012. Production of Donor-Derived Offspringby Allogeneic Transplantation of
Spermatogonia in the Yellowtail (Seriola
quinqueradiata). Biol. Reprod. 86: 1-11.
Authors established a method for the allogeneictransplantation of yellowtail spermatogonia and the
production of donor-derived offspring. Donor cells(i.e. type A spermatogonia) were collected fromimmature (10-month-old) yellowtail males, labeled49Genetic and Physiological Studies of Health and Fitness in Aquaculture-Reared
California Yellowtail, Seriola dorsalis
associated with pedigree. In addition, sustained
swimming capacity and oxygen consumption rates
were measured for randomly sampled juvenile
yellowtail (15-20 cm fork length) as additional fitness
measures and compared to the performance of wildcaught juveniles. Analyses are ongoing, but genetic
analyses of juveniles sampled from HSWRI show
significant differences in parental contribution
between offspring with deformities, growth variation,and the randomly sampled offspring. Aquaculturereared yellowtail also showed significantly lower
maximum sustainable swimming speeds and higher
oxygen consumption rates than wild caught
individuals, indicating reduced aerobic fitness infarm-raised fish. Understanding the genetic andphysiological processes contributing to yellowtail
seed stock quality is of considerable value to hatcheryand aquaculture facilities rearing this species.
Information gained in this study can be used to
inform hatchery decisions and potentially facilitate
the culture of yellowtail at a larger and more costeffective scale.

Catherine Purcell1, Nicholas Wegner1, Mark
Drawbridge2, Kevin Stuart2, John Hyde1
1

National Marine Fisheries Service,8901 La Jolla Shores Dr, 92037-1508 La Jolla, CA

2

Hubbs Sea World Research Institute,2595 Ingraham Street, San Diego, CA 92109

Corresponding author: catherine.purcell at noaa.gov
Keywords: Seriola, aquaculture, respiration,
parentage analysis, swim tunnel, deformity

ABSTRACTThe California yellowtail (S. dorsalis) is a likely
candidate for the future development and expansion
of offshore commercial aquaculture in the southern
California region. However, rearing methods for thisand other Seriola species are still unreliable, resulting
in highly variable survival rates and prevalence ofphysical malformations. The causes of, and solutions
for, the variability and abnormalities in the offspringremain largely unresolved and are likely associatedwith several factors, such as nutritional imbalances (inthe larvae, juveniles, and/or broodstock), culture
conditions (e.g. light, temperature, salinity), andgenetic background of the parents. To better
understand the connection between individual broodfish and abnormal offspring, and to gain insight into
possible causes of growth variation and deformities,
we conducted genetic analyses of juvenile fishexhibiting deformities and extreme size variation.
Juvenile Seriola dorsalis (43 to 50 days post hatching)were collected from three production runs at HubbsSea World Research Institute (HSWRI) in thesummer/fall of 2012. Three different types of juvenile
fish were collected: 1) small and large fish
(representing juveniles with different growth rates, 2)
deformed fish (this included various skeletal or soft
tissue abnormalities), and 3) randomly sampled fish(not sorted). Genetic samples from the 21 broodstockindividuals were also collected. Genetic markers (16nuclear microsatellites already optimized for thisspecies) were used to evaluate parent-progeny
relationships to test whether observed physical traits(e.g., size differences, various types of deformities) are

INTRODUCTION

Producing high quality and healthy fish larvae is key
to facilitating successful aquaculture ventures, butwith the small size and tremendous changes that
occur between the hatching and juvenile stages, this
aspect of the culture process is very difficult tocontrol. On the west coast of the United States, thenative California yellowtail, Seriola dorsalis (formerly
S. lalandi), is considered a serious aquaculture
candidate due to its high market demand and value.
Seriola species are cultured globally, and collectively
the Seriola production is valued at over one billion
U.S. dollars annually (FAO 2012). Hatcheryproduction of these species is rapidly growing but has
been hindered by a propensity for deformities andgrowth heterogeneity developed during larval andearly juvenile stages that limit production capacityand efficiency. Reportedly more common in reared
than wild fish (Fraser and de Nys 2005),
malformations in the larval and juvenile stages
directly impact commercial aquaculture by reducing
the market value of fish and requiring additional
labor for manual sorting to remove these individuals(Nagano et al. 2007; Karahan et al. 2013). In addition50

to market value and labor costs, deformities can also
lead to diminished growth rates, poor feed conversion
rates, and reduced survival (Fraser and de Nys 2005).

METHODS

Spawning dynamics and parentage analyses
Whole juvenile fish (F1 generation) between 43 and 50
days post hatching (dph) were sampled during three
production runs at HSWRI in June, August, andSeptember of 2012. Samples of these fish included arandomly sampled group, juveniles with extreme size
variation (very small or large), and juveniles withdifferent categories of deformities. Size of the fishvaried by production run. Small fish averaged 20.3(+/- 1.9) mm, 29.5 (+/-1.4) mm, and 21.3 (+/- 1.5)mm, while large fish averaged 36.3 (+/- 2.9) mm, 53.6(+/-4.2) mm, and 49.5 (+/- 5.1) mm in June (43dph),
August (50 dph), and September (43 dph) production
runs, respectively. Approximately 1,450 juvenile fish
were sampled and included in the analyses. A total of
986 juveniles were examined for variability in the
production runs, with 225, 176, and 585 fish sampled
in June, August, and September, respectively. For thesize comparisons, 191 large fish and 222 small fish
were compared to a randomly collected sample of 245
fish. A total of 195 juveniles were identified as having
deformities; these deformities included: head
deformities (n=3), incomplete operculum (n=125), jaw
deformities (n=44), skeletal deformities (n=13), andother non-specified malformations (n=10). The
parentages of the juveniles with deformities were also
compared to a randomly collected sample of 142
juvenile fish. The broodstock fish were also sampled
for genetics during yearly health checks using small,
non-lethal fin clips.

These deformities have been reported for a variety of
aquaculture species including Seriola species (Cobcroft
et al. 2004; Roo et al. 2010). Deformity rates for S.
dorsalis at HSWRI have ranged from 25-40%
depending on the cohort (Stuart and Drawbridge2012), and mass culture in S. rivoliana has also beenbottlenecked by unreliable production of juveniles as
the result of poor spawns and low hatchery survival(Roo et al. 2010). Cranial malformations are common
and include jaw deformities, incomplete or
malformed opercula, and misshapen skulls. Other
types of deformities include shortened (or perch-like)bodies and skeletal abnormalities. Additional internal
deformities have been detected in several older
juvenile yellowtail (4-6 months) reared at theSouthwest Fisheries Science Center (SWFSC),
including vertebral compression, vertebral fusion,and extra-ossification of vertebrae in the caudal
region.
Extreme variation in larval/juvenile growth rates isanother key consideration in production. Variable
growth rates lower the survival of smaller larvae/juveniles as a result of cannibalistic predation andphysical trauma from larger fish but also increasemortality in larger fish from choking on their smaller
cohort members. Growth heterogeneity among larvae
is already apparent a few days post-hatching andbecomes more pronounced over time. Without size
grading, growth heterogeneity results in larvae andearly juveniles of the same age being at different
developmental stages, often requiring different feeds
and culture conditions.

Genomic DNA was extracted from the samples using
Chelex® resin according to the manufacturer’s
protocol. The broodstock population was genotyped
using 15 microsatellite markers developed for Seriola(Porta et al. 2009; Renshaw et al. 2007, 2006; Ohara etal. 2005, 2003; Nugroho and Taniguchi 1999). Juvenile
yellowtail were genotyped with the same 15
microsatellites or a subset of nine microsatellites
following parentage analysis optimization. Samples
were analyzed on an ABI 3730XL Genetic Analyzer(Applied Biosystems) and fragment data wereanalyzed using GENEMAPPER 4.0 (Applied
Biosystems) and scored visually.

Many factors influence the health, fitness, andsurvival of larval/juvenile fish; these factors include
non-genetic or genetic parental effects on larvae,
environmental conditions, and any number of culture
methods (Trippel et al. 2005; Brown et al. 2011; Nuñez
et al. 2011). In an effort to improve California
yellowtail culture, we are investigating several factorsthat may be responsible for variable physical fitness
in the larval and juvenile stages. Spawning dynamics
of the broodstock population, and parentage analysesof juvenile fish with physical abnormalities andgrowth heterogeneity were examined using molecular
microsatellite markers. Relative aerobic fitness
between cultured and wild juveniles was also
investigated using key physiological measures.

Swim tunnel experimentation
Once large enough (15-20 cm FL), the health andaerobic fitness of aquaculture-reared yellowtail wereassessed in comparison to individuals of the same
size collected from the wild by examining maximum
sustainable swimming speed and oxygen
consumption rates at varying swim speeds. This wasdone using a variable speed, 5.4 l swim tunnel
51

saturation, at which point the system was flushed (toreturn the oxygen saturation level to near 100%) andthen MO2 measurements were repeated. Typically, 2-3
measurements could be made during each 30 min
velocity step. These rates were averaged and counted
as a single consumption rate for each fish at eachswimming speed. Regression lines were determined
for MO2 vs. swimming speed and used to extrapolate
to a swimming speed of 0 cm s-1 to determine resting
metabolic rate. Metabolic rates for all fish were
adjusted to a temperature of 18°C for comparison
using Q10 = 2.

respirometer (Loligo Systems, Tjele Denmark) with a
30x7.5x7.5 cm working section. Fish were placed in
the swim tunnel and allowed to swim steadily at apreferred, low, sustainable speed (typically about
20-25 cm s-1) with a continuous inflow of filteredseawater for at least 1 hour prior to experimentation.
The maximum sustainable swimming speed (criticalswimming speed, Ucrit) was determined by increasing
the velocity of the swim tunnel water by 4-10 cm s-1
steps every 30 minutes until the fish could no longer
maintain steady swimming (defined as no longer
being able to stay off the screen at the back of theworking section). Ucrit was determined by theequation:

Wild-caught fish tested for Ucrit and metabolic rate incomparison to aquaculture-reared individuals were
captured by hook and line from local waters (<20 km
from shore) typically associated with drifting kelp.
Upon transfer to the Southwest Fisheries Science
Center these fish were housed in identical tanks andwere fed the same diet as aquaculture-reared
individuals. Wild caught fish were held 2-4 months
in captivity before swim tunnel experimentation.

Ucrit = Ui + [(Ti / Tii) Uii)]		 (1)where Ui is the highest swimming speed maintained
for the full 30 minutes, Ti is time swimming at thehighest velocity before fatigue, Tii is time step lengthof each swimming speed (30 min), and Uii is thevelocity step (4-10 cm s-1). Swim tunnel water velocity
was corrected for solid blocking effects of the fish
according to Bell and Terhune (1970).

RESULTS AND DISCUSSION

Spawning dynamics and parentage analyses
To help make the other analyses more informative, we
identified and/or confirmed the sexes of thebroodstock population. We used biparentally
inherited microsatellites to identify the two parents,
and then used maternally inherited mitochondrial
sequences to identify the maternal parent. This could
be done for any broodstock fish for which we hadsampled at least one of their offspring. With thisinformation, we were able to look at spawning
dynamics in the broodstock population. Among thejuveniles sampled across the three production runs,
fewer females than males participated in the
spawning events (Fig. 1). The contribution of males
also appeared to be more evenly distributed among
the broodstock fish, while certain female broodstockindividuals accounted for most of the production in
the three sampled runs.

Oxygen consumption rates (MO2) were determined foreach fish at each swimming speed using a fiber optic
oxygen sensor connected to a Fibox 3 fiber optic
oxygen transmitter (PreSens Precision Sensing GmbH,Regensburg Germany). Shortly (1-2 min) after theeach step increase in swimming speed, therespirometer was temporarily closed to the inflow
and outflow of fresh seawater and the rate of oxygen
consumption was observed until the oxygen level ofthe swim tunnel water reached approximately 80%

Figure 1. Contributions (inpercent) of individual
broodstock fish to juvenile
offspring collected fromthree production runs
(n=986). Female
broodstock are on the leftof the figure, males are on
the right.

52

When parental contribution was examined for each of
the three production runs, it appears that only two
females contributed to each event, with one
contributing more dominantly (Fig. 2). Again, male
contribution to offspring is spread out among agreater number of fish, although contribution is noteven among the males.

For example, certain males (e.g., Brd637-20 and
Brd637-04) largely contributed to head deformities
and skeletal, jaw, and operculum deformities,
respectively (Fig. 4). However, the female parental
contributions to deformities under the incomplete
opercula category were not found to be significantly
different from the random sample (data not shown).

Figure 2. Contributions (in percent) of individual broodstock fish to juvenile
offspring collected for three different production runs: June (n=225),
August (n=176), and September (n=585). Female broodstock are on theleft of the figure, males are on the right.

Figure 4. Contributions (in percent) of male broodstock fish to juvenile
offspring with various deformities (head deformities (n=3), incomplete
operculum (n=125), jaw deformities (n=44), skeletal deformities (n=13)) incomparison to a random sample of juveniles (n=142). Male parental
contribution was significantly different (Chi-square P< 0.001) for all
deformities compared to the random juvenile sample.

Individual broodstock fish contribution to offspringexhibiting size heterogeneity was also investigatedusing parentage analyses. For each sex, broodstock
contribution to large or small offspring was compared
to a randomly sampled collection of juveniles. Certain
female broodstock fish, for example, did significantly

We also examined if parental broodstock pairs
contributing disproportionately to offspring
deformity pattern was detected when examining
parental broodstock pairs. Certain broodstock pairs
produced more deformed offspring (all deformities
combined together) than others (Fig. 5) (no statistical
test performed). While certain males may
disproportionately contribute to deformed offspring(e.g., Brd637-04), they appear to do worse when
paired with particular females (e.g., Brd637-04 with
Brd637-05, and Brd637-04 with Brd637-12, shown
below) (Fig. 5).

Figure 3. Contribution (in percent) of female broodstock to large (n=191)
and small-bodied (n=222) offspring compared with a random sample ofjuveniles (n=245).

(Chi-square P < 0.001) disproportionally contribute tosmall (e.g., Brd637-05 and Brd637), and to large
offspring (e.g., Brd637-09) (Chi-square P < 0.001) (Fig.
3). Contributions of individual broodstock fish tojuvenile offspring exhibiting various deformities werealso examined. Again, particular parents did appear
Figure 5. Contributions (in percent) of parental broodstock pairs to juvenile
to disproportionately contribute to various
offspring with deformities (all deformities combined) (n=195) compared to a
deformities. Male parental contribution was
random sample of juveniles (n=142). The broodstock pairs listed here are
significantly different (Chi-square P< 0.001) for all
sorted by individual male fish.
deformities compared to the random juvenile sample.
53

For a broadcast spawning species,
such as yellowtail, genetic analyses
are required to understand parental
contribution to positive and negative
traits in F1 populations used forcommercial growout and can provide
important insight into optimizing thecomposition of the broodstock
population. However, it is important
to note that although genetic tools
provide useful metrics to examine
physical fitness, it does notnecessarily mean that there is a
genetic component to this fitness.
Any number of environmental
factors or physical factors in the fish (e.g., age, health,nutritional status, and the genetic makeup of theindividual) can influence the growth heterogeneityand deformities.

Figure 7: Oxygen consumption (MO2) at varying swimming speeds (U) in 10
aquaculture-reared (yellow) and seven wild caught (blue) juvenile
yellowtail, Seriola dorsalis, adjusted to 18°C using Q10 = 2. Swimming
speeds are standardized in fork lengths per second.

82.06 ± 8.62 cm s-1 (4.90 ± 0.69 FL s-1), which was not
significantly slower than that determined for sevenwild caught individuals of similar size (17.7 ± 1.11 cm,
95.16 ± 14.21 cm s-1, 5.36 ± 0.63 FL s-1). The
relationships between MO2 and swimming speed for
all aquaculture-reared and wild caught yellowtail are
shown in Figure 7. Extrapolation to a swimming
speed of 0 cm s-1 suggests that the resting metabolic
rate of aquaculture-reared yellowtail (8.00 mgO2 min-1kg-1) is much higher than that of wild caught fish (4.18
mgO2 min-1 kg-1). These higher metabolic costs
indicate that aquaculture-reared fish likely havehigher feed and water quality requirements. Thus,
the physiological conditioning of young juveniles inaquaculture (e.g., raising fish under constant flow
regimes to force continuous swimming) could
potentially be used to increase juvenile fitness andpotentially reduce production costs.

Swim tunnel experimentation
To examine the fitness of juveniles without apparent
deformities, physiological measures of maximum
sustainable swimming speed and oxygen
consumption were obtained in comparison to wildcaught juvenile yellowtail (Fig. 6). The mean
maximum sustainable swimming speed for 10
aquaculture-reared yellowtail (17.3 ± 1.04 cm FL) was

Figure 6: Representative images of aquaculture-reared (A) and wild caught(B) yellowtail swimming in the swim tunnel respirometer. Note themorphological differences in the fish.

54

REFERENCES

ANNOTATED BIBLIOGRAPHY

Bell, W.H. and L.D.B. Terhune. 1970. Water tunnel
design for fisheries research. Fish. Res. Bd.
Canada. Tech Report No. 195:1-69.
Brown, E.J., M. Bruce, S. Pether, and N.A. Herbert
2011. Do swimming fish always grow fast?
Investigating the magnitude and physiologicalbasis of exercise-induced growth in juvenile
New Zealand yellowtail kingfish, Seriolalalandi. Fish Physiol. Biochem., 37:327-336.
Cobcroft, J., et al. 2004. Jaw malformation in cultured
yellowtail kingfish (Seriola lalandi) larvae. New
Zeal J Mar Fresh Res 38:67-71.
FAO. 2012. The State of World Fisheries andAquaculture. Rome. 209 pp.
Fraser, M.R., and R. de Nys. 2005. The morphology
and occurrence of jaw and operculum
deformities in cultured barramundi (Lates
calcarifer) larvae. Aquaculture 250:496-503.
Karahan, B., et al. 2013. Heritabilities and correlationsof deformities and growth-related traits in theEuropean sea bass (Dicentrarchus labrax, L) in
four different sites. Aqua Res 44:289-299.
Nagano, N., et al. 2007. Skeletal development anddeformities in cultured larval and juvenile
seven-band grouper, Epinephelus
septemfasciatus (Thunberg). Aquacult Res
38:121-130.
Nuñez, J., D. Castro, C. Fernández, R. Dugué, F.
Chu-Koo, F. Duponchelle, C. García, and J.-F.
Renno. 2011. Hatching rate and larval growthvariations in Pseudoplatystoma punctifer:maternal and paternal effects. Aquaculture
Research, 42: 764-775.
Roo, F., et al. 2010. Occurrence of skeletal deformities
and osteological development in red porgy
Pagrus pagrus larvae cultured under different
rearing techniques. J Fish Biol 77:1309-1324.
Stuart, K. and M. Drawbridge. 2012. Spawning andlarval rearing of California yellowtail (Seriolalalandi) in Southern California. Bull Fish Res
Agency 35:15-21.
Trippel, E.A., G. Kraus, and F.W. Köster. 2005.
Maternal and paternal influences on early life
history traits and processes of Baltic cod Gadus
morphua.

Brown, E.J., M. Bruce, S. Pether, and N.A. Herbert.
2011. Do swimming fish always grow fast?
Investigating the magnitude and physiologicalbasis of exercise-induced growth in juvenile
New Zealand yellowtail kingfish, Seriolalalandi. Fish Physiol. Biochem., 37: 327-336.
The focus of this study was to investigate the effect of
exercise on growth of cultured New Zealandyellowtail kingfish, Seriola lalandi at different watertemperatures. The authors also examined potential
sources of physiological efficiency to additionally
improve exercise-induced growth. Two growth trials
that exposed fish to various exercise levels(determined by current speed) were kept at 14.9° and
21.1°C. Lengths and weights were measured for eachfish, along with oxygen consumption rates (later
converted to metabolic rates) using a respirometer.
Results of these trials showed that long term exercise
yielded a 10% increase in growth but only in low
flows (0.75 BL s-1) and at a water temperature of
21.1°C. Experiments using a swim flume respirometer
indicated that exercise training had no effect on
metabolic scope or critical swimming speeds but it
did improve swimming efficiency. This study was ofinterest in working to improve fitness for cultured
Seriola lalandi.
Nuñez, J., D. Castro, C. Fernández, R. Dugué, F.
Chu-Koo, F. Duponchelle, C. García, and J.-F.
Renno. 2011. Hatching rate and larval growthvariations in Pseudoplatystoma punctifer:maternal and paternal effects. Aquaculture
Research, 42: 764-775.
The authors evaluated paternal and maternal effects
on hatching and growth on initial stages and during
an early dry feed adaptation period in P. punctifer
larvae. P. punctifer are a commercially important
catfish species from South America that are ofparticular importance for aquaculture production. It
was hypothesized that parental effects may influencelarval heterogeneity, which is responsible for highlevels of cannibalism and therefore mortality in theculture setting. Artificial reproduction methods wereused to set up factorial breeding crosses in this study,
for a total of nine families of full siblings. Results
showed that parentage significantly influences
growth and dry feed adaptation capabilities during
the first 26 days post fertilization. This study
highlights the importance of parentage and/or
parentage-interactions on the fitness and survival of55

larval offspring.
Trippel, E.A., G. Kraus, and F.W. Köster. 2005.
Maternal and paternal influences on early life
history traits and processes of Baltic cod Gadus
morhua.
The roles of each gender in various life-history traits
and overall fitness of their larval offspring hasreceived increasing attention in aquaculture research.
While maternal effects have been more commonly
studied, the effects of males are not well-documented.
The authors investigated the effects of bothbroodstock parents and bi-parental interaction on
fertilization rate, hatching rate, larval traits, yolk
utilization, and growth of Baltic cod during thenon-feeding larval phase using a crossing design.
They found that parental influences varied in theirimportance during different embryogenesis stages.
Females had the greatest effects during early and lateembryonic development, and male-female interaction
was most important during mid-embryonic stages.
Maternal effects influenced several traits andprocesses, however, paternal effects were only seen inpaternal-maternal interactions. This research isrelevant in our parentage studies of growth variationand deformities in the California yellowtail.

56

Demands for Infertility of Cultured Marine Fishes and Study of UV Irradiation at
Developmental Stages In Japanese Flounder
The hatching rate decreased as the intensity of UV
irradiation increased. The microscopic observation of
the gonads revealed the abnormal gonads in the
UV-irradiated group. The expression of vasa and sycp3
mRNA was observed in the gonads of the control
group indicating the presence of germ cells. On theother hand, the expression of vasa and sycp3 mRNA
was not detected in the gonad area of some of the UV
irradiated larvae. These results indicate that germ
cells may be deleted by UV irradiation of fertilized
eggs resulting in flounder that are infertile.

Toshiya Yamaguchi and Koichi Okuzawa
National Research Institute of Aquaculture,
Fisheries Research Agency, Tsuiura, Kamiura, Saiki,
Oita 879-2602, Japan
Corresponding author: tyamaguchi at affrc.go.jp
Keywords: infertility, ultraviolet, vasa, Japaneseflounder

ABSTRACTINTRODUCTION

Various kinds of marine fish species are produced inJapan. There has been increasing needs for thetechnique to sterilize farmed fish, because thesterilization of cultured fish could protect the
improved strains, prevent undesired gonadal
development that often result in the deterioration ofmeat quality, and prevent possible negative genetic
impact on wild populations. In this way, sterilizationof farmed fish is beneficial not only to the aquacultureindustry but also for the environmental conservation.
However, the applicable techniques for sterilization
have not been established in cultured marine fish.

Germ cells play an important role transmitting
genomic information to the next generation. It is
known that germ cells develop into sperm in males
and eggs in females and this differentiation begins
during early embryonic development and concludes
in adulthood with gamete differentiation. It is known
that germ cells develop into sperm in males and eggs
in females and this differentiation begins during
during early embryonic development and concludes
in adulthood with gamete differentiation. These germ
cells are derived from primordial germ cells (PGCs)
that segregate from the somatic cell lineage during
early embryogenesis of organisms Saffman and Lasko
1999). PGCs proliferate and migrate to the developing
gonad and become germline stem cells (Lin 1997).
When an organism reaches sexual maturity thesegerm cells follow a unique pathway ofspermatogenesis or oogenesis and differentiate into
sperm or eggs respectively (Saffman and Lasko 1999).

Germ cells play an important role in transmitting
genomic information to the next generation. It is
known that germ cells develop into sperm in males
and eggs in females and this differentiation begins
during early embryonic development and concludes
in adulthood with gamete differentiation. In thisstudy we investigate using ultraviolet (UV)
irradiation on fertilized egg of Japanese flounder,
Paralichthys olivaceus to block germ cell differentiation.

UV and X-ray irradiation is known to damage DNA
damage, arrest cell cycles and cause apoptosis(Iwamoto et al. 1999). Irradiation with X-rays caneffectively reduce the number of endogenous PGCs inchickens (Nakamura et al. 2012). Moreover,ultrasound can reduce the total number of epididymal
sperm by depleting spermatocytes and spermatidsfrom testes (Tsuruta 2012). Methods to use irradiation
to remove germ cells have been reported inexperimental animals such as rats and chickens,
however, these methods have not been reported incultured fish.

We examined the effects of irradiation on the hatchingrate of fertilized eggs. The UV was applied by a UV
cross linker. Hatched larvae were reared until 55 days
after hatching (dah), and the juveniles wereembedded in paraffin, sectioned, stained using
hematoxylin-eosin, for gonadal observations using
light microscopy. We also examined the expressions ofboth vasa and sycp3 (germ cell marker) mRNA in
gonads using RT-PCR to indicate the presence of germ
cells in the gonads.

57

percent survival of all groups is lowered in proportion
to the intensity of the ultraviolet irradiation. Survivalin the UV irradiated treatment at 4hpf was higher
than the other groups (Fig. 1). These results indicatethat the 4 hours post-fertilization 4cell stage embryo
may have a relative resistance to UV irradiation.

The Japanese flounder (Paralichthys olivaceus) is ateleost fish that has been cultured worldwide. In theJapanese flounder germ cell meiosis marker sycp3
cDNA has been isolated and the mRNA is expressedin the undifferentiated gonad. In early developmental
stages such as during embryonic cleavage the germ
cell marker sycp3 mRNA expression has not been
investigated because sycp3 mRNA expression is
initiated around 50~60 dah during sexual
differentiation.. Therefore, in this study weinvestigated expression of the germ cell marker vasa
mRNA in larvae hatching from flounder embryos
treated with UV irradiation prior to hatching.

MATERIALS AND METHODS

Animals
Fertilized eggs were obtained from natural mating
and spawning of Japanese flounder reared at 16 °C.

Figure 1. The survival rate of the UV irradiated embryos. The vertical axis isfor percent survival, and the horizontal axis is the intensity of UV irradiation.
Solid bar indicates the UV irradiated group at 4 hours after fertilization.
Striped bar indicates UV irradiated group at 12 hours after fertilization,
Dotted bar indicates UV irradiated group at 28 hours after fertilization, andwhite bar indicates UV irradiated group at 52 hours after fertilization.

UV irradiation
The UV was applied by a UV cross linker (UltraViolet Products, Germany). The intensity of UV
ranged from 0 to 200 mJ (millijoule) and the timing of
irradiation were set at 4, 12, 28, and 52 hours postfertilization. Hatched larvae were reared until 55 dah.

To investigate effects of UV irradiation on theflounder’s gonad the UV irradiated larvae werereared until 55 dah when the gonads are still sexually
undifferentiated (Kitano et al. 1999). The juvenile
gonads were sectioned, hematoxylin-eosin stained,
and observed using light microscopy. This experimentresulted in two normal gonads in the control group(Fig. 2A), one normal gonad in the 4hpf 10mJ UV
irradiated group (Fig. 2B) and no normal gonads
could be observed in the 52hpf 5mJ group (Fig. 2C).
The UV irradiation treatment during developmental
stages induced abnormal gonad development inJapanese flounder.

Histological analysis
Japanese flounder juveniles at 55 dah were fixed in
Bouin’s solution at 4 °C overnight, dehydrated in
graded ethanol, embedded in paraffin, and seriallysectioned at 5 µm thickness.
The sections were used for hematoxylin-eosin
staining, immunochemical staining or in situ
hybridization. The immunochemical staining and insitu hybridization were performed as described
previously (Yamaguchi et al. 2007).
RT-PCR analysis
One microgram total RNA extracted
from the juveniles using ISOGEN(Nippon gene, Tokyo, Japan) was
reverse-transcribed using RNA PCR
Kit (Applied Biosystems) at 42 °C for
30 min. The RT-PCR was performed
as described previously (Yamaguchi
et al. 2007).

Figure 2. Effect of UV irradiation on the flounder’s gonad. A: control (noUV irradiation). B: 10mJ UV irradiation at 4hpf. C: 5mJ UV irradiation at
52hpf. The circle indicates the gonad.

RESULTS AND DISCUSSION

We examined the effects of UV irradiation of fertilized
eggs on the hatching rate. The intensity of UV ranged
from 0 to 200 mJ (Joule) and the timing of irradiation
were set at 4, 12, 28, 52 hours post-fertilization. The58

To investigate the rate of abnormality of gonads in UV
irradiated flounders embryos were irradiated with theintensity of 10mJ at the 4-cell stage. The larvae werereared for 55 days after hatching for observation using
light microscopy. The samples were Bouin’s fixed,
sectioned, hematoxylin-eosin stained, and gonads
were observed. All larvae in the control group had
two normal gonads (Fig. 4A), whereas 20% of larvaein the 4hpf 10mJ UV irradiated group had one normal
gonad, and 65% of larvae had no normal gonads (Fig.
4B). This experiment conformed that UV irradiation at
developmental stages induced abnormal gonad
development in Japanese flounder.

Immunochemical staining or in situ hybridization
were used to investigate whether vasa gene and sycp3
genes were expressed in the germ cells. Vasa protein
and mRNA signals were found to be localized in thegerm cells. As a result, the vasa or sycp3 were used asa marker for germ cells in the gonad (Fig. 3A, 3B, and3C). The vasa mRNA has been demonstrated to be agerm cell marker in many fish species, such aszebrafish (Olsen et al. 1997), tilapia (Kobayashi et al.
2000), and medaka (Shinomiya et al. 2000). Thus, thevasa gene is highly conserved and studied between
vertebrate fish. SYCP3 encodes a component of thesynaptonemal complex, which is marker of meiotic
prophase (Di Cario et al. 2000). It has been reported
that sycp3 is involved in gonadal differentiation
during the sexual differentiation period Japaneseflounder (Yamaguchi et al. 2012). To investigate thepresence of germ cells in gonad tissue of UV
irradiated flounder, we performed RT-PCR for germ
cell specific marker vasa and sycp3 mRNA. UV
irradiated larvae were reared until 55 dah. Then the
gonads were isolated, and RT-PCR analysis was
performed. In the control group, the expression ofgerm cell specific markers vasa and sycp3 mRNA weredetected in all individuals. Whereas, in the UV
irradiated group the expression of vasa and sycp3
mRNA were not detected (Fig. 3D). These results
indicate that germ cells may be deleted by UV
irradiation of fertilized eggs.

In conclusion, in the most optimum treatment (UV
irradiation 4hpf 10mJ), normal gonad development
was been in 65% of the flounders. Moreover, theexpression of vasa mRNA is not confirmed in UV
irradiated flounders. These results indicate thepossibility that germ cells were destroyed by UV
irradiation and that that UV irradiation can be used toinduce sterilization in flounder.

Figure 4. The rate of abnormality of gonads in UV irradiated flounders. UV
irradiation intensity of 10mJ at the 4-cell stage. (A) Control receiving noradiation. (B) Gonadal status of 20 larvae at 55 dah observed with lightmicroscopy.

ACKNOWLEDGEMENTS

We thank Drs. H. Usuki, M. Awaji and Y. Kazeto in
Research Center for Aquatic Breeding, National
Research Institute of Aquaculture, and members ofFisheries Research Agency for their helpful advice.

Figure 3. Expression pattern of the vasa or sycp3 gene in UV irradiatedJapanese flounder. Immunochemical staining of vasa protein in juvenile
flounder at 55dah (A). In situ hybridization analysis of vasa mRNA (B) or
sycp3 (C) mRNA in juvenile flounder at 55dah. Arrowheads indicate vasa or
sycp3 signal. (D) RT-PCR for germ cell specific marker vasa and sycp3
mRNA. The ef-1a (elongation factor 1 alpha) is internal control.

59germ cells of the mouse embryo. Int J Dev Biol.
44:241-244.
Yamaguchi T, Kitano T. 2012. High temperatureinduces cyp26b1 mRNA expression and delays
meiotic initiation of germ cells by increasing
cortisol levels during gonadal sex
differentiation in Japanese flounder. BBRC 419:
287-92.

REFERENCES

E.E. Saffman, F. Lasko. 1990. Germline developmentin vertebrates and invertebrates. Cell. Mol.
Life Sci. 55: 1141–1163.
H. Lin. 1997. The tao of stem cells in the germline.
Annu. Rev. Genet 31: 455–491.
Iwamoto K, Shinomiya N, Mochizuki H. 1999.
Different cell cycle mechanisms between
UV-induced and X-ray-induced apoptosis in
WiDr colorectal carcinoma cells. Apoptosis.
4(1): 59-66.
Nakamura Y, Usui F, Miyahara D, Mori T, Ono T,
Kagami H, Takeda K, Nirasawa K, Tagami T. J
2012. Reprod Dev. 58(4): 432-7.
James K Tsuruta, Paul A Dayton, Caterina M Gallippi,
Michael G O’Rand, Michael A Streicker, Ryan
C Gessner, Thomas S Gregory, Erick JR Silva,
Katherine G Hamil, Glenda J Moser and DavidC Sokal Therapeutic ultrasound as a potential
male contraceptive: power, frequency andtemperature required to deplete rat testes ofmeiotic cells and epididymides of sperm
determined using a commercially available
system. 2012. RB & E. 10: 7.
Yamaguchi T, Yoshinaga N, Yazawa T, Gen K, Kitano
T. 2010. Cortisol is involved in temperaturedependent sex determination in the Japaneseflounder. Endocrinology. 151: 3900-3908.
Kitano T, Takamune K, Kobayashi T, Nagahama Y,
Abe S. 1999. Suppression of P450 aromatasegene expression in sex-reversed males
produced by rearing genetically female larvae
at a high water temperature during a period ofsex differentiation in the Japanese flounder(Paralichthys olivaceus). J Mol Endocrinol.
23:167-176.
L.C. Olsen, R. Aasland, A.A. Fjose. 1997. Vasa-likegene in zebrafish identifies putative
primordial germ cells. Mech. Dev. 66: 95–105.
T. Kobayashi, H. Kajiura-Kobayashi, Y. Nagahama.
2000. Differential expression of vasa
homologue gene in the germ cells during
oogenesis and spermatogenesis in a teleostfish, tilapia, Oreochromis niloticus. Mech. Dev.
99: 139–142.
A. Shinomiya, M. Tanaka, T. Kobayashi, Y. Nagahama,
S. Hamaguchi. 2000. The vasa-like gene, olvas,
identifies the migration path of primordial
germ cells during embryonic body formation
stage in the medaka, Oryzias latipes. Dev.
Growth Differ. 42: 317–326.
Di Cario AD, Travia G, DeFelici M. 2000. The meiotic
specific synaptonemal complex protein SCP3
is expressed by female and male primordial

ANNOTATED BIBLIOGRAPHY

Yamaguchi T. and T. Kitano. 2012. High temperatureinduces cyp26b1 mRNA expression and delays
meiotic initiation of germ cells by increasing
cortisol levels during gonadal sex
differentiation in Japanese flounder.
Biochemical and Biophysical ResearchCommunications 419: 287-292.
The Japanese flounder (Paralichthys olivaceus) is ateleost fish that has an XX/XY sex determination
system. However, the XX flounder can be sexreversed to phenotypic males by rearing the larvae athigh or low water temperatures. Thus, sex in theflounder is determined by genotype plus temperatureeffects. Therefore, the flounder provides an excellent
model to study the molecular mechanisms underlying
temperature-dependent sex determination. We
previously showed that cortisol, the major
glucocorticoid produced by the interrenal cells in
teleosts, causes female-to-male sex reversal by directly
suppressing mRNA expression of ovary-typearomatase (cyp19a1), a steroidogenic enzyme
responsible for the conversion of androgens to
estrogens in the gonads. In the present study, we
found that exposure to high temperature during
gonadal sex differentiation upregulates the mRNA
expression of retinoid-degrading enzyme (cyp26b1)
concomitantly with masculinization of XX gonads
and delays meiotic initiation of germ cells. We also
found that cortisol induces cyp26b1 mRNA
expression and suppresses specific meiotic marker
synaptonemal complex protein 3 (sycp3) mRNA
expression in gonads during the sexual
differentiation. In conclusion, cyp26b1 expression in
gonads is in male-specific manner during gonadal sex
differentiation and delays meiosis onset not only inmouse, chick and amphibian, but also in a teleost with
TSD. Therefore, CYP26B1 appears to regulate germ
cell fate by controlling retinoic acid signaling in amanner common among vertebrates.

60

Yamaguchi T., N. Yoshinaga, T. Yazawa, K. Gen, andT. Kitano. 2010. Cortisol is involved intemperature-dependent sex determination in
the Japanese flounder. Endocrinology,
Endocrinology 151:3900–3908.
The Japanese flounder (Paralichthys olivaceus) is ateleost fish with an XX/XY sex determination system.
However, XX flounder can be induced to develop intoeither phenotypic females or males, by rearing at 18
or 27 °C, respectively, during the sex differentiation
period. Cytochrome P450 aromatase, the product of
the gene cyp19, is expressed in various tissues andplays an important physiological role in theregulation of estrogen biosynthesis. Recently, it has
been reported that, in nonmammalian vertebrates, theexpression of cyp19 mRNA in the ovary is very muchhigher than in the testis during the period of gonadal
sex differentiation. In this sutudy, we demonstrated
that cortisol causes female-to-male sex reversal bydirectly suppressing cyp19a1 mRNA expression via
interference with cAMP-mediated activation and thatmetyrapone (an inhibitor of cortisol synthesis) inhibits
27 °C-induced masculinization of XX flounder.
Moreover, cortisol concentrations in 27 °C-reared
juveniles were significantly higher than in
18℃-reared fishes during sexual differentiation. In
conclusion, this study has presented the evidence of
involvement of cortisol in TSD. Cortisol induced themasculinization of XX fish by directly suppressing
cyp19a1 mRNA expression via interference with thecAMP-mediated activation. These findings provide
new insights into to the elucidation of molecularmechanisms underlying environmental sex
determination, including TSD.

61

RNAseq Analysis of Early Larval Development in Seriola lalandi with
Emphasis on Differential Development of Digestive System in Fast and Slow
Growing Groups¹ National Marine Fisheries Service,8901 La Jolla Shores Dr, 92037-1508 La Jolla, CA

digestive system, highlighting progressions withlarval stage and variation between size classes. Fishin the smaller size class developed more slowly, withheavier reliance on amylase and lipases. By 17dph,
faster growing fish displayed both upregulated
alkaline and acidic proteinase metabolic systems.

² Iowa State Genome Informatics Facility,
Ames, IA 50011

INTRODUCTION

Catherine Purcell1, Andrew Severin 2, Vincent P.
Buonaccorsi³, Mark Drawbridge4, Kevin Stuart4,
John Hyde¹

Harvest rates of global capture fisheries haveplateaued with most fisheries operating at maximum
or declining harvest levels (FAO 2012). However, as
capture fisheries have plateaued, the demand for
seafood and fishery products continues to increase. To
satisfy this demand, aquaculture production has
become increasingly important, making it one of thefastest-growing animal food-producing sectors,
growing at an average annual rate of 8.8 percent sincethe 1980s (FAO 2012). Globally, aquaculture value
exceeds US $119 billion (FAO 2012) and contributes toalmost half of the fish consumed (Terova et al. 2013).
Despite this explosive growth, aquaculture
development in the U.S. has lagged behind othercountries and is currently ranked 13th in aquaculture
production (Langan 2008; FAO 2012).

³ Juniata College,
1700 Moore St. Huntingdon PA 16652
4

Hubbs Sea World Research Institute,2595 Ingraham Street, San Diego, CA 92109

Corresponding author: buonaccorsi at juniata.edu
Key words: Seriola lalandi, aquaculture, genomics,
RNAseq, growth

ABSTRACT

Seriola lalandi is considered a prime candidate foraquaculture development in southern California. In
the U.S., hatchery production of both S. lalandi and S.
rivoliana is rapidly growing but has been hindered bya propensity for deformities and growth heterogeneity
developed during larval and early juvenile stages that
limit the production capacity and efficiency. The
causes of and solutions for the growth variation andhigh deformity rates remain unresolved. Our aim
here is to characterize development of physiologicalsystems important to aquaculture and understanddifferences in gene expression between size classes
that correlate with size heterogeneity. We performed
an RNAseq experiment to characterize differences ingene expression between fast and slow growing larval
S. lalandi at 2, 7 and 17 days post hatch (dph). Eachdevelopmental stage was represented by threebiological replicates, and each biological replicate wascomprised of mRNAs drawn from a pool of 10
individuals. An average of 60 million reads per
replicate were obtained from Illumina HiSeq
sequencing and subjected to differential gene
expression (DGE) analysis. Genes of related function
were sorted into clusters, and those that were found athigh frequency in the DGE set were identified. Here,
we focus on patterns of development related to the

Seriola species (S. dumerili, S. lalandi, S. rivoliana, S.
quinqueradiata), collectively known as amberjacks, arefish of particular interest to the growing aquacultureindustry due to their high value, forming a billion
dollar plus component of the sashimi industry. In
many locations, one or more of these species comprisea large percentage of the total marine finfish inculture. In Japan, for example, Seriola spp. account for
greater than 60% of the total mariculture program(Aoki et al. 2014). Culture of these species has
traditionally relied heavily on harvesting andgrowout of wild juveniles, which can be
unpredictable in supply and puts excessive pressure
on natural populations (Ohara et al. 2005; Ozaki et al.
2013; Aoki et al. 2014).
In the U.S., two Seriola species are currently incommercial culture. The yellowtail, Seriola lalandi, is apelagic species that is globally distributed in tropicaland sub-tropical waters (Kolkovski and Sakakura2004). This species is commercially cultured in Japan,
Australia, New Zealand, Chile, and even in the
Netherlands using recirculating aquaculture systems
62pronounced over time. Without size grading, growthheterogeneity results in larvae and early juveniles ofthe same age being at different developmental stages,
often requiring different feed items and culture
conditions.

(Abbink et al. 2012; Aguilera et al. 2013; Blanco Garcia
et al. 2014). In the northeast Pacific, the range of S.
lalandi extends from southern Washington State to
Mazatlán, Mexico, and the species is seasonallyabundant in southern California, with spawning
reported from Pt. Conception, California, southaround the Baja California peninsula (Sumida et al.
1985, Stuart et al. 2013). This native species is
considered a prime candidate for aquaculture
development in southern California. For over 10 years,
Hubbs-SeaWorld Research Institute (HSWRI) has been
experimentally culturing S. lalandi and has had goodsuccess spawning and rearing this species. Since 2007,
they have been developing more intensive culture
techniques, with the aim of transferring the technology
to the commercial sector in order to help establish amarine fish farming industry in the U.S. (Rotman et al.
2013). Currently, HSWRI is the only S. lalandi hatcheryin the U.S. (Rotman et al. 2013); it is also one of a few
hatcheries world-wide practicing closed life-cycle
production and not relying on wild-caught seed stock.
As noted above, the related S. quinqueradiata now
provides 60% of Japanese aquaculture production(152,000 tons in 2009; Ozaki et al. 2013), but growth inproduction is limited in part by reliance on the captureof wild juveniles due to difficulties in reliable hatcheryproduction of juveniles.

Despite exhaustive efforts by multiple groups, there
has been limited success in overcoming many of these
difficulties, which have limited the growth of thisindustry. The causes of and solutions for the growthvariation and high deformity rates remain unresolved(Stuart and Drawbridge 2012). Some deformities andgrowth variation are believed to be linked tonutritional deficiencies (in the larvae and/orbroodstock), culture conditions (e.g. light,
temperature, salinity) and various environmental
stressors. However, the high variability in deformities
and growth among spawns despite identical culturingconditions suggests that there may be a strong effectof parental genetic background (Roo et al. 2010).
Studies examining parentage assignment of juvenile
S. lalandi at the Southwest Fisheries Science Center(SWFSC) have shown connections between particularparents and deformities in offspring, indicating a
potential genetic component to this variability(unpublished data).

Figure 1: Deformities and growth heterogeneity in cultured S. lalandi
juveniles: a.) jaw deformity, b.) incomplete operculum, c.) malformed
operculum, d.) skeletal deformity, e.) size variation at 31 days post
hatching.

In the U.S., hatchery production of both S. lalandi andS. rivoliana is rapidly growing but has been hindered
by a propensity for deformities and growthheterogeneity developed during larval and early
juvenile stages that limit the production capacity andefficiency (Fig. 1). Variable growth rates lower thesurvival of smaller larvae/juveniles as a result ofcannibalistic predation and physical trauma fromlarger fish but also increase mortality in larger fishfrom choking on their smaller cohort members (Fig.
1e). Growth heterogeneity among larvae is apparent
at only a few days post-hatching, becoming more

Discovering methods to reduce deformities and size
variation in offspring is critical to Seriola culture. Here,
we aimed to identify and study molecular changes
underlying the physiological variation in development
of digestive system between fast and slow growing
groups of larval fish using RNAseq analysis. We
obtained samples of fast and slow growing larval S.
lalandi, sequenced whole larvae mRNA pools,
obtained gene counts, analyzed over-arching changes
63

in gene expression, and focused on characterizingchanges in specific digestive enzymes as markers ofdevelopment and relative fitness among groups.

RESULTS AND DISCUSSIONCount data were obtained for a total of 47,964
different assembled transcripts. A total of
approximately 20,000 transcripts had significant
protein blast hits to the non-redundant (NR) NCBI
database, and 13,000 hit proteins within the Danio
proteome. While many more transcripts wereidentified as differentially expressed using NR thanDanio identifiers (Fig. 2), Danio identifiers facilitatedanalysis of gene clustering and overrepresentation
using DAVID. Seven clusters of differentiallyexpressed genes related to digestion were found to be
overrepresented in frequency relative to the genomic

MATERIALS AND METHODS

RNA was extracted from whole fast and slow
growing larval S. lalandi. The slow-growing fish were
4.1 to 4.7mm (2dph), 4.7 to 5.2mm (7dph), and 5.4 to
6.4mm (17dph) in length. The fast-growing fish were
4.8 to 5.2mm (2dph), 6.0 to 6.3mm (7dph), and 8.7 to
10.5mm (17dph). Fish in the fast growing 17dph
group were visually assessed as in the post-flexion
developmental stage. Eighteen cDNA libraries weresequenced using a total of 600 million 50 bp pairedend reads at Iowa State University with Illumina
HiSeq. The libraries included three replicates for small
and large fish at 2, 7, and 17 days post-hatch.
Sequence adaptors and low quality ends were
removed with Trimmomatic (Bolger 2014). Trimmed
reads were aligned to a draft genome sequence(Purcell et al. in press) using GSNAP (Wu and Nacu
2010). Reads that mapped uniquely to the genome
were utilized for downstream analysis of differential
expression. Raw count data for each gene model was
obtained using the HTSeq program along with a GFF
file that was generated using Trinity (Haas et al. 2009)
and PASA (Haas et al. 2008). QuasiSeq in R (Lund2012) was used to normalization and differential gene
expression analysis. Read counts were normalized forlibrary size using upper quartile normalization
methodology. Multiple time points were modeled asa fixed-effect independent variable to increase
statistical power. All models assumed a negative
binomial distribution of read counts. False discovery
rate (FDR) correction was performed using theq-value method (Storey 2003). Open reading frames
from assembled transcripts were identified using
BlastP against the Danio rerio genome.
DAVID (Huang et al 2009) was used to cluster
differentially expressed genes by their function andtest for overrepresented gene ontology terms related
to digestion in particular. To investigate
overexpressed categories in more detail, expression
patterns for differentially expressed genes related todigestion in other species were also examined.
Tableau (Seattle, WA) was used to visualize trends in
S. lalandi gene expression related to digestion.

Figure 2. Number of differentially expressed genes among all comparisongroups with alternative NR or Danio rerio identifiers. Nodes identify
comparison groups for DGE comparisons: large or small fish and days
post hatch (2, 7, or 17 dph), and vertices are labelled with number ofdifferentially expressed genes (NR, Danio rerio).

background in pairwise comparisons of fish fromdifferent stages and sizes (Table 1). Overrepresented
clusters related to alkaline peptidases and theirinhibitors, as well as carbohydrate metabolism.
Protease patterns were similar to those found in otherspecies (Zambonino-Infante et al. 2008; ZamboninoInfante and Cahu 2010), with a faster developmental
progression in fish from the larger group. The
primary alkaline peptidases, trypsin, chymotrypsin,and elastase, were high from the start of theexperiment and increased faster in larger fish (Table2). Peptidase inhibitors showed a similar pattern,with expression increasing faster in larger fish. These
are necessary to inactivate and control peptidases toprotect native tissue. Proteases characteristic of amature stomach were seen only in individuals fromthe large group. Acidic digestion was detected withconcurrent dramatic increases in pepsinogen andpepsin transcripts, along with transcripts forhydrogen chloride pump that is necessary toactivate pepsinogen.
64

Table 1. Seriola lalandi RNAseq overrepresented differentially expressed gene clusters related to digestion. Experimental column labels identify
comparison groups for DGE comparisons: large (Lg) or small (Sm) fish and days post hatch (2, 7, or 17 dph).

Overall, developmental patterns were similar to thoseseen in other marine fish. Fish in larger group
showed stronger, earlier increases in many protein
digestive enzymes. Larger fish showed less relianceon starch, and presumably more advanced protease,lipase, and carbohydrate profiles. This study
demonstrates utility of the RNAseq method intracking changes expected in development anddiscovering new patterns in enzyme systems related
to growth. Arrested development of slow-growing
fish digestive tract indicates that a single food is not
likely to be efficiently digested by both fast and slow
growing larvae. Until extreme growth rate
heterogeneity can be prevented, separation of fishfrom different growth stages and tailored feeding iswarranted until all fish are post-flexion indevelopmental stage. Future analysis will involve
looking for shifts in relevant regulatory gene
expression levels for “master control genes” andmutations in regulatory genes between groups.

Lipases were present early in both groups and showedvarying patterns over time and between groups.
Pancreatic bile salt activated lipase (BAL) is the major
digestive neutral lipase in teleosts for the digestion offat, secreted from the vertebrate pancreas into theintestine (Saele et al. 2010). Transcripts for BAL andphospholipases (not shown) showed no consistent
trends, with both pronounced increases and decreases
depending on the transcript (Table 2). Hepatic lipase,however, showed dramatic increases in large 17 dph
fish only. This enzyme is a major lipase involved inphospholipid and triglyceride metabolism (Connelly1999), and here likely indicates a more developed liverin the faster progressing group.
Amylase is a major metabolic enzyme for digestion of
starch. Its expression has been well studied and ischaracterized by high levels early, followed by adecreasing trend (Douglas et al 2000; ZamboninoInfante et al. 2010). Given the consistency among
species, these changes have been labelled “geneticallyprogrammed” (Zambonino-Infante et al. 2010).
Larger fish were further in their progression, withlower levels overall and faster decreases.
Nonetheless, there was evidence of a higher generalcarbohydrate metabolism in larger fish (data notshown), so they may be obtaining carbohydrates fromalternative sources, such as chitin from Artemia intheir feeding program.

65

Table 2. Differentially expressed transcripts related to digestion in Seriola lalandi. Median transcript counts over three biologicalreplicates per experimental treatment group are shown. Experimental groups include large or small fish at 2, 7, or 17 days post-hatch.

66

Kolkovski, S., and Y. Sakakura, Y. 2004. Yellowtail
kingfish, from larvae to mature fish –problems and opportunities. In Cruz Suárez,L., et al. Advances in Nutrición Acuícola VII.
Memorias del VII Simposium Internacional de
Nutrición Acuícola. 16-19 Noviembre, 2004.
Hermosillo, Sonora, México.
Langan, R. 2008. The role of marine aquaculture inmeeting the future demand for animal protein.
J Foodservice 19:227–233.
Lund, S.P., D. Nettleton, McCarthy D.J., and G.K.
Smyth 2012. Detecting differential expression
in RNA-sequence data using quasi-likelihood
with shrunken dispersion estimates. Stat Appl
Genet Mol 11(5) doi:10.1515/1544-6115.1826
Ohara, E., et al. 2005. Genetic linkage maps of two
yellowtails (Seriola quinqueradiata and Seriolalalandi). Aquaculture 244:41-48.
Ozaki, A., et al. 2013. Quantitative Trait Loci (QTL)
associated with resistance to a monogenean
parasite (Benedenia seriolae) in yellowtail(Seriola quinqueradiata) through genome wide
analysis. PLOS One 8:e64987.
Purcell, C.M., A. Severin, M. Drawbridge, K. Stuart,and J. Hyde (in press) The de novo draft
assembly of the yellowtail, Seriola dorsalis,genome. NOAA Tech Memo. NMFS.
Rotman, F., K. Stuart, and M. Drawbridge. 2013.
Intensive juvenile production of yellowtail
amberjack (Seriola lalandi) in Southern
California. In Rust, M., et al. (editors)
Hatchery Tech for High Quality Juv Prod:
Proceedings of the 40th U.S.-JP Aquacult PanelSymp, Honolulu, HI, Oct 22-23, 2012. U.S.
DOC, NOAA Tech. Memo. NMFS-F/SPO-136.
p75.
Sæle O, A. Nordgreen, P.A. Olsvik, and K. Hamre
2010. Characterization and expression ofdigestive neutral lipases during ontogeny ofAtlantic cod (Gadus morhua), Comparative
Biochemistry and Physiology Part A:
Molecular & Integrative Physiology, 157:
252-259.
Stuart, K., and M. Drawbridge, M. 2012. Spawningand larval rearing of California yellowtail(Seriola lalandi) in Southern California. Bull
Fish Res Agency 35:15-21.
Roo, F., et al. 2010. Occurrence of skeletal deformities
and osteological development in red porgy
Pagrus pagrus larvae cultured under different
rearing techniques. J Fish Biol 77:1309-1324.
Storey J.D., and R. Tibshirani. 2003. Statisticalsignificance for genome wide studies. Proc
Natl Acad Sci USA 100: 9440-9445.

REFERENCES

Abbink, W., et al. 2012. The effect of temperature andpH on the growth and physiological responseof juvenile yellowtail kingfish Seriola lalandi inrecirculating aquaculture
systems. Aquaculture, 330, 130-135.
Aguilera, E., G. Yany, and J. Romero 2013. Cultivableintestinal microbiota of yellowtail juveniles(Seriola lalandi) in an aquaculture system. Latin
American Journal of Aquatic Research, 41, 395403.
Aoki, J., et al. 2014. Construction of a radiation hybrid
panel and the first yellowtail (Seriola
quinqueradiata) radiation hybrid map using ananofluidic dynamic array. BMC Gen 15:165.
Blanco Garcia, A., G.J. Partridge, G. Flik, J.A. Roques,
and W. Abbink. 2014. Ambient salinity andosmoregulation, energy metabolism andgrowth in juvenile yellowtail kingfish (Seriolalalandi Valenciennes 1833) in a recirculating
aquaculture system. Aquaculture Research,early view online.
Bolger, A. M., Lohse, M., and B. Usadel 2014.
Trimmomatic: A flexible trimmer for Illumina
sequence data. Bioinformatics, btu170.
Connelly, P.W. 1999. The role of hepatic lipase inlipoprotein metabolism. Clinica Chimica Acta.
286: 243-255.
Douglas, S. E., Mandla, S. and Gallant J.W. 2000.
Molecular analysis of the amylase gene and its
expression during development in the winterflounder, Pleuronectes americanus. Aquaculture
190: 247-260.
FAO. 2012. The State of World Fisheries andAquaculture. Rome. 209 pp.
Huang, D.W., Sherman, B.T. and Lempicki, R.A. 2009.
Systematic and integrative analysis of large
gene lists using DAVID bioinformatics
resources. Nature protocols, 4(1), pp.4457. Haas, B.J., Salzberg, S.L., Zhu, W., Pertea,
M., Allen, J.E., Orvis, J., White, O., Buell, C.R.
and Wortman, J.R., 2008. Automatedeukaryotic gene structure annotation using
EVidenceModeler and the Program to
Assemble Spliced Alignments. Genome
biology, 9(1), p.R7.
Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr,M., Blood, P.D., Bowden, J., Couger, M.B.,
Eccles, D., Li, B., Lieber, M. and MacManes,
M.D., 2013. De novo transcript sequence
reconstruction from RNA-seq using the Trinity
platform for reference generation and analysis.
Nature protocols, 8(8), pp.1494-1512.
67

A total of 169,326,711 paired-end reads weregenerated, resulting in a transcriptome with 68,220
contigs. This study describes the clusters formed by
the differentially expressed transcripts and how gene
expression patterns changed over the developmental
periods. The approaches taken in this study and theexamination of different larval stages are of interestfor our work on larval yellowtail gene expression.

Stuart, K., F. Rotman, and M. Drawbridge. 2013.
Larval rearing advancements for yellowtail
amberjack in (Seriola lalandi) in Southern
California. In Rust, M., et al. (editors)
Hatchery Tech for High Quality Juvenile
Production: Proc 40th U.S.-JP Aqua PanelSymp, Honolulu, HI, Oct 22-23, 2012. U.S.
Dept. Commerce, NOAA Tech. Memo.
NMFS-F/SPO-136. p.69.
Sumida B., H. Moser, and E. Ahlstrom. 1985.
Descriptions of larvae of California yellowtail,
Seriola lalandi, and three other carangids fromthe eastern tropical Pacific: Chlorosconbrus
orqueta, Caranx caballus, and Caranx
sexfasciatus. CalCOFI 26:39-159.
Terova, G., et al. 2013. Molecular cloning and gene
expression analysis in aquaculture science: areview focusing on respiration and immune
responses in European sea bass (Dicentrarchus
labrax). Rev Fish Biol Fisheries 23:175-194.
Wu, T., and S, Nacu. 2010. Fast and SNP-tolerant
detection of complex variants and splicing inshort reads. Bioinformatics (Oxford, England),p.btq057v1.
Zambonino-Infante, J.L., E. Gisbert, C. Sarasquete, I.
Navarro, J. Gutierrez, and C.L. Cahau. 2008.
Ontogeny and physiology of the digestivesystem of marine fish larvae. In. Feeding anddigestive functions of fishes. pp. 281-348.
Zambonino-Infante, J.L., and C.L. Cahau. 2010. Effectof nutrition on marine fish development andquality. In. Recent advances in aquaculture
research. pp. 103-124.

Valente, L.M.P., K.A. Moutou, L.E.C. Conceiçāo, S.
Engrola, J.M.O. Fernandes, and I.A. Johnston.
2013. What determines growth potential andjuvenile quality of farmed fish species?
Reviews in Aquaculture, 5 (Suppl. 1):
S168-S193.
This review focuses on the research related to the
biology, physiology, and genetics that are relevant tolarval fish growth and quality. Specifically, this review
goes into great detail on understanding fish skeletal
muscle ontogeny, and the impact of that on larval andjuvenile fitness. Genes and gene pathways involvedin the development of these systems and in muscle
growth processes are described in detail, along withthe current state of knowledge from research on
non-salmonid fish species in aquaculture. This
research is of interest for our work on exploring
growth variation in larval Seriola lalandi using RNASeq data.
Srichanun M, Tantikitti C, Utarabhand P, Kortner TM.
2013. Gene expression and activity ofdigestive enzymes during the larval
development of Asian seabass (Lates calcarifer).
Comparative biochemistry and physiology,
Part B. 165:1-9.

ANNOTATED BIBLIOGRAPHY

Sarropoulou, E., H.K. Moghadam, N. Papandroulakis,
F. de la Gandara, A.O. Garcia, and P. Makridis.
2014. The Atlantic bonito (Sarda sarda, Bloch1793) transcriptome and detection ofdifferential expression during larvae
development. PLoS One, 9(2): e87744

The authors characterize gene expression and activity
patterns during larval development from 2 to 30days
post hatch (dph) in the Asian seabass. Increases anddecreases in key enzymes involved in digestion of
starches, lipids, and proteins were tracked and major
developments of the digestive system were noted.
This research is of interest for our work on exploring
major patterns of development in larval Seriola lalandi
using RNA-Seq data. Differences in development of
the digestive system between fast and slow growing
Seriola lalandi larvae may affect protocols forweaning off of live-food.

In this study the authors generated and assessed thetranscriptome and gene expression profile of fivedevelopmental stages of the Atlantic bonito.
Aquaculture on this species is just starting andalthough there were not many genetic resources
available for this non-model species, the authors
wanted to use molecular tools to help produce highquality juveniles for aquaculture production. Pooled
larval bonito were collected at 0 dph, 5 dph, 10 dph,
20 dph, and 30 dph. Samples were extracted andprepared for sequencing on the Illumina HiSeq 2000.
68

Exploring the Genetic Risks Posed to Natural Populations by Escaped Cultured
Marine Fish: A Reintroduction to the OMEGA Modelthreat to genetic integrity these escaped fish pose,several factors merit consideration, including but notlimited to the population genetic structure andphenotypic variability of the species in the wild; sizeof the local (affected) wild population relative to themagnitude, frequency, and survival rate of theescapees; age and growth characteristics of the wild
and cultured populations; encounter rate between
wild and cultured fish; management practices at thefarm, including broodstock selection and breeding
protocols; and the likelihood of genetic drift anddomestication resulting from farm practices.

Kristen M. Gruenthal ¹, Gregory R. Blair ², Jason D.
Volk ², Michael B. Rust ³¹ Northwest Fisheries Science Center,
National Marine Fisheries Service,
National Oceanic and Atmospheric Administration,
2725 Montlake Blvd E, Seattle WA 98112, USA² ICF International,
710 Second Avenue Suite 550,
Seattle, WA 98104, USA³ Office of Aquaculture,
National Marine Fisheries Service,
National Oceanic and Atmospheric Administration,
1315 East-West Highway,
Silver Spring, MD 20910, USA

Unfortunately, little information is available toreliably assign the risk to population fitness due toescapes. Because scientific data is lacking, escapestandards are largely theoretical or qualitative rather
than quantitative. To address this technical andscientific barrier and advance sustainable US marine
aquaculture, the NOAA Fisheries Office of
Aquaculture (OAQ) fostered a research initiative to
explore the interactions between wild fish andescapees and how to minimize the genetic impact. As
part of this initiative, the Offshore Mariculture
Escapes Genetics Assessment (OMEGA) model wasdeveloped by NOAA Fisheries and ICF International
to simulate, identify, and quantify the fitness risk
posed by escapees over time. Ultimately, OMEGA isintended to provide insight into factors affecting risk,
help identify research priorities, explore options fordesign or modification of culture programs, andinform policy and management decisions related tomitigation of genetic impacts. To realize theseobjectives, the OAQ is focused on fostering external
collaborations to develop model scenarios, evaluate
model parameters, and validate the model with datafrom current and planned domestic and international
marine aquaculture operations.

Corresponding author: kristen.gruenthal at noaa.gov
Keywords: domestication selection, fitness, genetic
load, offshore aquaculture

ABSTRACTThe rapid development worldwide of offshore marine
finfish aquaculture has raised concerns, at least inpart, because aquaculture programs have thepotential to create various fitness effects if cultured
fish escape and come in contact with wild
conspecifics. Physical contact alone may result infragmentation, competition, and disease transmission,
while reproduction between cultured and wild fish
may have negative genetic consequences for themixed (wild plus cultured) population. Genetic
impacts may include reduced genetic variability,
introduction of non-native or maladapted alleles,
swamping of the gene pool, inbreeding or
outbreeding depression, and domestication, each ofwhich can compromise the adaptive potential of themixed population, making it less fit or able to respondto environmental change.

INTRODUCTION

The rapid development of offshore marine finfish
aquaculture worldwide has raised concerns due, insignificant part, to the potential negative fitness
impact(s) escaped cultured fish may have on natural
populations (Tufto 2010; Waples et al. 2012). Fitness isinversely related to the level of domestication inescapees (Baskett et al. 2012), and fitness declines due
to domestication can occur rapidly in the wild (Araki

Fish escape for a variety of reasons, such as using
improper mesh size or the presence of holes fromnormal wear and tear in net pen cages; carelessness
during fish transfer among cages; washout from highwind and sea conditions during storms; and cagebreaches by large predators. When evaluating the69

et al. 2008). Cultured fish escape from aquaculture
holding systems for a variety of reasons. Operators
may use a poor mesh size for net pen cages or holes in
the nets may arise from normal wear and tear. Fishmay be lost during transfer among cages (e.g. forgrading, harvesting, etc.), due to high wind and sea
conditions during storms, or when predators breachcages. Even spawning of reproductively matureindividuals within cages may result in escaped
gametes and fertilized eggs.

(Volk et al. 2015). Volk et al. (in press) then described
how OMEGA was originally developed,
parameterized, and verified using a hypothetical US
domestic aquaculture program for sablefish(Anoplopoma fimbria). Here, we reintroduce OMEGA,
touching again on the model’s theoretical basis;
describe user inputs and model outputs; discuss
potential applications; and make a call for domestic
and international collaboration toward validation of
the model.

Non-genetic impacts of offshore aquaculture occur
because of spatial proximity and physical contact
between cultured and wild conspecifics and include
the fragmentation of populations, competition,disease transmission, and pollution (IWG-A 2014).
Genetic impacts occur due to gene flow when
individuals from cultured and wild populations
interbreed (Baskett et al. 2013). The introduction ofhatchery genetic backgrounds, which may include
non-native or maladapted alleles, can change thegenetic composition of the wild population,
potentially resulting in reduced genetic variability,
inbreeding or outbreeding depression, anddomestication (Tringali and Bert 1998; Tringali et al.
2007; Baskett and Waples 2012). Larger escape events
could even swamp the wild population withhatchery-based alleles. These impacts threaten theadaptive potential of the wild population and may
make it less able to respond to and survive
fluctuations in the environment (Tringali and Bert
1998), including both stochastic events and long-term
trends like climate change, ocean acidification, andwarming ocean temperatures.

DISCUSSIONOMEGA was developed to provide insight into
factors affecting risk, help identify research priorities,
explore options for the design or modification ofculture programs, and inform policy andmanagement decisions related to the mitigation ofgenetic impacts. To evaluate risk, several factors –describing threats to and vulnerabilities of the system– are important, including 1) the genetic structure andphenotypic variability of the species in the wild; 2)
local wild population size relative to the number andsurvival rate of escapees, as well as frequency ofescape events; 3) age and growth characteristics of thewild and cultured populations, including maturation
schedule and fecundity; 4) amount of contact between
wild and cultured fish; 5) hatchery practices,
including broodstock selection and breeding
protocols, which affect the likelihood and magnitude
of genetic drift and domestication. Once threats andvulnerabilities have been identified and quantified,risk (here, the probability of a negative fitness impact
due to escapes from aquaculture) can be assessed.
Subsequent risk reduction may then be fulfilled
through applied research, design and modification ofculture programs, and informed policy andmanagement decisions, aspects of which may be fed
back into OMEGA for further evaluation.

To address the issue of escapes from offshore
aquaculture operations, the NOAA Fisheries Office of
Aquaculture (OAQ) developed a research initiative
designed to explore the genetic interactions between
wild and cultured fish. As part of this initiative, theOAQ first sanctioned a comprehensive in-housereview of the genetic risks of marine aquaculture(Waples et al. 2012). The OAQ then solicited the helpof scientists at ICF International, Inc., (ICF) to develop
the Offshore Mariculture Escapes Genetics
Assessment (OMEGA) model. OMEGA is a Microsoft
Excel-based program designed to simulate the risk
posed to wild population fitness by escapes frommarine aquaculture programs. ICF scientists Greg
Blair and Jason Volk and collaborators originallyintroduced the OMEGA model at UJNR Aquaculture
2013 in Sapporo, Hokkaido, Japan, discussing the
program and its guiding principles, as well as therelated and precursory All-H Analyzer (AHA) model

Structure and inputs
OMEGA is organized around three interacting
components that incorporate the above factors: 1) theaquaculture operation itself, including the frequency
and magnitude of escape events, and the cultured
population characteristics, such as the number of fish,
broodstock source, maturation, and growth; 2) wild
population characteristics, such as abundance,
distribution, survival, age and size at maturity, age
composition, and age-specific harvest rates; and 3) the
potential for interaction between escapes and wild
conspecifics, which is affected by characteristics likethe location of pens relative to the wild populationand the survival rate, movement, and reproductive70

and subject to bell-shaped (Gaussian) selection; 3)mating is random, meaning the escapees and wild fish
do not sort by origin; 4) population size is large, so
drift, phenotypic plasticity, and other stochastic forces
are ignored; 5) changes in mean trait value aredeterministic, based on selection and gene flow; and 6)
selection does not reduce population size, variance, or
heritability of the trait over time. Ford (2002)
originally developed it to assess the effects ofsupportive breeding on wild population fitness, andthe model was first incorporated by an earlier Excelbased analytical tool called the All H Analyzer (AHA).
The AHA was used to explore ways to balance the“4-Hs” – habitat, hatcheries, harvest, and hydroelectriceffects – toward restoration and management of PacificNorthwest salmon populations.

success of escapees in nature. Under thesecomponents, OMEGA employs a modular format todescribe assumptions used to model the potential
interactions and impacts of escapees in the wild.
There are nine modules requiring user input. The
aquaculture operation and cultured population
component contains modules describing the fishculture program itself and escape scenarios, while thewild population component has modules for natural
production, growth and maturity, and harvest. Under
the interactions component are modules for the
relative survival of escapes, encounter rate, andfitness. Finally, there is an optional regulatory
standards module, which helps the user determine
whether the aquaculture program may be incompliance with or violation of certain regulations, if
they exist.

In terms of OMEGA, Ford (2002) underlies aquantitative two population analysis of differential
selection regimes and the effect of gene flow between
the populations on the mean trait value. Initially, eachof the two populations is exposed to a separate
environment (hatchery or wild) and, therefore,

Each module contains a variety of user inputs,
ranging from basic settings and preferences for saving
the workspace and running simulations to wild andcultured population data (e.g. Fig. 1). Currently, there
are 103 possible user input variables (Index of User

Figure 1. User input interface for cultured population characteristics.
Inputs include information, such as annual production goals, size of fish at
harvest, annual inventories, von Bertalanffy growth parameters, and the
proportion of natural origin broodstock.

Inputs available upon request). Some modules andinputs are optional, while other inputs represent
alternatives for which the user chooses one of two or
more options. Ultimately, appropriate default values
will be assigned for certain parameters, with theintent of making the model more user friendly, as wellas time efficient.
Theoretical basis
Once the model has been parameterized, OMEGA
employs a single trait phenotypic fitness model
developed by Ford (2002) to simulate risk. The
assumptions of Ford (2002) are: 1) a single trait isunder selection with different optimum values in thetwo environments; 2) the trait is normally distributed71

selective regime (domestication or natural). The mean
trait value of each population is equal to its
environmental (and fitness) optimum. In one scenario,
gene flow is unidirectional from the hatchery to thewild as escapees breed with the wild population. The
resulting case is a change in the wild population mean
trait value as it shifts toward the hatchery optimum. It
should be noted that it is assumed any shift away fromeither environmental optimum leads to a reduction in
fitness (Lande 2007; Tufto 2010). An alternative
scenario encompasses the first but also includes gene

larger and/or successive events resulting in higher
percentages, but gradually shrinking to <1% of the
population (Fig. 2).

flow from the wild population to the cultured
population as determined by the percent broodstock
sourced from the wild population. The resulting caseis a more nominal to nonexistent change in the wild
population mean trait value.

High chronic escapes coupled with several acuteescape events, in contrast, had a longer lasting impact
on population make-up. The high escape scenario
resulted in escapees comprising a significantly higher
proportion, averaging 5-7%. Correspondingly, fitness
in the wild was reduced (shifted toward the hatchery
optimum) more under the high escape scenario,
whereas the low escape scenario remained near thewild optimum (Fig. 3). These results are in keepingwith what is expected according to Ford (2002), as
well as results from related research (e.g. Baskett etal. 2013).

Outputs
OMEGA outputs the simulation results in graphicalformat, and the figures include total biomass andtotal spawning biomass, plus percent escapeebiomass and hatchery origin spawners; total harvestand percent wild yield; change in wild abundance
due to escapes; and the change in wild fitness due toescapes. The original verification scenarios, forexample, explored a low incidence of escapes versus
a high incidence with a high encounter rate for asablefish (Anoplopoma fimbria) hatchery program in
the Pacific Northwest (Volk et al. 2015). Over thedefault 100-year timespan of the simulations, the low
escapes scenario resulted in escapee percentage
peaking at ≤5% of the total population and thespawning biomass soon after each escape event, with

Future research
Aquaculture must simultaneously support
commercial interests; increase the availability of safeand nutritious fish, shellfish, and other products for
consumers; and protect wild populations (IWG-A

Figure 2. Simulation results for low escapes scenario. The number of escapees, based on escape event type, over time (here, 100 years) is in
the upper left panel; escapees as a proportion of the population is at lower left; trends in wild population fitness at upper right; and the percent
change in natural recruitment due to escapees is at lower right.

Figure 3. Simulation results for high escapes scenario. The number of escapees, based on escape event type, over time (here, 100 years) is in
the upper left panel; escapees as a proportion of the population is at lower left; trends in wild population fitness at upper right; and the percent
change in natural recruitment due to escapees is at lower right.

72

2014). OMEGA was designed to as a decision-making
tool to assist in the assessment and management ofmarine aquaculture operations such that they may
remain both commercially viable and environmentally
responsible. Toward that end, there are four key
questions that OMEGA may be used to address as
marine aquaculture moves forward:
1. How significant are the impacts of escapees
surviving to reproduce?
2. What are the effects of our decisions aboutaquaculture operations?
3. How do we define an acceptable risk to marine
resources?
4. How do we balance the dual goals of
economically viable aquaculture and conservationof marine resources?

tools like OMEGA, will enable informed discussion
about escapes and their risks to sustainability of wild
populations.

CONCLUSIONOMEGA v1.0 and its user guide are freely available
for download on the OAQ website at www.nmfs.
noaa.gov/aquaculture/science/omega_model_
homepage.html. However, the model is still a work inprogress. OMEGA requires further testing, including
a sensitivity analysis of model inputs, using multiple
real systems. The next steps toward finalizing the
OMEGA model package for broader use are 1) to
identify collaborators to develop case studies for
current and future aquaculture programs for marine
finfish species, 2) complete a sensitivity analysis ofcase studies, including determining acceptable default
parameters and proxies; and 3) develop an economic
analysis module.

First, in the shorter-term, we can use the model andthe best available scientific information to explore thesingle-generation effects of reproductively-matureescapees. Longer-term, we would couple the use of
OMEGA with monitoring and evaluation to improve
our understanding of the realized effects of theseescapees over time, both as more escape eventsoccur, as later generations of offspring of mixed
background also mature, and if and when hatcherypractices change.

The OMEGA team is extremely interested in pursuingpotential collaborations with Japanese scientists.
Japan supports several longer-term and prolificaquaculture programs for economically importanttaxa of marine finfish, from groundfish to pelagic,
such as olive flounder (Paralichthys olivaceus), red sea
bream (Pagrus major), and several yellowtail jacks(Seriola spp.). The OMEGA development and test
team would like to collaborate with one or more
Japanese researchers having an interest in modelingthe potential fitness effects of escapes from current
and planned marine aquaculture operations in the
country. Japanese researchers involved with these
programs represent excellent potential sources ofreal-world data (e.g. natural stock structure, life
history, demographic, and ecological information;
culture program history and scope; and regulatory
requirements) for parameterizing OMEGA,
developing model scenarios, and validating model
assumptions. Moreover, these scientists possess theknowledge and experience to assess model output,
which will help identify the model’s strengths andweaknesses.

Next, in terms of economic viability, many
aquaculture operators will eventually develop andimplement various genetic improvement programs,
including selective breeding, to increase theprevalence of desirable characteristics in theircultured populations. Moreover, selective breeding
programs have the opportunity to enhancesustainability by improving feed and labor utilization
efficiencies (Gjedrum 2012). In US marine
aquaculture at present, genetic modification and othermore manipulative technologies (e.g. cloning,transgenics) are either not commonly employed or
not permitted for offshore stocking in federal waters(e.g. Gulf of Mexico Aquaculture Fishery
Management Plan); conventional and marker assisted
selective breeding and hybridization, although stillrare are permitted (i.e., Atlantic salmon culture).
Evaluation (with OMEGA) of genetic improvement
programs is more likely to entail modeling various
scenarios involving domesticated, or even inbred,broodstocks; non-chemical sterilization (e.g. triploidy,
irradiation); or intraspecific hybridization. In terms of
the final two questions, defining “acceptable” requires
a qualitative systematic review of the best-available
science that, when coupled with decision support

Since OMEGA is freely available, support levels fromcollaborators may range from supplying data ormodel results only, to active involvement indeveloping various real and/or simulated
aquaculture program scenarios, to problem-solving
and decision-making for future aquaculture planning,
and up to mitigation planning within the Japanese
regulatory framework. Preferentially, the teams
would work closely with one another to develop the73

aquaculture programs. Bulletin of the Fisheries
Resource Agency 40:89-96.
Waples R.S., K. Hindar, and J.J. Hard. 2012. Genetic
risks associated with marine aquaculture.
NOAA Technical Memorandum NMFSNWFSC-119. US Department of Commerce
NOAA NMFS, Washington, D.C.

most mutually beneficial and comprehensive
evaluations of OMEGA, the species of interest, andthe current or proposed aquaculture program.

REFERENCES

Araki H, B.A. Berejikian, M.J. Ford, M.S. Blouin. 2008.
Fitness of hatchery-reared salmonids in thewild. Evolutionary Applications 1:342-355.
Baskett M.L., R.S. Waples. 2012. Evaluating alternative
strategies for minimizing unintended fitness
consequences of cultured individuals on wild
populations. Conservation Biology 27:83-94.
Baskett M.L., S.C. Burgess, R.S. Waples. 2013.
Assessing strategies to minimaize unintended
fitness consequences of aquaculture on wild
populations. Evolutionary Applications
6:1090-1108.
Ford M.J. 2002. Selection in captivity during
supportive breeding may reduce fitness in thewild. Conservation Biology 16:815-825.
Gjedrum T. 2012. Genetic improvement for thedevelopment of efficient global aquaculture: apersonal opinion review. Aquaculture 344349:12-22.
Interagency Working Group on Aquaculture (IWG-A).
2014. National Strategic Plan for Federal
Aquaculture Research (2014-2019). National
Science and Technology Council, Committee
on Science, www.whitehouse.gov/sites/
default/files/microsites/ostp/NSTC/
aquaculture_strategic_plan_final.pdf.
Lande R. 2007. Expected relative fitness and theadaptive topography of fluctuating selection.
Evolution 61:1835-1846.
Tringali M.D., T.M. Bert. 1998. Risk to genetic effective
population size should be an importantconsideration in fish stockenhancement
programs. Bulletin of Marine Science 62:641659.
Tringali M.D., T.M. Bert, F. Cross, J.W. Dodrill, L.M.
Gregg, W.G. Halstead, R.A. Krause, K.M.
Leber, K. Mesner, W. Porak, D. Roberts, R.
Stout, and D. Yeager. 2007. Genetic policy forthe release of finfishes in Florida. Florida Fish
and Wildlife Conservation Commission,Florida Fish and Wildlife Research Institute,Publication No. IHR-2007-1, St. Petersburg.
Tufto J. 2010. Gene flow from domesticated species towild relatives: migration load in a model ofmultivariate selection. Evolution 64:180-192.
Volk J.D., M.R. Rust, G.R. Blair, L.E. Mobrand, C.V.W.
Mahnken, and W.W. Dickhoff. 2015. Modelingintraspecific genetic effects for management of

ANNOTATED BIBLIOGRAPHY

Ford M.J. 2002. Selection in captivity during
supportive breeding may reduce fitness in thewild. Conservation Biology 16:815-825.
The author presents a single-trait phenotypic modelthat assumes different optimum trait values forhatchery and natural environments. Hence, due tothe various effects of genetic drift and domestication
selection in a hatchery environment, escaped cultured
fish may cause a potential loss of genetic fitness if
breeding occurs between them and wild conspecifics.
The Ford model describes how mean phenotype
values of the mixed population (captive plus wildfish) may shift relative to the optimum values for eachenvironment, based on the presence/absence andamount of gene flow (interbreeding) between thecultured escaped and wild fish. The overall fitness
effect depends on details, such as magnitude of thedifference in optimum trait value, trait heritability,
and selection pressure in the wild, as well as habitat
capacity, number of escapes, and population
demographics and dynamics. This model has beenused in several studies, most notably in salmonids, toexplore the potential fitness consequences ofaquaculture, whether from supplementation of wild
populations or the unintended straying of cultured
fish to wild populations.
Tringali M.D., T.M. Bert, F. Cross, J.W. Dodrill, L.M.
Gregg, W.G. Halstead, R.A. Krause, K.M.
Leber, K. Mesner, W. Porak, D. Roberts, R.
Stout, and D. Yeager. 2007. Genetic policy forthe release of finfishes in Florida. Florida Fish
and Wildlife Conservation Commission.
Florida Fish and Wildlife Research Institute,Publication No. IHR-2007-1, St. Petersburg.
The authors of this document provide acomprehensive overview, including key references, ofthe potential genetic effects aquaculture may have on
natural populations, such as a loss of fitness andviability, altered genetic diversity, and a reduction in
long-term adaptive potential. The document is also
intended to build a policy-based framework for74

genetic management planning for species subject tosupplementation in the state of Florida. Tringali et al.
(2007) describe the various types of purposeful
introductions that may occur, touching on escapesfrom commercial aquaculture, as well, and theauthors also provide descriptions of options forevaluating, monitoring, and mitigating the negative
genetic effects that may be seen in mixed populationsof wild and cultured conspecifics. Finally, thedocument provides a step-by-step template useful to(non-geneticist) aquaculture managers, both within
and outside of the state of Florida, to develop
responsible and sustainable genetic management
plans for supplemented species.
Waples R.S., K. Hindar, and J.J. Hard. 2012. Genetic
risks associated with marine aquaculture.
NOAA Technical Memorandum NMFSNWFSC-119. US Department of Commerce
NOAA NMFS, Washington, D.C.
The authors of this NOAA technical memorandum
intended to “provide managers with a better
understanding of the genetic effects of marine
aquaculture on natural populations, so that thesefactors can more effectively be incorporated into
informed decisions pertaining to federal marine
aquaculture policy and regulatory decisions.” Waples
et al. (2012) is a comprehensive overview of thegenetic issues and impacts to wild populations
associated with aquaculture. The document attempts
to synthesize relevant information and provide key
references, identify areas where additional research isneeded, provide a framework for assessing andbalancing risks, and give examples of how monitoringand evaluation might be done toward developing
responsible and sustainable aquaculture. While
Waples et al. (2012) focuses on commercial
aquaculture of marine finfish, it also discusses
information gathered from salmon hatcheries andmarine stock enhancement, which are related tomarine aquaculture, and provide arguably morecomprehensive scientific data due to their comparably
longer history in the USA.

75

Suspended Culture of Asari Clam, Ruditapes philippinarum, and Their Roles inthe Ecosystem
18, 2011. After the five month culture, average SL and
WW of the clams reached 33 to 35 mm and 8.3 to 9.7
g, and the survival rate was higher than 90%. Total
wet weight of clams in the containers having started
from 300 g reached 1,100 to 1,300 g. In terms of
substrates, 50% of Care-Shell contents or overmaintained the pore water pH of more than 8.0
during the culture period. The growth of the clams in
50 and 80% of Care-Shell contents seemed to be better
result, but not significant. The growth of Asari clamin suspended culture showed much higher than on
the tidal flat in Ise Bay. The results suggest thatsuspended culture of the clam has a potential forcommercial clam production both on the high growthand survival rates.
According to the filtration experiment, the clam canfiltrate 1.5 to 2.5 L/hr/g soft tissue dry weight. On
the other hand, chlorophyll concentration adjacent thenet pens of the red sea bream culture in Gokasho Bay,Mie showed in the range of 1.7-27.5 µg-Chl/L 2 - 8 m
beneath the surface. By the estimation of nitrogenbudget around the suspended culture of the clam, one
kg of the clam has ability to absorb 4 – 200 mgNitrogen/day depending on the chlorophyll
concentration. The harvesting the clam withincreased one kg corresponds to 3.4 g nitrogen
removal from the system. It is suggested that the
introduction of the suspended clam culture has
positive ecological impacts in terms of controlling
water quality and biological production.

Junya Higano1*, Nariaki Inoue¹, Natsuki Hasegawa¹,
Yuka Ishihi¹, Yoshimi Fujioka¹, Masahiro Kuno²,
Daisuke Asao³, Megumu Yamaguchi4, Yoshitaka Imai4,
Setsuo Kobayashi4¹ National Research Institute of Aquaculture,
Fisheries Research Agency²Mie Prefectural Fisheries Research InstituteShiroko 1-6277-4, Suzuka-shi, Mie 510-0243, Japan³Toba-Isobe Fisheries Cooperative
4-2360-16 Toba, Toba-shi, Mie, Japan
4

Care Shell Co., Ltd.

Corresponding author: higa at fra.affrc.go.jp
Keywords: Asari clam, Ruditapes philippinarum,
growth, suspended culture, oyster culture raft

ABSTRACT

Asari clam, Ruditapes philippinarum, is one of the mostimportant fisheries species in Japan. Most of theclam’s production depends on wild catch although
the culture production in tidal flats is occupied only 3
to 4% of the total production. The suspended cultureof Asari clam is not prevalent yet in Japan and anyother countries. In order to develop the practicalmethod of the suspended culture, the culture
experiment was performed at an oyster culture raft in
Ohnoura Bay, Toba City, Mie Prefecture. Prior to theculture experiment, for the collection of natural seedof Asari clam, 60 x 30 cm Nylon mesh bags with 2x3
mm mesh size were set on the tidal sand flat inOhnoura Bay. Fine gravels and Care-Shells (oyster
shell processed materials) were put in mesh bags. The
mesh bag could collect more than 100 individuals/
bag with the medium size of 20 mm in shell length
after several month. For suspended culture, theseseeds were put in plastic containers (41x31x14 cm). In
each container 150 seeds (average shell length was 22mm, wet weight was 2.0 g) were put with fine gravels
and Care-Shells (oyster shell processed materials) in
five different ratio of these substrates, namely 0, 20,
50, 80, or 100 % Care-Shell contents, setting to 6 cm inthickness. The containers were suspended in 2 mfrom the water surface. The culture started on April

ANNOTATED BIBLIOGRAPHY

Fujiwara M., S. Tsuji, M. Tanaka, Y. Imanishi and M.
Nakanishi. 2008. Growth of Manila clam
Ruditapes philippinarum on hanging cultureusing containers. Bull. Kyoto. Inst. Ocean.
Fish. Sci. 30:49-53. (in Japanese)
In order to examine the growth of the manila clam
Ruditapes philippinarum, a hanging cultivation
experiment using containers was conducted in Kunda
and Maizuru Bay. We covered the upper part of theculture containers with a fine mesh net, and put
anthracite soil (particle size: 2 - 3 mm) into thecontainers for bed material. The wild juvenile clams
used in this experiment were collected from hanging
cockle culture containers in Kunda Bay. We could
recognize from the time of discovering juvenile clams
76

whether they were autumn or summer broods.
Autumn broods grew well from spring to summer,
but hardly grew in autumn. Summer broods grewwell from the first autumn to the summer of thefollowing year, but hardly grew in autumn of the next
year. The mean shell length of 1-year-olds was 32 - 42
mm in autumn broods, and 45 mm in summer broods.
These results revealed markedly high growth rates
not reported to date.

reported. In the survey point in Ise Bay, it wasobserved that juveniles immediately after settlement
rarely survived after the flood in June throughAugust, while juveniles occurred in autumn survived
once in a few years. These survivors were exploited
soon after their shell length reached at 25 mm, theminimum size allowed to catch, in 2 years.
Catch per unit effort (CPUE) of hard clam andfreshwater clam recovered when the society of local
fishermen became aware of the importance ofregulating their catch and protecting fishing grounds
in the light of field surveys. As contrasted with casesof these 2 species living in or around rivers, thespatial scale necessary for the fishing management of
Asari clam should be much larger. In 2009, fishingcooperatives along the bay were organized to conduct
resource surveys and activities to protect fishinggrounds from undesirable predators. It has been alsoplanned to enhance the quality of some fishinggrounds in the bay by creating artificial tidal flats andadjusting the ground height to the known suitablelevel for the habitat of Asari clam.

Mizuno T., T. Maruyama and J. Higano. 2009.
Transition and Prospect of Asari clam
(Ruditapes philippinarum) Fisheries in Ise Bay,
Mie Prefecture. Bull. Mie Pref. Fish. Res. Inst.
17:1-21. (in Japanese)
The authors took an overview on the history of theclam fishery in the western-side region of Ise Bay, alarge bay (2,342 km2 with 660 km coastline) in thecentral part of Japan. Major commercial clams
produced in the bay are Asari clam Ruditapes
philippinarum living in shallow coastal areas,
freshwater clamCorbicula japonica and hard clam
Meretrix lusoria in brackish-water.

Goulletquer, P., J.M. Deslous-Paoli, M. Héral. 1989.
Ecophysiologie et bilan energétique de lapalourde Japonaise d’élevage Ruditapes
philippinarum. Jour. Exp. Mar. Biol. Ecol.
132:85-108.

The annual yield of these 3 species decreased in
response to fishing efforts and environmental
changes. Until early 1960s, tidal flats at river mouths
covered in excess of 6,000 ha, and eelgrass beds grew
there as well as in other parts along the coastline.
After the annual catch of Asari clam peaked at ca.
15,000 tons in the late 1960s, it decreased rapidly
when coastal developments destroyed tidal flats andeelgrass beds during 1970s. In this period, thestructure of coastal fisheries changed then laver
culture and fisheries on clams and pelagic fish were
continued. The second peak of Asari yield was seen
in 1980s when clams in the subtidal zone were also
exploited. Aged fishermen recruited into clam
fisheries during 1990s avoiding intensive labors in thelaver culture. In 2000s, the 70% of fishermen are
working in clam fisheries, and the yield of Asari clam
became stagnant in 1,000 to 3,000 tons due to thestrong fishing pressure.

Rates of filtration and respiration both follow anonlinear model based on temperature of the form:
with maximal values at 15 and 20 °C, respectively.
Quantities of seston varying from 0 to 30 mg · 1−1 haveno effect in reducing the filtration rate > 8 mg · 1−1,
ingestion is regulated by the production ofpseudofaeces. Maximal assimilation efficiency is ≈78%, but this is considerably reduced when themineral content of the water increases. Assimilation
efficiency for the Manila clam is reduced at both high(> 10 mm3· h−1) or low (< 2 mm3· h−1) values ofingested ration. The estimated value of growthefficiency (75%) and values of growth efficiency
derived from the model K1 = 33%, K2 = 51% areoptimized when ingested volumes are between 1 and
2 mm3. Standard metabolism is estimated as 0.11 ml
O2 · h−1. Zero growth efficiency occurs at a ration levelof 2 J · h−1 for an adult. The individual energy budget
shows that production is dependent more on
temperature than on the energy value of the food.
Comparison of calculated and measured production
reveals differences resulting from the higher levels of
seston found in the field. In particular, during thewinter when the mineral content of the seston is highIn the early 2000s, very few Asari clam larvae, lessthan 200 individuals/m3 in the main reproductive
period (spring and autumn in this region), wereobserved at the river mouth in the southern part ofthe bay. This abundance of larvae was contrastingly
smaller than that in Mikawa Bay adjacent to Ise Bay
where stable annual yields and high abundances oflarvae, thousands of individuals in 1 m3, were77

(90 mg · 1−1), there is a continuous loss of weight. This
results from a lower assimilation efficiency together
with production of pseudofaeces. Excretion of organic
nitrogen varies throughout the year, ammonia
representing no more than a mean of 29.8% of thetotal nitrogen excretion.

by intertidal macrozoobenthos and are comparable tothose found on dense assemblages (800 to 2000 g ash
free DW m-2) of the most investigated mussel, Mytilus
edulis. In addition, nutrient fluxes through bivalve
excretion varied strongly within a few months, up to
10-fold (R. philippinarum) and 100-fold (M. senhousia)
between April 1994 and August 1994, as related to thetemporal change of bivalve standing stock. The extent
of nutrient regeneration through diffusive flux wascomparable to that reported for other intertidal zones
of Japan and in the eutrophic Seto Inland Sea, withina range of 0.2 to 1.5 mmol NH4+-N m-2 d-1 and 0.01 to
0.05 mmol PO43--P m-2 d−1, thus more than 1 order ofmagnitude lower than that due to the excretory
activity by R. philippinarum and M. senhousia. Our
results indicate that the dominant bivalves, R.
philippinarum and M. senhousia, play a major role in
the processes of benthic nutrient regeneration within
the intertidal zone, rapidly and efficiently recycling
the inorganic forms of N and P available to primary
producers. This study highlighted the importance ofconsidering the temporal scaling of intertidal
macrozoobenthos distribution in the quantification of
the processes of benthic nutrient regeneration in thesehighly variable sytems.

Magni P., S. Montani, C. Takada and Tsutsumi H.
2000. Temporal scaling and relevance ofbivalve nutrient excretion on a tidal flat of the
Seto Inland Sea, Japan. Mar. Ecol. Prog. Ser.
198:139-155
Using an indirect and integrated approach, wequantified the magnitude and temporal variability of
the contribution of macrozoobenthos to the upward
flux of inorganic nitrogen and phosphorus on a sandy
tidal flat of the Seto Inland Sea, Japan. From April
1994 to April 1996, we conducted monthly fieldsurveys on the abundance and faunal composition of
the macrozoobenthic communities inhabiting thelower part of the intertidal zone. Subsequently, we
carried out 24 h day/night laboratory experiments on
the nutrient excretion rate by various size-classes ofthe dominant species. We then obtained the animal
nutrient excretion over a 2 yr period, multiplying thespecies-specific excretion rates by the actual animal
biomass found on the tidal flat. Additionally, for all
different seasons, we calculated the upward diffusive
flux from the sediments from the vertical profiles ofnutrient concentrations in the porewater. On the flat,
the bivalves Ruditapes philippinarum (Veneridae) and
Musculista senhousia (Mytilidae) were dominant,making up 86 ± 5.6% when the total biomass exceeded
100 g DW (dry weight) m-2. From our laboratory
experiments, the mean (day and night) nutrient
excretion rates at 20 to 22 °C by the 2 bivalve species
were 18.9 µmol NH4+-N g-1 DW h−1, 4.8 µmol (NO3- +NO2-)-N g−1 DW h−1 and 3.3 µmol PO43--P g−1 DW h−1.
In addition, NH4+-N excretion by R. philippinarum, but
not that by M. senhousia, was significantly higher
during the day than during the night. This occurred
while the food (Thalassiosira sp.) offered in spikes was
rapidly taken up irrespective of the concentration(within a field relevant spring-summer range of 10 to60 µg l−1 chl a) and in day/night treatments. The
release of nitrogen (N) and the release of phosphorus(P) were highly correlated with each other, for both R.
philippinarum and M. senhousia, with an N/P ratio of
7.8 ± 3.0 and 9.9 ± 3.5, respectively. In the field, thebivalve excretion rates of nutrients were calculated to
be up to 35.2 mmol NH4+-N m-2 d−1, 8.8 mmol (NO3- +NO2-)-N m-2 d-1 and 5.8 mmol PO43--P m-2 d-1. These
values rank in the upper range of nutrient excretion
78

Interpreting the Microbial Ecology Found within Marine Fish

composition of the intestinal contents of individual
fish. Initial results confirm that fish microbiomes havedistinct taxonomic signatures unique from theenvironment, including the presence of key hostassociated taxa such as segmented filamentous
bacteria (SFB), the phyla Tenericutes, and the phyla
Spirochaetes. Furthermore, differences between thecommunities associated with the epithelial and thelumenal regions indicate the importance of spatialorganization within the gut environment. These
results demonstrate early steps and methods tocharacterize marine fish gut microbiomes within an
ecological framework. Ultimately, this knowledge canbe applied to understanding the importance of the gut
microbiome to fish health in aquaculture andecosystems management.

Jessica M. Blanton, Eric E. AllenUCSD Scripps Institution of Oceanography,
9500 Gilman Drive, La Jolla, CA 92093
Corresponding author: jmblanton at ucsd.edu

ABSTRACTThe natural microbial communities found withinvertebrate digestive tracts are composed of amultitude of different microorganisms, collectively
known as the microbiome. Recent work in humans
and model organisms has shown that the microbiome
is a dynamic and vital element of host health—contributing to nutrition and digestion processes, aswell as immune system development. Technologicaladvances in sequencing now allow us to look at thesecommunities as a whole, promoting the use of
ecological theory to understand how these fishmicrobiome systems are structured. Here we
investigate the microbiome of wild fishes by looking
at species local to the Southern California Bight: thesport fish and aquaculture target Seriola lalandi(California yellowtail) the forage fish Scomber
japonicus (Pacific chub mackerel) and the omnivorous
Atherinops affinis (Topsmelt silverside). Using highthroughput sequencing, we analyze the microbial

79Development of Integrated Multi-Trophic Aquaculture Using Sea Cucumber

particulate N waste (i.e. feces and leftover feed) at thestocking density of 35 ind/m²; the stocking weight of
Elkhorn sea moss line culture was 10 kg; culturing
period was 200 days.

Satoshi Watanabe¹, Masashi Kodama², Joemel G.
Sumbing³, Ma. J. H. Lebata-Ramos³¹ National Research Institute of Aquaculture,
Fisheries Research Agency, 422-1 Nakatsuhamaura,
Minamiise, Mie, 516-0193, Japan

Keywords: integrated multi-trophic aquaculture,
nitrogen budget, sandfish (Holothuria scabra), milkfish(Chanos chanos), Elkhorn sea moss (Kappaphycus
alvarezii)

It was estimated that milkfish culture cumulatively
produced 145 kg of particulate N, and milkfish andsandfish together produced 60 kg of NH4-N in 200
days of culture. Daily assimilation rate of theparticulate N by sandfish ranged 3.4 - 12.4%, and 4.3%of the particulate N was estimated to be removed bysandfish in 200 days of culture. Daily absorption rateof NH4-N by Elkhorn sea moss increased
exponentially with time and reached 100% after 125days of culture. Cumulative NH4-N was estimated to
be depleted after 162 days of culture. For complete
utilization of particulate N by the end of culture,
sandfish stocking density should be 805 ind/m²,
which is 200 times as high as that in existing sandfish
aquaculture operations.

ABSTRACTINTRODUCTION² Japan International Research Center for Agricultural
Sciences, 1-1 Owashi, Tsukuba, Ibaraki,305-8686, Japan³ Aquaculture Department, Southeast Asian Fisheries
Development Center, Tigbauan, 5021, Philippines
Corresponding author: swat at affrc.go.jp

In Southeast Asian countries, aquaculture production
continues to increase. Environmental deterioration
associated with water and sediment eutrophication byaquaculture effluent has been problematic, sometimes
resulting in disease outbreaks and fish kills due tohypoxia and hydrogen sulfide poisoning. Integrated
multi-trophic aquaculture (IMTA) is one of thepromising measures for sustainable aquaculture. In
this study, a box model estimation of nitrogen (N)budget based on experimental data and values fromliterature was made for a system of sandfish(Holothuria scabra) in sea cage IMTA with milkfish(Chanos chanos) and Elkhorn sea moss (Kappaphycus
alvarezii).

In Southeast Asian countries, aquaculture production
continues to increase. Environmental deterioration
associated with water and sediment eutrophication byaquaculture effluent has been problematic, sometimes
resulting in disease outbreaks and fish kills due tohypoxia and hydrogen sulfide poisoning. To minimize
the effect of intensive aquaculture production on thesurrounding environment and to potentially increase
economic income, integrated multi-trophic
aquaculture (IMTA) has been practiced in several
temperate regions of the world and is increasingly
getting attention in Southeast Asian countries
including the Philippines.
IMTA is the polyculture of organisms in different
trophic levels (i.e. primary producer, primaryconsumer, secondary consumer, etc.). Typically,
combinations of fed species (e.g. finfish), inorganic
extractive species (e.g. seaweed) and organic
extractive species (e.g. benthos) are used (Troell et al.
2009; Abreu et al. 2009, 2011). IMTA mitigates theeffects of cultivation on the environment throughrecycling of nutrients (excess feeds, feces andmetabolites) from organisms in higher trophic levels
by organisms in lower trophic levels.

Information on stocking density, stocking size,
mortality, growth, feed ration, feed assimilation,
NH4-N production and NH4-N absorption of these
species was obtained from a series of experiments andexisting literature. In the production system 26 g
milkfish were cultured in a 5 x 5 x 4 m cage at thestocking density of 36.7 ind/m³ with the initial
feeding ration of 10% of body weight which was
gradually decreased to 4% over time; 10 g sandfish
were cultured in a cage with the same bottom area asmilkfish cage hanged under the milkfish cage to trap80

sampling. Mortality was recorded daily and monthly
survival was recorded.

Sea cucumbers have been intensively harvested forprocessing into bêche-de-mer (dried product) in many
Southeast Asian countries for export to the Chinese
market (Carpenter and Niem 1998; Conand 2004;
Hamel et al. 2001; Uthicke et al. 2004), causing
depletion of wild stocks in many species. There has
been interest in developing hatchery and aquaculture
techniques for sandfish, Holothuria scabra, one of themost valued tropical sea cucumber species (Battaglene
et al. 1999; Purcell and Kirby 2006). There has also
been interest in developing IMTA methods using sea
cucumbers (Ahlgren 1998; Slater and Carton 2007; Pittet al. 2004; Watanabe et al. 2012; Watanabe et al. 2013).

A logistic growth curve was fitted to the BW data ofmilkfish from DOC 1 to DOC 200 (i.e. 61 to 260 daysof age): BW (g) = 400.5 / (1 + e-0.0202 × (t-192.0)) (r² = 0.996),
where t is age in days. Daily BW growth rate (g/d)
was calculated from the logistic growth curve (i.e.
BWt – BWt-1). Daily survival rate was estimated bylinear interpolation between the monthly samplingdata.
The relationship between BW and ammonium-N(NH4-N) excretion rate of milkfish was estimated byregression analysis of data reported by SumagaysayChavoso (2003): NH4-N (mg/kg/d) = 1.49×10³ ×
BW-0.47 (n = 4, r² = 0.99), where BW is fresh bodyweight of milkfish (g).

This paper introduces an example of a box model
analysis to estimate the nitrogen budget based on
experimental data and values from literature for a
production system of sandfish in sea cage IMTA withmilkfish (Chanos chanos) and Elkhorn sea moss
(Kappaphycus alvarezii) which are both commonly
cultured in the Philippines. The results help in
deciding stocking density and weight of these species
in IMTA.

Daily BW growth pattern of sandfish followed theGompertz model reported by Watanabe et al. (2014):
BW (g) =1961 × e^(-22.5 × e^(-0.00661 × t)), where t is
age in days. The relationship between BW and NH4-N
excretion of sandfish followed the equation reported
by Kodama et al. (2015): NH4-N (g/d) = (0.0153 ×
BW0.81)/1000.

MATERIALS AND METHODSFor the box model analysis of nitrogen (N) budget inpolyculture of milkfish, sandfish and Elkhorn sea
moss, data from our own experiments and literature
information were used. In this closed-box model,
milkfish feed was the sole N input source to thepolyculture system. Sandfish consumed excess feed
and feces (particulate N) from milkfish culture.
Elkhorn sea moss absorbed ammonium (NH4)excreted from milkfish and sandfish for growth.
Unused N accumulated in the system; neither
decomposition nor denitrification was taken into
account for the model calculations.
Milkfish were reared in a floating net cage 5 × 5 × 4 m(length × width × depth) at Igang Marine Station(IMS) of Aquaculture Department, Southeast Asian
Fisheries Development Center (SEAFEC/AQD).
Milkfish fingerlings (26 g, 61 days of age) were
stocked at 36.7 ind/m³ (3670 ind, i.e. standard
stocking density in the Philippines) on November 21,
2011. A commercial milkfish diet (Oversea Feeds,
Cebu, Philippines) was fed to the fish throughout therearing period. Daily feeding ration was as follows:
days of culture (DOC) 1 - 77: 4% of the mean bodyweight, DOC 78 - 161: 3.5%, and DOC 162 - 200: 3%.
Body weight (BW) was measured monthly to thenearest 1 g under anesthesia with 2-phenoxyethanol
by random sub-sampling of 300 individuals. Amountof feed was calculated and adjusted after each

Sandfish was assumed to be cultured in a cage with afloor hung underneath the milkfish cage at thestocking size of 10.5 g and stocking density of 35 ind/
m². The sandfish cage had the same bottom area as
the milkfish cage (25 m²), and excess feed andmilkfish feces (particulate N) accumulate 100% in thesandfish cage. Mortality of sandfish was assumed tobe linear at the rate of 20%/year (Agudo 2006).
The whole body dry matter content of milkfish (n = 3)
and sandfish (n = 10) obtained from the sea cucumber
hatchery of SEAFDEC/AQD was measured after
drying in a 50 °C oven for two weeks. N content of themilkfish whole body (n = 10), milkfish feed (n = 3) andsandfish whole body (n = 10) was analyzed using an
EA-1108 elemental analyzer (Carlo Erba, Italy) at
National Research Institute of Fisheries Science, Japan.

81

Daily particulate N production of milkfish (PNPM,
excess feed and feces) was calculated as: PNPM (g/d/cage) = (Feed-N – (growth-N + NH4-N)) × Nmilkfish,
where growth-N is the amount of N gained in one dayof body growth, and Nmilkfish is the number of milkfish.
Daily N requirement of sandfish (NRS) was obtainedas: NRS (g/d/cage) = ((growth-N + NH4-N)/0.8) ×
Nsandfish, where 0.8 is apparent digestibility coefficientof animal protein in sandfish (Orozco et al. 2014) andNsandfish is the number of sandfish.

Growth rate and NH4-N requirement of Elkhorn sea
moss were assumed to be 4.6%/d (specific growthrate) and 0.03 mg/g fresh plant, respectively (Rui etal. 1990). Fresh weight of Elkhorn sea moss on a
specific day of culture (Wd) was calculated as: Wd =e(0.046+lnWd-1). Stocking weight of Elkhorn sea moss was
10,000 g (10 kg).

RESULTS AND DISCUSSION

The mean milkfish whole body dry matter content
was 51.8% ± 4.1 (mean ± SD, n = 3), and the mean N
content was 11.2% ± 1.5 (dry wt/wt, n = 10). The
mean whole body dry matter content of sandfish was
18.4% ± 5.6 (n = 10), and the mean N content was 7.0%
± 2.9 (n = 10). The mean N content of milkfish feedwas 7.0% ± 0.23 (n = 3).
Milkfish grew from 26 g to 320 g on average in the
200-day culture period. Feed conversion rate (FCR) of
milkfish culture was high (2.9), and it was estimatedthat 66% of N in feed was wasted as excess (uneaten)
and feces, and 7% was excreted as NH4-N during 200
DOC (Fig. 1). In other words, 145 kg of particulate N
and 60 kg of dissolved N were calculated to be wasted
and released to the environment from a small (5 × 5 ×4 m) milkfish cage over 200 days. This is considered
to have a high impact on environmental
eutrophication. Not only is waste from milkfish
culture harmful to the environment, but it is also
uneconomical for the fish farmers.

Figure 1. Nitrogen budgetof milkfish aquaculture.

Figure 2. Growth (A), daily body-N gain (B), daily NH4-N excretion (C) and daily N requirement (D) of sandfish during 200 days ofpolyculture with milkfish and Elkhorn sea moss.

82

Milkfish sea cage culture usually has a higher FCR(around 2) than pond culture (around 1.5 or less)where natural feeds are available (SumagaysayChavoso and San Diego-McGlone 2003). The FCR of
2.9 obtained in this study was very high perhaps
owing to the unusual experimental rearing season
that started in November. Milkfish are usuallystocked at the onset of the dry season around Marchor April in the Philippines. The growth rate ofmilkfish in this study was slower than those reported
for marine pens and comparable to those reported forextensive pond culture (FAO web page). Although
there is a possibility that the amount of wasted N may
be lower during the regular culturing season,
intensive milkfish culture may have detrimental
effects on the surrounding environment, especially insemi-closed embayment with low water exchange.

sandfish was estimated to sustain growth of 1068 g
and 76 g of Elkhorn sea moss respectively.
Under the polyculture scheme, the recycle rate of
NH4-N excreted by milkfish and sandfish by Elkhorn
sea moss increased exponentially with DOC andexceeded 100% by DOC 125 (Fig. 4A), and cumulative
NH4-N was expected to be depleted on DOC 162 (Fig.
4B). Thus, elkhorn sea moss seemed to have highpotential to assimilate NH4-N. However, in the wild
sea cage conditions, these estimations may not holdtrue since NH4–N excreted from the animals isquickly diluted and transported by the current.
Therefore, the amount of Elkhorn sea mosssustainable by the NH4-N excretion from milkfish
and sandfish might have been overestimated. On theother hand, availability of naturally occurring
dissolved inorganic N in the seawater should also be
incorporated in the model analysis. The specificgrowth rate of Elkhorn sea moss reported by Rui andChaoyuan (1990) was based on a 36-day rearing
experiment, and the growth rate may not stay
constant for 200 days. The effect of stocking density
on growth rate (Thirumaran and Anantharaman2009) should also be included in the model.

Based on the Gompertz model (Watanabe et al. 2014),
sandfish was calculated to grow from 10 g to 481 g in
200 DOC (Fig. 2A). Moisture content of sandfish was
84%, and the N content was 7.0% (dry wt/wt). Basedon a linear mortality condition (Agudo 2006), thenumber of sandfish decreased from 870 to 784 in the
200-day period. Based on these, daily body N gain of
sandfish is shown in Fig. 2B, and daily NH4-N
production is shown in Fig. 2C. Daily N requirement
is obtained as the sum of growth and excretion shownin Fig. 2D.

Cumulative particulate N did not greatly differ
between monoculture and polyculture (Fig. 4D) due
to the low particulate N recycle rate by sandfish,
which fluctuated between 3.4% and 12.4% due to the
stepwise milkfish feeding regime (Fig. 4C). The total
amount of particulate N removed by sandfish during
the 200 DOC was 4.3% of the total particulate N
accumulation from milkfish culture. Thus, although
sea cucumbers are known to play an important role
in recycling of nutrients of sediments (Uthicke 1999),
particulate N recycling rate by sandfish did not seemto be able to keep up with particulate N accumulationfrom milkfish culture.

Growth and N requirement of Elkhorn sea moss areshown in Fig. 3A and Fig. 3B, respectively.
Box model calculations were based on the above
parameters. On DOC 200 for instance, one milkfish(320 g, 260 days of age) provided enough particulate
N to support the N requirement of 5.8 sandfish (481 g,
420 d). NH4-N excretion from one milkfish and one

Figure 3. Growth (A) and N requirement (B) of Elkhorn sea moss during
200 days of polyculture with milkfish and sandfish.

83

Figure 4. NH4-N recycle rate (A), cumulative NH4-N (B), particulate N
recycle rate (C) and cumulative particulate N (D) in monoculture of milkfish
and polyculture of milkfish, sandfish and Elkhorn sea moss.

designed in such a way that water exchange rate ishigh enough for particulate N level to remain low.
The highest organic matter level in the sediment
tolerable by sandfish is 1.8% when aquaculture feed isthe only source of organic matter (Novilla,
unpublished data).

Based on the box model, stocking weight of Elkhorn
sea moss at which NH4-N from milkfish and sandfish
culture on DOC 200 is completely absorbed would be
2351 g, and the stocking density of sandfish (10 g,weight at stocking) should be 805 ind/m2 tocompletely consume particulate N from milkfish
culture on DOC 200. In existing sandfish aquaculture
operations, however, stocking densities are much
lower: e.g. 4 ind/m² (20 g) in New Caledonia and 0.73
ind/m² (50 g) in Vietnam (Agudo 2006). The model
estimated stocking density of 805 ind/m² seems toohigh to be practical.

This study focused only on estimation of the N
budget in the IMTA system. Since sandfish culture
methods in sea cages have not been well established,
more rearing experiments should be done to build on
the data resulting from this study.

IMTA is a method to recycle wasted nutrients into
harvestable commodities. Although sandfish may not
be an effective bioremediator for removing nutrientsfrom milkfish culture effluents, IMTA using sandfish
may still be economically feasible due to its highcommercial value. Sandfish culture cages should be

84

REFERENCES

Abreu M.H., D.A. Varela, L. Henríquez, A. Villarroel,
C. Yarish, I. Sousa-Pinto, and A.H.
Buschmann. 2009. Traditional vs. integrated
multi-trophic aquaculture of Gracilaria chilensis
C. J. Bird, J. McLachlan & E. C. Oliveira:productivity and physiological performance.
Aquaculture 293: 211–220.
Abreu M.H., R. Pereira, C. Yarish, A.H. Buschmann,and I. Sousa-Pinto. 2011. IMTA with Gracilaria
vermiculophylla: Productivity and nutrient
removal performance of the seaweed in aland-based pilot scale system. Aquaculture
312: 77–87.
Agudo N. 2006. Sandfish hatchery techniques.
Secretariat of the Pacific Community Noumea,
New Caledonia, http://wwwx.spc.int/
coastfish/Reports/Worldfish/Sandfish_
hatch_tech.pdf. pp. 44.
Ahlgren M.O. 1998. Consumption and assimilation of
salmon net pen fouling debris by the red sea
cucumber Parastichopus californicus:
implication for polyculture. Journal of World
Aquaculture Society 29:133-139.
Battaglene S.C., J.E. Seymour, and C. Ramofafia. 1999.
Survival and growth of cultured juvenile sea
cucumbers, Holothuria scabra. Aquaculture 178:
293-322.
Carpenter K.E. and V.H. Niem. 1998. The living
marine resources of the Western Central
Pacific, volume 2. Cephalopods, crustaceans,holothurians and sharks. FAO species
identification guide for fishery purposes. FAO,
Rome. pp.1396
Conand C. 2004. Present status of world sea cucumber
resources and utilization: an international
overview, In A. Lovatelli, C. Conand, S.
Purcell, S. Uthicke, J.F. Hamel, A. Mercier(eds.), FAO Fisheries Technical Paper 463
Advances in sea cucumber aquaculture andmanagement, United Nations Food andAgriculture Organization, Rome, p. 13-23.
FAO. Aquaculture Feed and Fertilizer Resources
Information System, Milkfish Chanos chanos,
http://www.fao.org/fishery/affris/speciesprofiles/milkfish/milkfish-home/en/
Hamel J.F., C. Conand, D.L. Pawson, and A. Mercier.
2001. The sea cucumber Holothuria scabra(Holothuroidea: Echinodermata): its biology
and exploitation as beche-de-mer. Advances inMarine Biology 41: 129-202.
Kodama M., J.G. Sumbing, M.J.H. Lebata-Ramos, andS. Watanabe. 2015. Metabolic ratecharacteristics and sediment cleaning potential
85of the tropical sea cucumber Holothuria scabra.
JARQ 49: 79-84.
Orozco Z.G.A, J.G. Sumbing, M.J.H. Lebata-Ramos,
and S. Watanabe. 2014. Apparent digestibility
coefficient of nutrients from shrimp, mussel,
diatom and seaweed by juvenile Holothuria
scabra Jaeger. Aquaculture Research 45: 1153–
1163.
Pitt R., N.D.Q. Duy, T.V. Duy, H.T.C. Long. 2004.
Sandfish (Holothuria scabra) with shrimp(Penaeus monodon) co-culture tank trials. SPC
Beche-de-mer Information Bulletin 20: 12–22.
Purcell S.W., and D.S. Kirby. 2006. Restocking the sea
cucumber Holothuria scabra: sizing no-takezones through individual-based movement
modeling. Fisheries Research 80: 53-61.
Rui L., L. Jiajun, and W. Chaoyuan. 1990. Effect ofammonium on growth and carrageenan
content in Kappaphycus alvarezii (Gigartinales,
Rhodophyta). Hydrobiologia 204/205: 499503.
Slater M.J., and A.G. Carton. 2007. Survivorship andgrowth of the sea cucumber Australostichopus(Stichopus) mollis (Hutton 1872) in polyculture
trials with green-lipped mussel farms.
Aquaculture 272: 389-398.
Sumagaysay-Chavoso N.S. 2003. Nitrogen andphosphorus digestibility and excretion ofdifferent-sized groups of milkfish (Chanoschanos Forsskal) fed formulated and natural
food-based diets. Aquaculture Research 34:
407-418.
Sumagaysay-Chavoso N.S., and M.L. San DiegoMcGlone. 2003. Water quality and holding
capacity of intensive and semi-intensive
milkfish (Chanos chanos) ponds. Aquaculture
219: 413-429.
Troell M, A. Joyce, T. Chopin, A. Neori, A.H.
Buschmann, and J.G. Fang. 2009. Ecologicalengineering in aquaculture - Potential for
integrated multi-trophic aquaculture (IMTA)in marine offshore systems. Aquaculture 297:
1–9.
Uthicke S. 1999. Sediment bioturbation and impact of
feeding activity of Holothuria (Halodeima) atraand Stichopus chloronotus, two sedimentfeeding holothurians, at Lizard Island, Great
Barrier Reef. Bulletin of Marine Science 64:
129-141.
Uthicke S., D. Welch, and J.A.H. Benzie. 2004. Slow
growth and lack of recovery in overfished
Holothurians on the Great Barrier Reef:
evidence from DNA fingerprints and repeatedlarge-scale surveys. Conservation Biology 18:1395-1404.

shrimp, mussel, diatom and seaweed byjuvenile Holothuria scabra Jaeger. Aquaculture
Research 45:1153–1163.

Watanabe S., M. Kodama, J.M. Zarate, M.J.L. Ramos,
and M.F.J. Nievales. 2012. Ability of sandfish(Holothuria scabra) to utilise organic matter inblack tiger shrimp ponds. In C.A. Hair, T.D.
Pickering and D.J. Mills (eds.), Asia-PacificTropical Sea Cucumber Aquaculture, ACIAR
Proceedings 136, Canberra, Australia, p. 113 –120.
Watanabe S., M. Kodama, J.G. Sumbing, and M.J.H.
Lebata-Ramos. 2013. Diet-tissue stable isotopic
fractionation of tropical sea cucumber,
Holothuria scabra. JARQ 47: 127–134.
Watanabe S., J.G. Sumbing, and M.J.H. Lebata-Ramos.
2014. Growth pattern of tropical sea cucumber,
Holothuria scabra, under captivity. JARQ 48:
457-464.

The ability of Holothuria scabra to digest nutrients,
such as organic matter (OM), protein andcarbohydrate from animal and plant feed ingredientswas investigated. Four test feeds prepared by mixing
sand with single ingredients from animal sources(shrimp and mussel) and plant sources (diatom andseaweed) were fed to H. scabra to estimate apparent
digestibility coefficient (ADC). The total assimilated
nutrient (TAN) increased with ADC, whereas
ingestion rate (IR) varied slightly among the feeds
suggesting that ADC might be a good indicator of
nutrient availability to H. scabra. The ADCOM ofshrimp and mussel was significantly higher than thatdiatom and seaweed: 86.2%, 77.1%, 55.1% and 32.3%
respectively. ADCprotein was similar for shrimp(88.7%), mussel (84.8%) and diatom (75.2%), but
significantly lower in sea- weed (34.4%).
ADCcarbohydrate was similar in mussel (58.5%) anddiatom (58.3%) as well as in seaweed (31.6) andshrimp (28.0%). ADCprotein was relatively higher
than ADCcarbohydrate suggesting that H. scabra
generally digests more protein than carbohydrate.
Furthermore, results indicated that nutrients fromanimal-based feeds are more efficiently digested by H.
scabra; thus, animal ingredients rich in easily
digestible protein could potentially provide an
efficiently balanced diet for H. scabra fed with diatom
containing high easily digestible carbohydrate.

ANNOTATED BIBLIOGRAPHY

S. Watanabe, J. G. Sumbing and Ma. J. H. LebataRamos (2014) Growth pattern of tropical sea
cucumber, Holothuria scabra, under captivity.
Japan Agricultural Research Quarterly 48:457464.
The growth of the juvenile sea cucumber, Holothuria
scabra, was studied under captivity to elucidate thegrowth variation pattern and determine the best-fit
growth model to estimate age- and size-specificgrowth rates. Individual growth was extremely
variable, with some individuals below the mean
initial weight and some expanding their original bodylength (L) and weight (W) by up to 6.4 and 156 times,
respectively; during 84 days of culture starting at 127
days of age. Some of the smallest individuals showeda higher condition factor than larger individuals in
the presence of ample food, indicating that lack offood may not be the only impediment to growth.
Among the three growth models compared (vonBertalanffy, Gompertz and logistic), the Gompertz
model was considered optimal to express H. scabra
growth; both in L and W. The age- and size-specificdaily growth rate for L and W up to 365 days of age,as estimated by the Gompertz model, had a range of
two and nine orders of magnitude in L (0.035 – 0.96mm/day) and W (3.4 × 10-7 – 3.5 g/day), respectively.
Use of the Gompertz model over the linear model,
which tends to overestimate growth rates, isencouraged to estimate the growth of H. scabra moreaccurately.

S. Watanabe, M. Kodama, J. G. Sumbing and Ma. J. H.
Lebata-Ramos (2013) Diet-tissue stable
isotopic fractionation of tropical sea cucumber,
Holothuria scabra. Japan Agricultural Research
Quarterly 47:127-134.
To provide a basis for a stable carbon and nitrogenisotope ratio (δ13C / δ15N) analysis to determine theassimilated organic matter in sea cucumber, Holothuria
scabra, diet-tissue fractionations were experimentally
determined by mono-feeding rearing with diatom.
While δ15N fractionation of the whole body wall
(2.4‰) was similar to the commonly accepted value(2.6 – 4‰), δ13C fractionation of the body wall (4.2‰)
showed considerable discrepancy with the commonly
accepted value (0 – 1‰) due to the high content (35%dry wt/wt) of calcareous spicules (CaCO3) in the
body wall, which had significantly higher δ13C(-8.6‰) than the organic fractions. Computationalelimination of spicules based upon spicule content
and spicule δ13C reduced the δ13C fractionation ofZ. G. A. Orozco, J. G. Sumbing, Ma. J. H. LebataRamos and S. Watanabe (2014) Apparent
digestibility coefficient of nutrients from86the body wall to 1.5‰, close to the common value.
δ13C fractionation after spicule removal by aciddecarbonation and subsequent rinsing (3.2‰) did notagree with the common value, and δ15N fractionation
was significantly elevated by decarbonation. δ15Nand δ13C fractionations of the intestine (1.5 and 2.2‰,
respectively) did not agree with the common values.
Since δ13C and δ15N of the feces did not differ
significantly from those of the diet, feces may be used
to determine ingested organic matter in the wild.

S. Watanabe, M. Kodama, J. M. Zarate, Ma. J. H.
Lebata-Ramos and M. F. J. Nievales (2012)
Ability of sandfish (Holothuria scabra) to utiliseorganic matter in black tiger shrimp ponds.
ACIAR Proceedings 136:113-120.
Due to frequent viral disease outbreaks, a large
proportion of shrimp aquaculture in South-East Asian
countries has switched from black tiger shrimp(Penaeus monodon) to P. vannamei, an exotic species
originally imported from Latin America. One of thecauses of disease outbreaks is thought to be poor
water and sediment conditions in the shrimp ponds,
which may aggravate disease symptoms. To obtainbasic information for co-culture methods of black
tiger shrimp and sandfish (Holothuria scabra) for
possible mitigation of shrimp-pond eutrophicationand prevention of disease outbreaks, basic laboratory
experiments were conducted at the Southeast Asian
Fisheries Development Center - Aquaculture
Department in Iloilo, the Philippines. A feeding trialof juvenile sandfish showed that they do not growwell with fresh shrimp feed on hard substrate.
Another trial indicated that sand substrate enhances
the growth of juvenile sandfish fed with shrimp feed.
A feeding trial using shrimp tank detritus, shrimp
faeces and Navicula ramosissima (a benthic diatom) as
food sources showed that sandfish grew fastest withthe faeces, followed by detritus and N. ramosissima.
Dissolved oxygen consumption and acid-volatilesulfur levels in the shrimp tank detritus were reducedby sandfish feeding. This suggests that sandfish arecapable of growing with organic matter in shrimp
ponds, and can bioremediate shrimp-pond sediment.

S. Watanabe, J. M. Zarate, J. G. Sumbing, Ma. J. H.
Lebata-Ramos and M. F. Nievales (2012) Sizemeasurement and nutritional condition
evaluation methods in sandfish (Holothuria
scabra Jaeger). Aquaculture Research 43:940948.
The aims of this study were to establish an accurate
size measurement method and a nutritional condition
evaluation method of Holothuria scabra (Jaeger).
Although 0.5% KCl and 0.05% MgSO4 did not induce
anaesthesia, 2% menthol-ethanol for 20 min was
found to be effective and harmless. The
anaesthetization significantly reduced the coefficientof variation of the mean body length and weight by
68% and 43% respectively. During starvation, bodysize and weight decreased concomitantly, resulting inan unchanged condition factor (body weight/
volume), suggesting that the condition factor cannot
be used as an index of nutritional condition. Protein,cholesterol and carbohydrate concentrations in the
body fluid were analysed to study the relationshipwith starvation. As the protein and cholesterol
concentrations initially increased and then decreased
during the starvation period, it is difficult to use themas an index of nutritional condition. The carbohydrate
concentration showed a gradual one-fold increaseduring 10 days of starvation, and it may be used as aproxy for nutritional condition; however, further
physiological studies are needed. Body fluid densityand volume relative to body size gradually increased
and decreased, respectively, during starvation. Thesemethods may be used to correctly monitor theconditions of H. scabra in studies for aquaculture andstock enhancement techniques.

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