Aquaculture Technologies
for Invertebrates

Proceedings of the Thirty-sixth
U.S.-Japan Aquaculture Panel
Symposium
Durham, New Hampshire
October 29-30, 2007
andMilford, Connecticut
November 2, 2007

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

National Marine Fisheries Service
NOAA Technical Memorandum NMFS-F/SPO-99

Aquaculture Technologies
for Invertebrates

Proceedings of the Thirty-sixth
U.S.-Japan Aquaculture Panel
Symposium
Durham, New Hampshire
October 29-30, 2007
andMilford, Connecticut
November 2, 2007

Robert Stickney*, Robert Iwamoto, and Michael Rust, editors
Northwest Fisheries Science Center
2725 Montlake Boulevard East
Seattle, Washington 98112* Texas A&M University

Texas Sea Grant Program
2700 Earl Rudder Freeway South, Suite 1800
College Station, Texas 77845

NOAA Technical Memorandum NMFS-F/SPO-99
July 2009

U.S. Department of Commerce
Gary Locke, Secretary
National Oceanic and Atmospheric Administration
Dr. Jane Lubchenco, Under Secretary for Oceans and Atmosphere
National Marine Fisheries Service
Dr. James W. Balsiger, Assistant Administrator for Fisheries

Suggested citation:
Stickney, R., R. Iwamoto, and M. Rust (editors). 2009. Aquaculture Technologies
for Invertebrates: Proceedings of the Thirty-sixth U.S.-Japan Aquaculture Panel
Symposium, Durham, New Hampshire, October 29–30, and Milford, Connecticut,
November 2, 2007. U.S. Dept. Commerce, NOAA Tech. Memo. NMFS-F/SPO-99,
73 p.
A copy of this report may be obtained from:
Northwest Fisheries Science Center
2725 Montlake Boulevard East
Seattle, Washington 98112
Or online at:
http://spo.nmfs.noaa.gov/tm/
Reference throughout this document to trade names does not imply endorsement by
the National Marine Fisheries Service, NOAA.

ii

Table ofContents

Aquaculture Overview
The Basic Plan for Fisheries and Current Status of Aquaculture
Research in Japan
Kazumasa Ikuta

1

Overview of NOAA’s Aquaculture Program
Michael Rubino

2

Crustacean Culture
Application of Pedigree and Relatedness Analyses Based on
Microsatellite DNA and Mitochondrial DNA Markers to the
Stock Enhancement and Population Studies on the Kuruma
Prawn (Marsupenaeus japonicus)
Takuma Sugaya, Masahiro Kato, Kazuhisa Teruya, and Keiichi
Mushiake3

Marine Ornamental Aquaculture
Junda Lin

7

Nutritional Significance of n-3 Highly Unsaturated Fatty Acids
for Larval Survival and Development in Mass Production ofBrachyuran Crab Larvae
Shigeki Dan, Katsuyuki Hamasaki, Takayuki Kogane, Tadao
Jinbo, and Takashi Ichikawa

8

The Chesapeake Bay Blue Crab (Callinectes sapidus):A Multidisciplinary Approach to Responsible Stock
Enhancement
Oded Zmora, Yonathan Zohar, Anson H. Hines, andEric G. Johnson

15

An Outline of the Research Project, Development of Seed
Production Technology in Japanese Spiny Lobster
Hideaki Aono, Keisuke Murakami, and Masahiko Awaji

17

Mollusk Culture
Shellfish Aquaculture Research on Oysters and Clams at
Haskin Shellfish Research Laboratory
John Kraeuter

18

Improvements in Yield of the Pacific Oyster (Crassostrea
gigas) by Selective Breeding
Chris Langdon, Ford Evans, and Alan Barton

19

Synthesis of Vitellogenin in the Pacific Oyster (Crassostrea gigas)
Toshie Matsumoto

20

iii

Disease Diagnostics and Treatment of Hemic Neoplasia in the Softshell
Clam (Mya arenaria)

25

The Japanese Monitoring System on Shellfish Toxins and Developmentof Liquid Chromatography–Mass Spectrometry of Lipophilic Toxins
Associated with Diarrhetic Shellfish Poisoning for Improvement of theMonitoring System

26

Bivalves as Biofilter: Efficient, Profitable, and Tasty as Well!

31

Considerations on the Fine-scale Topography in Sand Flats, Habitat
Heterogeneity, and Refugia for Clams

32

Methods, Economics, and Commercialization of Open Ocean Mussel
Culture in the Northeast United States

43

Bivalve Mollusk Culture on the U.S. West Coast

44

S. Anne Böttger and Charles W. Walker

Toshiyuki Suzuki, Yutaka Okumura, and Takashi Kamiyama
Muki Shpigel

Hajime Saito, Hideyuki Takahashi, Akihiko Matsuda, Hisami Kuwahara, Yuka Ishihi,
Tomoko Sakami, and Junya Higano

Richard Langan
Bill Dewey

Echinoderms and Other Species
Relationship between Gametogenesis and Food Quality in Sea Urchin
Gonads45

An Overview of the Culture of Marine Invertebrates in Maine

55

Polyculture of Red Abalone (Haliotis rufescens) and Pacific Dulse(Palmaria mollis) in a Land-based Recirculation System

56

Wave Induced Flow in Seawater Exchange Structures for Improving
Seawater Quality

57

Novel Culture Feed for Short-neck Clam Using Single-cell Material from
Porphyra

65

Tatsuya Unuma and Charles W. Walker
Dana L. Morse

Chris Langdon and Ford Evans

Yoshihiro Ohmura

Takao Yoshimatsu, Alok Kalla, Nakib Dad Khan, Toshiyoshi Araki, and Shuichi
Sakamotoiv

Milford Laboratory Minisymposium Presentations
Culture Systems and Habitat Evaluation Branch Overview—Molluscan
Aquaculture

66

Bay Scallop (Argopecten irradians irradians) Husbandry Program

67

Growth and Stress Response of Bivalve Mollusks in Response to Different
Microalgal Diets and Arachidonic Acid Supplementation

68

Biotechnology Branch Overview on Probiotic Bacteria and Harmful Algal
Bloom Research

69

The Importance of Nutrients, Light Duration, Light Intensity, and pH when
Growing Large Volumes of Phytoplankton

70

Phytoplankton Ecology and Aquaculture

71

Cross-field Technologies in Aquaculture72

An Overview of Genetic Studies on Commercial Species of Bivalves

73

Ronald Goldberg

James C. Widman Jr.

Lisa Milke, Shannon Meseck, Gary Wikfors, Monica Bricelj, and Christopher Parrish

Gary Wikfors

Shannon Meseck
Judy Yaqin Li

Barry C. Smith

Sheila Stiles, Joseph Choromanski, and Dorothy Jeffressv

vi

Introduction

The U.S.-Japan Cooperative Program in Natural Resources (UJNR) was establishedon January 29, 1964. Since its creation, the UJNR has evolved to become one ofthe oldest and most effective cooperative agreements between Japan and the UnitedStates. In 1969, the UJNR Aquaculture Panel was created as a vehicle for scientistsof both countries to meet and discuss aquaculture research accomplishments, needs,
and priorities, as well as provide opportunities for cooperative research and scientificexchange.
Under UJNR Aquaculture Panel auspices, U.S. and Japanese scientists have met
annually since 1971 without interruption. The venue for these meetings has alternatedbetween the two countries. The 36th annual meeting and symposium were held in
October 2007 at the University of New Hampshire, Durham, and a minisymposium
was held in November at the Northeast Fisheries Science Center’s Milford
Laboratory, Milford, Connecticut. By prior agreement of the U.S.-Japan panels,
contributors to the proceedings were requested to submit manuscripts in the form ofbrief papers or abstracts.
Hiroshi Nakano, Japan Panel Chairman
Robert Iwamoto, United States Panel Chairman

vii

The Basic Plan for Fisheries and Current Status of Aquaculture Research in Japan
Kazumasa Ikuta
Fisheries Research Agency
National Research Institute of Aquaculture
Aquaculture Systems Division
Minamiise, Mie 516-0193, Japanikutak at fra.affrc.go.jp
Keywords: aquaculture research, sustainable production
Abstract

Alterations to the structure of the fisheries industry have become necessary in Japan due to low levels of capture fisheries production, an aging population, environmental concerns, and increased worldwide demand for marine food products. In accordance with these developments, the Fisheries Agency,
Ministry of Agriculture, Forestry, and Fisheries, established the new Basic Plan for Fisheries in 2007.
Under this plan, measures to realize the improvement of food self-sufficiency and global competitiveness of the industry are given priority, such as the promotion of stock management in Japan’s exclusive economic zone and on the high seas, development of ecosystem-based sustainable aquaculture,establishment of new distribution systems, promotion of the export of marine products to expanding
international markets, and utilization of multiple functions of fisheries communities.
Concerning aquaculture technology, the plan calls for the promotion of sustainable production basedon the responsible use of aquaculture grounds and development of multiaquaculture technology employing low environmental-loading feed. Development of large-scale aquaculture systems and offshore
aquaculture technology should also be important in achieving more efficient usage of sea areas. Innovation of alternative protein sources for feed, that is, plants or agricultural waste products, is urgently
required to deal with the shortage of fish meal. The Fisheries Research Agency (FRA) has recently initiated projects on blue-fin tuna and kanpachi (amber jack) aquaculture in accordance with these research
schemes. In this paper, current aquaculture research in the FRA was introduced.

Ikuta

Overview of NOAA’s Aquaculture Program
Michael Rubino
NOAA Aquaculture Program
1315 East-West Highway
SSMC3-Route: F-AQ Room 13117
Silver Spring, Maryland 20910
Michael.Rubino at noaa.gov
Keywords: sustainable aquaculture, fisheries management
Abstract

As a federal agency under the U.S. Department of Commerce, the National Oceanic and Atmospheric
Administration (NOAA) is focused on creating domestic seafood supplies to meet the growing demand
for all seafood products and on working with international partners to foster development of sustainable aquaculture. Currently, 80% of the seafood that Americans consume is imported, and at least half of
those imports are farmed seafood.
Spurred on by the growth of aquaculture worldwide, the role of aquaculture in meeting consumer
seafood demand, and the enhancement needs of commercial and recreational fisheries, aquaculture
continues to attract international attention from researchers, fisheries managers, policy makers, and the
public.
Over the past three years, the NOAA Aquaculture Program has successfully focused U.S. attention
on marine aquaculture as a vital tool for fisheries management and additional domestic seafood production. NOAA is also interested in continuing to advance an international dialogue on the role of aquaculture in seafood supply—a dialogue that includes a science-based examination of the benefits and challenges of aquaculture production.
This presentation provided an overview of NOAA’s rich tradition of marine aquaculture researchand the importance of nurturing important international partnerships, such as the U.S.-Japan Natural
Resources Panel on Aquaculture.

2

RubinoApplication of Pedigree and Relatedness Analyses Based on Microsatellite DNA and
Mitochondrial DNA Markers to the Stock Enhancement and Population Studies on
the Kuruma Prawn (Marsupenaeus japonicus)
Takuma Sugaya, Masahiro Kato, Kazuhisa Teruya, and Keiichi Mushiake
Fisheries Research Agency
National Research Institute of Aquaculture
Kamiura Station
Tsuiura, Kamiura, Saiki
Oita 879-2602, Japantsugaya at affrc.go.jp
Keywords: kinship, Penaeidae, genetic diversity, stock effectiveness, resource management
Abstract

The availabilities of pedigree and relatedness analyses with microsatellite DNA (MS-DNA) andmitochondrial DNA markers for stock enhancement and population studies on the kuruma prawn (Marsupenaeus japonicus) are discussed based on our current research. Among experimentally reared full-sib
groups, kinships were completely described on an unweighted pair group method with arithmetic mean
(UPGMA)–based dendrogram constructed from the MS-DNA analysis. Released juveniles were successfully distinguished by pedigree analysis in a mark-recapture experiment, showing the usefulness of the
DNA markers as genetic tags. Moreover, relatedness analysis revealed the existence of closely relatedgroups in wild populations of the shrimp, showing that the analysis can provide detailed information
about their genetic variability.

Introduction

The kuruma prawn (Marsupenaeus japonicus)
is a marine shrimp that is widely distributed in thetemperate and tropical zones of the world (Dall et al.
1990). While this shrimp is one of the most famous
fishery animals in Japan, the yield rapidly declined
during the late 1960s. That led to the establishmentof stock enhancement programs resulting in theannual release of approximately 200 million hatchery-reared individuals for about 30 years, mainly
in southern Japan (Hamasaki and Kitada 2006).
However, it has been difficult to assess the precise
effectiveness of the program because of the lack ofsuitable tags to distinguish introduced shrimp fromwild ones at all life history stages. In addition, themassive releases led to a concern about the conservation of genetic variability in the wild population.
Microsatellite DNA (MS-DNA) is made up of
tandem repeats of very short nucleotides. These
repetitive regions of nuclear DNA exhibit high variability due to length differences (Tauz 1989). Mitochondrial DNA (mtDNA) also shows high variability because of its nucleotide substitutions (Brown etal. 1979). In particular, the control region that does
not code for proteins or RNA shows high variability

Sugaya et al.

with regard to relaxed functional constraints (Brown
1985). Those markers can provide sufficiently rare
genotypes that can not only identify individuals
taken randomly from wild populations, but also estimate the genetic relatedness among individuals withunknown pedigree (Finch et al. 1996, Cronin et al.
1999). Therefore, DNA markers should be useful forthe assessments of the effectiveness and the genetic
influences of stock enhancement of kuruma prawns.
Recently, we have tried to apply the pedigree
and relatedness analyses with MS-DNA and mtDNA
markers to the stock enhancement and population
studies. In this paper, we discuss the availability of
the analyses in the stock enhancement program of
kuruma prawn based on our current studies.

Pedigree Analysis of Kuruma Prawns by
MS-DNA Markers

The feasibility of MS-DNA markers for pedigree
analysis of kuruma prawns was tested on sevenexperimentally reared full-sib groups (Sugaya et al.
2002a). The genetic distances among individuals
were estimated by the inverse of genetic identities
among individuals (Nei 1987), which were calcu-

3Figure 1. Genetic relationshipamong seven full-sib groups:
Group 1 = l
Group 2 = p
Group 3 = i
Group 4 = r
Group 5 = oGroup 6 = o, andGroup 7 = n.

lated from multilocus
genotypes of five MSDNA loci. The averagesof the distances were0.392 ±0.161 withinsiblings and 0.783 ±0.138
between siblings. Moreover, each sibling wasclearly distinct on an
unweighted pair groupmethod with arithmetic
mean (UPGMA)–based
dendrogram constructedfrom the identities
among individuals(Figure 1). These results
demonstrate the availability of the MS-DNA
markers for pedigree
analysis estimating genetic relatedness amongindividuals in shrimp
populations of unknown pedigree.

The Detection of Released Shrimp
in a Stocking Area

The feasibility of the DNA markers as tags forreleased shrimp was examined with a mark-recapture experiment in Saiki Bay along the eastern coastof Kyusyu island in southern Japan (Figure 2). Twohundred thousand juveniles of 5 cm total length
were produced from 6 wild females. They were released in late July and early August in the bay. From
August to November, 539 prawns were sampled byexperimental trawling and gill net fishing by commercial fishermen. The pedigree analysis by the
MS-DNA and mtDNA markers detected 51 offspringfrom the six females in the samples. The contributions of the offspring ranged from 2 to 17.2% on amonthly basis (Figure 3). From that data we canconclude that the microsatellite and mitochondrial
DNA markers are useful as tags for released kuruma

4

prawns in stocking programs. In addition, the relatedness analysis of the samples revealed the existenceof many related shrimp in the bay. Although mean
genetic relatedness (rxy) of Queller and Goodnight(1989) estimated by the five MS-DNA markers wasalmost zero, the relatedness among individuals sharing common haplotypes of mtDNA markers werefrom 0.126 to 0.458 in each month (Figure 4).
In the bay, approximately 500,000 hatcheryreared juveniles, derived from 100 to 200 wild
females caught near the stocking area, were released
annually by the local government and a fisheryunion. The relatedness analysis is considered tohave detected those released prawns. This may
show the possibility of the analysis of large-scale
stocking effectiveness estimation.

Sugaya et al.

Analysis of Genetic Relatedness Structureof Wild Kuruma Prawns

The genetic relatedness (rxy) among wild
shrimp individuals was examined in four localities
around Japan (Figure 5) with MS-DNA and mtDNA
markers (Sugaya et al. 2002b). Average rxy among
310 individuals within each locality calculated fromthe information of the five MS-DNA loci ranged
from 0.059 to 0.063. The values from Kagoshima andKumamoto were significantly higher than simulated
values estimated on the assumption of randommating (permutation test, p < 0.05). The frequenciesof rxy in Kumamoto and Kagoshima were high(permutation test, p < 0.05, Figure 6). Furthermore,
the relationship between rxy and the distributionof the haplotypes of mtDNA markers examinedby an UPGMA-based dendrogram showed that
individuals sharing a common haplotype tended to
be closely related in Kagoshima (Figure 7).
The results showed the existence of full-sib level
kinships in the localities because the theoreticalvalues of rxy were approximately 0.5 among the fullsibs. In the East China Sea, including Kumamoto
and Kagoshima, 40–90 million kuruma prawns havebeen released annually. Since those shrimp are generally reared from a limited number of parents, therelease might explain the closely related individuals detected in those two localities. However, theseresults do not necessarily show the gene flow amongwild and released populations, as we cannot deter-mine whether the closely related individuals wereborn in the wild or in a hatchery. Hence the magnitude of gene flow should be examined to reveal thegenetic effects of release in the future.

Figure 3.
Distribution of
released prawns
detected bypedigreeanalysis in eachmonth.

Figure 4. Relatedness distributions among the individuals with
common haplotypes (dotted line) and among those with different haplotypes (solid line) each month.

Figure 2.
Location of
Saiki Bay.

Sugaya et al.

Figure 5.
Sampling location ofwild kuruma prawn.

5

References

Brown, W. M. 1985. The mitochondrial genome ofanimals. In R. J. MacIntyre (ed.), Molecular evolutionary genetics, p. 95–130. Plenum Press, New York.
Brown, W. M., M. J. R. George, and A. C. Wilson.
1979. Rapid evolution of animal mitochondrialDNA. Proc. Natl. Acad. Sci. 76:1967–1971.
Figure 6. Distribution of relatedness among individuals calculated from the data of MS-DNA in localities of Kumamoto andKagoshima. Relatedness less than zero was regarded as zero.
Observed value = o, simulated value ±95% confidence interval(CI) = l, and significant difference = *.

Cronin, M., R. Shideler, J. Hechtel, C. Strobeck, and
D. Paetkau. 1999. Genetic relationships of grizzly
bears (Ursus arctos) in the Prudhoe Bay region of
Alaska: Inference from microsatellite DNA, mitochondrial DNA, and field observations. J. Hered.
90:622–628.
Dall, W., and B. J. Hill. 1990. The biology of thePenaeidae. Adv. Mar. Biol. 27:1–489.
Finch, M. O., and D. M. Lambert. 1996. Kinship andgenetic divergence among populations of tuatara,
Sphenodon punctatus, as revealed by minisatellite
DNA profiling. Mol. Ecol. 5:651–658.
Hamasaki, K., and S. Kitada. 2006. A review of kuruma prawn Penaeus japonicus stock enhancement inJapan. Fish. Res. 80:80–90.
Nei, M. 1987. Genetic distance between populations. In M. Nei (ed.), Molecular evolutionary genetics. Columbia University Press, New York.
Queller, D. C., and K. F. Goodnight. 1989. Estimating relatedness using genetic markers. Evolution
43:258–275.
Sugaya, T., M. Ikeda, and N. Taniguchi. 2002a. Relatedness structure estimated by microsatellite DNA
and mitochondrial DNA polymerase chain reactionrestriction fragment length polymorphisms analysis
in the wild population of kuruma prawn Penaeus
japonicus. Fish. Sci. 68:793–802.
Sugaya, T., M. Ikeda, H. Yagi, and N. Taniguchi.
2002b. Inheritance mode of microsatellite DNA
markers and their use for kinship estimation in kuruma prawn Penaeus japonicus. Fish. Sci. 68:793-802.

Figure 7. A UPGMA-based dendrogram created from the genetic
relatedness among individuals calculated from the data of MSDNA. Bold lines show the branches that join common haplotypes 1 to 64.

6Tauz, D. 1989. Hypervariability of simple sequences
as a general source for polymorphic DNA markers.
Nucleic Acids Res. 17:6463-6471.

Sugaya et al.

Marine Ornamental Aquaculture
Junda Lin
Florida Institute of Technology
Melbourne, Florida 32901
jlin at fit.edu
Keywords: aquarium trade, Lysmata, seahorses, Hippocampus
Abstract

Many species of marine animals belonging to different taxonomic groups are collected from the wild
for the aquarium trade. Direct and indirect impacts of the collection, especially the use of explosives andtoxins (e.g., cyanides), have caused grave concerns. In recent years, efforts have been made to understandthe biology of some of the species and develop cultivation technology to reduce wild collection while
sustaining the aquarium industry trade. My laboratory has worked on developing aquaculture protocols
for marine ornamental crustaceans (primarily shrimp, but also crabs and lobsters) and, in more recent
years, seahorse species.
Our focus has been on several Lysmata spp., popular in the aquarium industry. The caridean shrimp
species have a unique (among decapod crustaceans) reproductive system, protandric simultaneous hermaphrodite. The shrimp, which first matures as a male, may change through several transitional stages
to simultaneous hermaphrodite that can function as both a male (during intermolt) and female (during
postmolt).
Spawning of these shrimp in captivity is relatively easy, especially for the Lysmata spp., as they are
simultaneous hermaphrodites. A female can produce several hundred to several thousand eggs during
each spawning and carries the embryos under her abdomen until the larvae hatch 10 to 20 days later.
Within several to 48 hours after hatching, the female molts, is receptive to mating, and spawns (with orwithout mating) again. The larvae are composed of many (9–13) zoea stages. Artemia nauplii can beused as the sole food for all life stages of Lysmata spp.
The biggest challenge for commercial culture of marine ornamental shrimp is the long and variable
larval durations, due to mark time molting. Shortening larval cycle through nutrition and culture system
improvement is the key for successful commercial production of these ornamental shrimp species. Improving diets of broodstock, and especially of larvae, may also accelerate the rates of larval development
and increase the potential of commercial aquaculture.
Seahorses (genus Hippocampus) are a group of fish that have fascinated people for centuries for their
unusual upright body form, unique reproductive system (males give birth and are the major caretakersof the offspring) and healthcare value. More than 20 million individuals are collected annually for use
in traditional Chinese medicine and aquarium trade. Overfishing has led to placement of all 34 seahorse
species under Convention on International Trade in Endangered Species protection since 2004. Unlike
for Chinese medicine, tank-raised seahorses are preferred in the marine aquarium market (and demandhigher prices). In recent years, efforts have been made to develop protocols for culturing seahorse species that are popular in aquarium trade.
We are in the process of developing aquaculture protocols for the lined seahorse (H. erectus), one of
the most popular aquarium species and native along the Atlantic coast of North America from Nova Scotia to Florida and throughout the Caribbean. Pairing, mating, and copulation behavior were observed.
Gestation time and brood size were 17.33 ±2.94 days and 272.33 ±66.45 juveniles/brood, respectively. The
highest growth rate and survivorship of the juveniles during the first 9 weeks occurred at 28–29°C among
the temperatures tested (24–33°C).

Lin

7Nutritional Significance of n-3 Highly Unsaturated Fatty Acids for Larval Survival andDevelopment in Mass Production of Brachyuran Crab Larvae
Shigeki Dan
Fisheries Research Agency
Seikai National Fisheries Research Institute
Ishigaki Tropical Station
Ishigaki, Okinawa 907-0451, Japansdan at fra.affrc.go.jp
Katsuyuki Hamasaki
Tokyo University of Marine Science and Technology
Minato, Konan
Tokyo 108-8477, Japan
Takayuki Kogane
Fisheries Research Agency
National Center for Stock Enhancement
Yashima Station
Takamatsu, Kagawa 761-0111, Japan
Tadao Jinbo
Fisheries Research Agency
National Center for Stock Enhancement
Minamiizu Station
Irozaki, Shizuoka 415-0156, Japan
Takashi Ichikawa
Fisheries Research Agency
National Center for Stock Enhancement
Akkeshi Station
Akkeshi, Hokkaido 088-1108, Japan
Keywords: brachyuran crab, seed production, n-3 HUFA, survival rate, larval morphogenesis
Abstract
To evaluate the n-3 highly unsaturated fatty acid (HUFA) requirement by larvae of the swimming
crab (Portunus trituberculatus), two mud crab species (Scylla serrata and S. paramamosain), snow crab
(Chionoecetes opilio), and horsehair crab (Erimacrus isenbeckii), we conducted two types of larval rearing
experiments using small vessels and large larval production tanks. In small rearing vessels, n-3 HUFA
in live foods improved larval survival of the swimming crab and mud crabs. On the other hand, in largetanks, larval mass mortality of S. serrata frequently occurred during the metamorphosis to megalops due
to abnormal molting by morphologically advanced last stage zoea. These last stage zoea have morphological features similar to those of megalops, such as large chelipeds.
The excess dietary n-3 HUFA is concluded to cause this hypermorphogenesis of larval S. serrata. The
phenomenon was also observed for the swimming crab and the mud crab S. paramamosain. Horsehaircrab larvae fed unenriched Artemia showed high survival rates to the first crab stage in small rearing
vessels. In contrast, enrichment with n-3 HUFA, especially docosahexaenoic acid (DHA) in live food,
improved the survival of snow crab larvae in small rearing vessels. However in large tanks, DHA con-8

Dan et al.

tent of snow crab larvae largely decreased because of the low content of DHA in live food. We concluded
strategies for optimal n-3 HUFA enrichment in live food are specific for each species, requiring development for each crab species.

Introduction

Hatchery technologies of commercially important brachyuran species such as the swimming crab
(Portunus trituberculatus), mud crabs (Scylla serrata
and S. paramamosain), snow crab (Chionocetes opilio),
and horsehair crab (Erimacrus isenbeckii) have beenstudied for stock enhancement programs by the
Fisheries Research Agency (FRA) of Japan (Hamasaki 2003, Hamasaki et al. 2002a, 2002b, 2006, 2007,
Suprayudi et al. 2002, 2004, Arai et al. 2004, 2007,
Jinbo et al. 2005, Kogane et al. 2005, 2007a, 2007b).
While the swimming crab and two mud crabs arewarm water species, snow crab and horsehair crab
inhabit cold water.
Between 1993 and 2005, approximately 50 million juveniles and several million juveniles of thefirst crab stage for swimming crab and mud crabs
have been produced annually for restocking purposes (Fisheries Agency et al. 1995–2007). Accordingly, hatchery technologies have been developed
for those warm water species. However, larval massmortality has frequently occurred during larval
production (Hamasaki et al. 2002a, Arai et al. 2004).
Reliable techniques are needed for the efficient mass
production of large numbers of juveniles. On theother hand, large-scale larval production techniques
remain undeveloped for snow crab and horsehaircrab (Hamasaki et al. 2006, 2007, Kogane et al. 2007a,
2007b).
The n-3 highly unsaturated fatty acids (n-3
HUFA) such as eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) are essential fatty
acids (EFA) for marine fishes (Watanabe et al.
1989a, 1989b, Izquierdo et al. 1989, Takeuchi et al.
1990, 1994, 1998, Furuita et al. 1996, 1998). It is wellknown that n-3 HUFA affect larval survival, growth,and stress tolerance of marine fish larvae and juveniles. However, the effectiveness of n-3 HUFA
for larval brachyuran crabs as EFAs has not beenstudied in detail. Since the early 1990s, we haveinvestigated this issue to improve and develop mass
production technologies for brachyuran crabs.
We conducted larval rearing experiments for
swimming crab, mud crabs, horsehair crab, andsnow crab using small vessels and large larval
production tanks with live foods containing various
levels of n-3 HUFA and examined the significanceof dietary n-3 HUFA for larval development and

Dan et al.

survival of the species of interest. In this paper, wesummarize the results of our experiments and discuss future prospects for the development of massseed production techniques of brachyuran species
from the point of view of n-3 HUFA requirement bylarvae.

Materials and Methods

Rearing experiments in small vessels. Larvae ofeach species were reared in 1–2 L plastic beakers
with gentle aeration (Anger 2001). The beakers were
set in shallow water baths in which temperatures
were regulated by heaters, chillers, or both. Thirty
newly hatched larvae were stocked in each beakerwith seawater and prey (rotifers, Brachionus plicatilis
sp. complex, or Artemia sp.). The larval developmental stages were determined when they weretransferred daily using a largemouth pipette into
new beakers with new seawater and prey. Rotifers
and Artemia nauplii were enriched with emulsified
oils containing different levels of n-3 HUFA (mainly
EPA and DHA).
Rearing experiments in large production tanks.
Larvae of the mud crab S. serrata were reared in
200,000 L tanks and snow crab in 20,000 L tanks.
Newly hatched larvae were stocked in the flowthrough tanks with aeration at a density of 10,000–
40,000 individuals/1,000 L. Rotifers were supplied
daily to the tanks to maintain the designated concentrations in the larval rearing water. Artemia nauplii
were used as prey from the third zoeal and firstzoeal stages for mud and snow crabs, respectively.
Phytoplankton (Chlorella vulgaris, Nannochloropsissp.) were also added to the tanks as food for therotifers and Artemia. The rotifers were enriched withbrewer’s yeast or phytoplankton containing cellularn-3 HUFA before being fed to the larvae. Larval
survival rates were estimated by the volumetricmethod. The length of chelae and carapace of fifth
stage zoea of mud crabs were measured under amicroscope and the percent ratio of relative chela
length to carapace length (termed the relative chela
length in this study) was calculated as an index of
the degree of morphogenesis in fifth stage zoea.

9Fatty acid analysis. The n-3 HUFA contents oflarvae and prey used in each experiment were determined. Lipids were extracted by the chloroformmethanol (2:1, v/v) method (Folch et al. 1957). Total
lipids were saponified with KOH. The saponifable
matter was then esterified by BF3 in methanol.
Fatty acid methyl esters were diluted in hexane andanalyzed with a gas chromatograph equipped witha silica capillary column. Fatty acid methyl esters
were identified by comparing retention times against
standards.

frequency of serious abnormal molting (type A)
linearly increased against the relative chela length.
As can been seen in Figure 4, the upper limit of relative chela length should be less than 50% for normal
molting of last stage zoea into megalops (Hamasaki
et al. 2002a).
To examine what factors affect larval morphogenesis in terms of larval n-3 HUFA requirement,
the relationship was determined between n-3 HUFA
content in rotifers sampled from the larval rearing

Results and Discussion

Effect of dietary n-3 HUFA on larvae of swimmingand mud crabs. Larvae of the swimming crab andthe two mud crab species fed rotifers and Artemia
enriched with n-3 HUFA showed higher survival
rates than larvae fed unenriched live foods in therearing experiments using small vessels (Takeuchi etal. 1999, Takeuchi et al. 2000, Suprayudi et al. 2004)(Figure 1). Thus it was determined that dietary n-3
HUFA greatly improves the survival rates of larvaeof the swimming crab and mud crabs.
Based on the results of the rearing experiments
using small vessels, we enriched the rotifers and Artemia with n-3 HUFA for feeding larvae of the same
species in large production tanks. Larval mass mortality of the mud crab S. serrata has been frequently
observed during metamorphosis to megalops due
to abnormal molting of morphologically advanced
last stage zoea (Hamasaki et al. 2002a, 2002b). The
last stage zoea have morphological features similar
to those of megalops, including large chelipeds
compared with normal last stage zoea (Figure 2).
Morphologically advanced last stage zoea often failto molt to normal megalops. The abnormal molting
patterns by morphologically advanced last stage
zoea were classified by Hamasaki et al. (2002a) as
follows (Figure 3): type A, serious abnormality (larva
has not shed its exuvia completely and died duringmolting); type B, partial abnormality (larva has shed
its exuvia but was unable to shed the integumentof the chelipeds, walking legs, or both); and type C,
slight abnormality (larva has shed its exuvia except
for the integument of one cheliped).
To elucidate the relationship between morphogenesis of zoea and abnormal molting, percentagesof larvae showing abnormal molting were plottedagainst the relative chelae lengths as an index of thedegree of morphogenesis of fifth stage zoea (Figure4). Almost all larvae with a relative chela length
exceeding 50% failed to molt successfully and the10

Figure 1. Survival rates to each larval stage of Portunus trituberculatus, Scylla serrata,and S. paramamosain reared in 1-L beakers
with rotifers and Artemia nauplii that were either unenriched (p)or enriched with n-3 HUFA (o), and n-3 HUFA contents in rotifers
and Artemia used in the experiments. In the all experiments, rotifers were used as prey from first zoeal and second zoeal stages,
and Artemia were used from third zoeal to megalopal stage.
(Takeuchi et al. 1999, Takeuchi et al. 2000, Suprayudi et al. 2004.)

Figure 2. Photographs show the fifth stage zoea in the productionof S. serrata. Photo A, the fifth stage zoea which have morphological features similar to those of megalops. Photo B, a normal
megalopa. (Modified with permission from Hamasaki et al. 2002a.)

Dan et al.

Figure 3. Photographs show the molting patterns during themetamorphosis to megalops in the production of S. serrata. TypeA, serious abnormality (larva has not shed its exuvia completely
and died during molting); Type B, partial abnormality (larva has
shed its exuvia but was unable to shed the integument of thechelipeds or walking legs or both); Type C, slight abnormality(larva has shed its exuvia except for the integument of one cheliped). (Modified with permission from Hamasaki et al. 2002a.)tanks with phytoplankton that contained different
supplementary levels n-3 HUFA in their cells andrelative chela length of last stage zoeas (Figure 5).
The relative chela lengths increased with increasingn-3 HUFA content in rotifers and the upper limit
content of n-3 HUFA in rotifers in larval rearing
tanks of S. serrata that prevented larval mass mortality during the metamorphosis to megalops wasaround 0.6% (Hamasaki et al. 2002b). That upper
limit in rotifers was similar to the n-3 HUFA content
of enriched rotifers but less than that of enriched
Artemia that were provided in the rearing experiments using beakers by Suprayudi et al. (2004) (seeFigure 1). The optimum level of n-3 HUFA content
in Artemia should be determined in the large production tanks.
Larval mass mortality of morphologically advanced last stage zoeas has also been observed in the
production of the swimming crab and the mud crab
S. paramamosain (Arai et al. 2004, 2007). However,the upper limit content of n-3 HUFA in rotifers has
yet to be determined.
We found that dietary n-3 HUFA markedly
affects the survival and morphogenesis of swimming
crabs and mud crabs. We concluded that the relative
chela lengths of last stage zoeas reflect the nutritional condition of larvae based on the positive correlation between the n-3 HUFA content of prey fed to S.
serrata larvae and relative chela length of the larvae.
Furthermore, this relationship can be used as a
useful index to evaluate the effects of environmental
conditions on larval development and survival as
found in our rearing experiments of S. serrata larvae(Dan and Hamasaki unpubl. data).

Figure 4. Relationships between the relative chela length of fifth
stage zoeas to the carapace length and the percentage showing
abnormal molting during the metamorphosis to megalops in the
production of S. serrata. Symbols indicate the types of abnormal
molting as shown in Figure 3. The line indicates linear approximation of type A. (Modified with permission from Hamasaki etal. 2002a.)

Figure 5. Relationships between the fatty acid contents in the rotifers and the relative chela length of fifth stage zoea to the carapace
length of S. serrata. (Modified with permission from Hamasaki et al.
2002b.)Dan et al.

11

Survival rates of S. serrata larvae reared in 1 L
beakers at different salinity levels between 10 and
35 ppt with an interval of 5 ppt were low at 10, 15,30, and 35 ppt conditions. Based on that result, it
might be natural to conclude that the optimal salinity for survival and development of S. serrata larvae
was 20–25 ppt. However, the relative chela lengthsof last stage zoeas increased with increasing salinity and they exceeded the critical level of 50% at 30
and 35 ppt, so the larvae failed to undergo normal
metamorphosis to megalops at those salinity levels.
Based on these observations, it is clear that hypermorphogenesis caused the larval mass mortality at
30–35 ppt. Therefore, we recommend that larvalmorphogenesis should be examined to evaluate therearing experiments with swimming crab and mud
crabs.
Effect of dietary n-3 HUFA on larvae of horsehairand snow crabs. Horsehair crab larvae were rearedin 2 L beakers with Artemia unenriched and enrichedwith n-3 HUFA (Jinbo unpubl. data). Horsehaircrab larvae showed high survival rates to the firstcrab stage even when fed unenriched Artemia. On
the other hand, the snow crab larvae that were fed
rotifers enriched with n-3 HUFA showed higher
survival rates than larvae fed unenriched rotifers.
In addition, larvae that were fed rotifers containing
mainly DHA showed the highest survival rate (Kogane unpubl. data). We also analyzed the n-3 HUFA
compositions in rotifers and Artemia supplied to thelarval rearing tanks and snow crab larvae reared inlarge production tanks. Rotifers and Artemia contained minimal DHA. On the other hand, first stage
zoea contained the highest amount of n-3 HUFA,
mainly composed of EPA and DHA (Figure 6).
The DHA content of larvae decreased with growththrough second stage zoea to megalops stages andlarval mass mortality occurred (Figure 7) during metamorphosis to megalops (Kogane et al. 2007b). Thiswas probably due to the low DHA content in the live
food. Consequently, we conclude that improving the
DHA content in live food is one of the primary ways toimprove the mass production of snow crab.
Our recent research demonstrated that dietaryn-3 HUFA is essential to improve larval survival anddevelopment in the production of swimming crab, mudcrabs, and snow crab. However, the requirement bylarvae of brachyuran crabs for n-3 HUFA varied withspecies. So, strategies for n-3HUFA enrichment in live
food require individual verification for each species infuture research.

12

Figure 6. The n-3 HUFA content in rotifers and Artemia nauplii
supplied to the larval rearing tanks and larvae of snow crab
(Chionocetes opilio) reared in large production tanks. (Based ondata from Kogane et al. 2007b.)

Figure 7. Survival rates of larvae (o) and percentage composition of megalops (p) of the snow crab (Chionocetes opilio) rearedin large seed production tanks. (Modified with permission from
Kogane et al. 2007b.)

Acknowledgments

The authors thank the staff of the Akkeshi,
Obama, Tamano, and Yaeyama stations of the FRA
National Center for Stock Enhancement, especiallyK. Murakami and M. Ashidate for their kind hospitality and cooperation throughout the experiment.

Dan et al.

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Hamasaki, K., T. Kogane, T. Jinbo, and S. Dan. 2006.
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Jinbo, T. Unpubl. data. Larval rearing experiments.
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Erimacrus isenbeckii (Crustacea, Decapoda, Brachyura) reared in the laboratory. Nippon Suisan Gakkaishi 71:563–570.
Kogane, T. Unpubl. data. Larval rearing experiments. (Available from T. Kogane, Fisheries
Research Agency, National Center for Stock Enhancement, Yashima Station, Takamatsu, Kagawa
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Kogane, T., S. Dan, and K. Hamasaki. 2007a. Improvement of larval rearing technique for mass seedproduction of snow crab Chionoecetes opilio. Fish. Sci.
73:851–861.

13

Kogane, T., K. Hamasaki, and S. Dan. 2007b. Improvement of larval survival rates by water agitation and medication in the seed production of snow
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Effect of temperature on survival and developmental period of larval snow crab Chionoecetes opilio(Brachyura: Majidae) reared in the laboratory. Nippon Suisan Gakkaishi 71:161–164.

Watanabe, T., M. S. Izquierdo, T. Takeuchi, S. Satoh,and C. Kitajima. 1989. Comparison between eicosapentaenoic and docosahexaenoic acids in terms ofessential fatty acid efficacy in larval red seabream.
Nippon Suisan Gakkaishi 55:1635–1640.

Suprayudi, M. A., T. Takeuchi, and K. Hamasaki.
2004. Essential fatty acids for larval mud crab Scylla
serrata: Implications of lack of the ability to bioconvert C18 unsaturated fatty acids to highly unsaturated fatty acids. Aquaculture 231:403–416.
Suprayudi, M. A., T. Takeuchi, K. Hamasaki, and J.
Hirokawa. 2002. The effect of n-3 HUFA content
in rotifers on the development and survival of mudcrab, Scylla serrata, larvae. Aquacult. Sci. 50:205–212.
Takeuchi, T., Z. Feng, K. Yoseda, J. Hirokawa, and T.
Watanabe. 1994. Nutritive value of DHA-enrichedrotifer for larval cod. Nippon Suisan Gakkaishi
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Takeuchi, T., Y. Ishizaki, T. Watanabe, K. Imaizumi,
and K. Shimizu. 1998. Effect of different DHA
content in rotifers on the DHA requirement of larval
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2000. The necessity and suitable feeding schedule of
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Takeuchi, T., N. Satoh, S. Sekiya, T. Shimizu, and T.
Watanabe. 1999. Influence of the different feedingschedule of food organisms for larval swimming
crab Portunus trituberculatus. Nippon Suisan Gakkaishi 65:804–809.
Takeuchi, T., M. Toyota, S. Satoh, and T. Watanabe.
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Dan et al.

The Chesapeake Bay Blue Crab (Callinectes sapidus):A Multidisciplinary Approach to Responsible Stock Enhancement
Oded Zmora and Yonathan Zohar
University of Maryland Biotechnology InstituteCenter of Marine Biotechnology
701 East Pratt Street
Baltimore, Maryland 21202
zmorao at umbi.umd.edu
Anson H. Hines and Eric G. Johnson
Smithsonian Environmental Research Center
647 Contees Wharf Road
Edgewater, Maryland 21037
Keywords: blue crab, Chesapeake Bay, stock enhancement, juvenile

Abstract

Blue crab (Callinectes sapidus) catches, the major remaining harvest of the Chesapeake Bay (morethan $100 million in value), dropped over 70% from record highs in the early 1990s. Overfishing andenvironmental degradation led to a sustained decline of 84% in the blue crab breeding stocks, which
in turn resulted in historically low levels of juvenile recruitment and in nursery habitats being undercarrying capacity. This situation makes the Chesapeake Bay blue crab an excellent candidate for stockenhancement efforts that target replenishment of the declining breeding stocks. A multidisciplinary
program was therefore developed to 1) study the basic biology and life cycle of the blue crab, 2) develop
hatchery and nursery technologies for the mass production of blue crab juveniles, and 3) assess thepotential of using hatchery juveniles to enhance blue crab breeding stocks and, in turn, bay-wide abundance and harvests.
Understanding the environmental regulation of the reproductive cycle led to full photothermal control of the timing of ovulation and hatching of wild-caught inseminated females. Intensive larval rearing(60–140 larvae/liter), utilizing microalgae species of high nutritional value and omega-3 enriched rotifers
and brine shrimp larvae (Artemia nauplii), resulted in 30–80% survival from hatch to megalopae in 3 to 4
weeks. Megalopae were reared to 20 mm juveniles (mean carapace width) at lower densities (5–20/liter)in four weeks at survival rates ranging from 10 to 30%, depending on rearing density.
During 2002 to mid-2007, in excess of 400,000 hatchery-reared juveniles were experimentally released
into nursery habitats of the Chesapeake, both in upper and lower bay waters. All released juveniles
were individually tagged with coded microwire, elastomer tags, or both and monitored to study survival,
growth, migration patterns, field performance, and enhancement. Simultaneous releases and monitoringof hatchery and wild crabs demonstrated that performance of hatchery-reared juveniles did not differ
from wild juveniles in most variables, including survival, growth, feeding, and habitat use, despite someminor differences in morphology and behavior.
Enhancement doubled to tripled the wild population in release sites, and survival from release tosexual maturity averaged 15% (range 6–26%). Survival varied among years, depending on environmental
conditions such as salinity and temperature, and was inversely dependent on stocking density. Optimal
juvenile release size was found to be 20 mm and above, which corresponds with change in dispersal
behavior of smaller juveniles.
We also examined optimal release sites, habitats, density, and timing. Hatchery crabs grew rapidly
to maturity, and were observed mating in as few as two months after release. Inseminated female crabs
migrated in the fall along the bay’s main deepwater channel from nursery and mating habitats to thespawning sanctuary in the lower bay, suggesting that hatchery crabs can contribute to the spawning

Zmora et al.

15

stock as soon as several months postrelease. This, together with evidence showing the effectiveness ofthe spawning sanctuary, suggests the importance of implementing protected migratory corridors linking
nursery habitats with spawning grounds.
In summary, using a multifaceted approach, we demonstrated the feasibility of releasing hatcheryproduced juvenile crabs to restore the dwindling blue crab breeding stocks. Working with watermen,
the production of juvenile crabs is now being scaled up to allow for larger releases and to optimize release strategies, which will be ultimately recommended to and implemented by the Chesapeake crabbing
industry.

16

Zmora et al.

An Outline of the Research Project, Development of Seed Production Technology inJapanese Spiny Lobster
Hideaki Aono
National Research Institute of Fisheries Science
Nagai, Yokosuka, Kanagawa 238-0316, Japan
aochan at affrc.go.jp
Keisuke Murakami
Minamiizu Station
National Center for Stock Enhancement
Irouzaki, Minamiizu, Shizuoka 415-0156, Japan
Masahiko Awaji
National Research Institute of Aquaculture
Minamiise, Mie 516-0193, Japan
Keywords: spiny lobster, seed production, diet

Abstract

The Japanese spiny lobster (Panurilus japonicus) is a commercially important crustacean in Japan.
Since the lobster fishery is fully exploited, development of the seed production and rearing techniques ofthe lobster has been eagerly desired. However, specific biological characteristics of phyllosoma, such as
their peculiar body form, protracted lifespan (about one year), and pelagic open ocean life, have hinderedsignificant progress in culture. To overcome these problems and produce large numbers of juveniles
through larval culture, a research program for development of seed production technology in the Japanese spiny lobster was started in 2005. The project team consists of two subteams, one to improve anddevelop diets and the other to improve and develop rearing methods of phyllosoma.
In order to improve and develop diets, we are investigating natural diets of lobster phyllosoma by
molecular methods. Improvement of dietary value of brine shrimp (Artemia) and mussel gonads, which
are known to be effective as foods for phyllosoma, and development of artificial feed are in progress. We
are also attempting to develop methods to evaluate and control conditions of phyllosoma in the rearing
tank by monitoring expression of DNA responsible for biodefense, molting, and digestion. The survival
rate of phyllosoma has been gradually increasing year by year by incorporating these results into culturing methods.

Aono et al.

17Shellfish Aquaculture Research on Oysters and Clams
at Haskin Shellfish Research Laboratory
John Kraeuter
Rutgers University
Institute of Marine and Coastal Science
Haskin Shellfish Research Laboratory
Port Norris, New Jersey 08349
Keywords: aquaculture, oysters, clams, genetic structure
Abstract

Haskin Shellfish Research Laboratory has been working on various aspects of shellfish aquaculture
for more than 100 years. Most current genetic work is mapping genes for disease resistance in easternoysters (Crassostrea virginica) using our 50-year breeding program that has developed disease resistantlines. These studies augment our development of tetraploid stocks of C. virginica, and continued development of strains of eastern oyster resistant to diseases caused by MSX (Haplosporidium nelsoni) andDermo (Perkinsus marinus). Information from long-term studies suggests that native stocks have developed some resistance to MSX disease. This data has encouraged a program of large-scale restoration ofoysters in Delaware Bay using classic shelling techniques. This restoration program is being coupledwith a study to develop an understanding of disease transmission in these populations using field studies and modeling.
We are involved in efforts to identify species of oysters along the coast of China. To date approximately 16 distinct genetic entities have been identified. While this is not direct aquaculture research, it
has shown that oyster culturists in China prefer to use Hong Kong oysters (C. hongkongensis) rather than
Suminoe oyster (C. ariakensis). The reason for this decision is not clear.
We are working on a model of the genetic structure of oysters. The model is at the level of the allele,but tracks a representative selection of chromosomes each with a selection of alleles. When completed
this could be utilized to model such things as the probability of generating a reduced genetic complement through inbreeding, how difficult it might be to increase growth by selective breeding, or how
aquaculture populations might affect the genetics of nearby natural populations.
In addition, our hatchery has been providing disease resistant lines of oysters to growers in a number of states, and assisting in developing rack and bag oyster culture in New Jersey. Clam investigations
include identifying the effects of the disease QPX on aquaculture of five strains of the hard clam (Mercenaria mercenaria). Work to date has shown that there are interactions between strains and the disease
and latitude. Strains of southern origin (Florida and South Carolina) were more susceptible to the disease than those from Virginia, and the New Jersey and Massachusetts strains were the least susceptible
when planted in either Virginia or New Jersey.
We are also evaluating the effects of the alga that causes brown tide (Aureococcus anophagefferens) on
hard clam larval and juvenile growth and survival, and adult feeding. Laboratory and field experiments
have shown that blooms of this alga reduce or stop feeding in clams and scallops. We have been able tomodel the reduced growth of seed and adults and are currently adding a larval model based on lipid, protein, and carbohydrate levels in the food. Ultimately, the larval model will be added to our existing hardclam numerical population model. Additional work on clams focuses on Manila clam (Ruditapes philippinarum) and modeling the combined effects of the dinoflagellate Alexandrium and brown ring diseaseon growth and reproduction in France.
In other studies, we have just begun a small project investigating the potential to use aquaculture toenhance the population of horseshoe crabs (Limulus polyphemus) in Delaware Bay, and an industry sponsored effort on the potential for the use of triploid bay scallops (Argopecten irradians irradians).

18

KraeuterImprovements in Yield of the Pacific Oyster (Crassostrea gigas) by Selective Breeding
Chris Langdon, Ford Evans, and Alan Barton
Oregon State University
Department of Fisheries and Wildlife
Coastal Oregon Marine Experiment Station
Hatfield Marine Science Center
Newport, Oregon 97365
chris.langdon at oregonstate.edu
Keywords: broodstock, Pacific oyster, yield
Abstract

The Molluscan Broodstock Program (MBP), funded by the U.S. Department of Agriculture, was initiated in 1995. Six cohorts of 50 to 60 families were initially produced from about 600 founder broodstockoysters collected from several naturalized populations on the U.S. West Coast and Canada. Cohorts were
evaluated at commercial test sites and top-yielding families from each cohort used as broodstock to produce the next generation.
Two complete selection cycles were completed in 2003 and an average 16.7% improvement in yield
was obtained per generation of selection. Improvements in yield were mainly a result of increased survival. Most of the highest yielding families were derived from founder broodstock obtained from Pipestem Inlet, Vancouver Island, Canada. Yields of the highest yielding families were more than three times
greater than those of families from nonselected broodstock.
MBP will continue the selection program using a rotational breeding scheme among all lines as well
as more focused selection on the Pipestem Inlet line. We will also include shell shape and color in theselection program.
MBP works closely with the West Coast oyster industry in order to provide commercial hatcheries
with improved broodstock. We have increased production of improved broodstock oysters by creating
inbred lines. These inbred lines can be crossed in hatcheries to produce high-yielding families.

Langdon et al.

19Synthesis of Vitellogenin in the Pacific Oyster (Crassostrea gigas)
Toshie Matsumoto
Fisheries Research Agency
National Research Institute of Aquaculture
Minami-ise, Watarai
Mie 516-0193, Japan
mtosie at fra.affrc.go.jp
Keywords: estrogen receptor, bivalve, oyster, vitellogenin, follicle cells, immunolocalization
Abstract

Vitellogenins are precursors of the major yolk protein (vitellin) in oviparous vertebrates and invertebrates. In mollusks, a full-length cDNA encoding vitellogenin was cloned from the Pacific oyster(Crassostrea gigas) and its amino acid sequence was deduced. The deduced primary structure of vitellogenin in the Pacific oyster was shown to be similar to vitellogenins of fish, crustacean, and nematodespecies, especially in the N-terminal region. Vitellogenin mRNA expression was detected only in theovary. To determine the distribution of oyster vitellogenin mRNA expression in the ovary, we performedin situ hybridization using DIG-labeled RNA probes. A strong signal was detected in follicle cells. It is
concluded that follicle cells are the site of vitellogenin synthesis. In all vertebrate species, estradiol-17ß
(E2) is involved in many regulatory steps controlling reproduction. In the Pacific oyster, E2 is detected in
the ovary and its content shows a synchronous profile with gonadal maturity.
To investigate estrogen signaling in the vitellogenesis, a cDNA encoding the Pacific oyster estrogen
receptor (cgER) was cloned. The phylogenetic analysis indicated that the cgER is an ortholog of the othermollusk estrogen receptors (ERs). Reporter gene assay revealed that cgER is unresponsive to estrogen.
This result is similar to those of other mollusk ERs. We examined the localization of cgER in the oyster
ovary at the vitellogenic stage using anti cgER peptide antiserum. The immunohistochemical study indicated that cgER was mainly localized in the nuclei of follicle cells—the site of vitellogenin synthesis—in
the oyster ovary. This result suggests that cgER could work as a nuclear receptor. Our results will facilitate further research to understand the vitellogenesis in the oyster.

Introduction

In marine bivalves as in most oviparous animals, a large amount of yolk protein (vitellin) is accumulated in oocytes during ovarian maturation. In
vertebrates such as fish, amphibians, and birds, yolk
protein is synthesized from a precursor, vitellogenin(Vg) produced by the liver, and is transported to the
oocytes via the blood circulation system. In bivalve
mollusks, a few biochemical studies of yolk proteins
have been carried out (Osada et al. 1992, Suzuki etal. 1992). In the oyster, vitellin has been isolated andcharacterized (Suzuki et al. 1992, Li et al. 1998), but
the characterization of Vg has not been reported,and information on bivalve vitellogenesis remains
limited. To identify the genes associated with vitellogenesis, we cloned and sequenced more than 100
cDNA fragments using mRNA differential display
and found that one of the isolated genes was similar to Vg. We cloned the full-length of the Pacificoyster (Crassostrea gigas) Vg cDNA and examined the20

expression of its mRNA.
Estrogen receptors (ERs) are members of thenuclear receptor superfamily, which have a number of common features, and their proteins can bedivided into several distinct domains (Krust et al.
1986). The DNA-binding domain (C domain) andthe ligand-binding domain (E domain) are highly
conserved between species (Mangelsdorf et al. 1995).
The ER is a transcription factor that directly binds
to a specific DNA sequence, the estrogen responsive element (ERE, consensus = 5'-AGGTCAXXXTGACCT-3', Umesono and Evans 1989) and regulates
the transactivation of estrogen target genes (Schwabe
et al. 1993) such as Vg.
In fish, as in other oviparous vertebrates, it isclearly established that the Vg gene expression isregulated by estradiol-17ß (E2) via ER (Pakdel et al.
1991). In marine bivalves, the presence of steroids
was reported in the 1960s (Botticelli et al. 1961).

MatsumotoEstrogens have been detected in blue mussels (Mytilus edulis) and softshell clams (Mya arenaria) and areconsidered to have various physiological functions
in bivalves (Reis-Henriques et al. 1990, Stefano et al.
2003, Gauthier-Clerc et al. 2006). In the oyster andscallop, E2 has been detected in the ovary, and its
contents show a synchronous profile with gonadal
maturity on the basis of HPLC, suggesting that E2
plays a role in reproductive events (Matsumoto et al.
1997). In addition, E2 treatment in vivo and in vitro
stimulated vitellogenesis in oysters (Li et al. 1998)
and scallops (Osada et al. 2003, 2004). These findings suggest that E2 may be involved in the controlof vitellogenesis mediated by ER.
In invertebrates, ER has been cloned from mollusks, the California sea slug (Aplysia californica)(Thornton et al. 2003), intertidal snail (Thais clavigera)(Kajiwara et al. 2006), and common octopus(Octopus vulgaris) (Keay et al. 2006), but no ER orother steroid hormone receptors have yet beencloned in bivalves. We isolated the oyster ER homologue, which is highly similar to ERs of other species, and detected the presence of an ER in the nuclei
of follicle cells using an antibody against synthetic
oyster ER peptide.

Oyster Vg

In order to identify the expressed genes in theovary, transcripts derived from the ovaries during
the spawning season and the spent gonads containing no oocytes were examined by mRNA differential display. During this procedure, we identified acDNA of approximately 1.2 kb in length. Sequence
analysis of the cDNA fragment followed by a database search revealed a similarity to Vg. Subsequently the 5' and 3' ends of this cDNA were obtained by
5' and 3' RACE. The sequence of oyster Vg cDNAis submitted to DDBJ/EMBL/GenBank with the accession number AB084783 (Matsumoto et al. 2003).
The cDNA consisted of 5,023 bp and had an ORF(open reading frame) that encoded 1,583 amino acidresidues with a predicted molecular mass of 179,191
Da. The deduced amino acid sequence contained aconsensus cleavage site, R-X-R-R (Arg828 to Arg831),
capable of undergoing processing by endoproteases of the subtilisin family (Barr 1991) that havealso been reported in Vgs of insects and crustaceans(Chen et al. 1994, Yano et al. 1994, Tsutsui et al. 2000,
Okuno et al. 2002).
The overall serine content of the oyster Vg(11.9%) was higher than in the mosquito (10.1%) andMatsumotothe silkworm Vg (9.8%), which possess polyserine
clusters. The deduced amino acid sequence contained 56.2% of serine residues in the region between
Ser1149 and Ser1180, and this region may be thepolyserine domain in Pacific oyster Vg. This protein
possessed six potential N-linked glycosylation sites,
which is conserved in vertebrate and invertebrate
Vgs. The deduced amino acid sequence has 19–35%
identity with scallop, nematode, fish, and kuruma
prawn Vgs in the N-terminal and the central region.
The C-terminal region showed no significant similarity to any of the other reported species. Thus this region appears to possess unique properties comparedwith Vg of the other known species.
The levels of Vg mRNA in various tissues fromfemale oysters were measured by reverse transcription-mediated PCR. Vg mRNA expression was
detected only in the ovary. To determine the distribution of oyster Vg mRNA expression in ovary, we
performed in situ hybridization using DIG-labeled
RNA probes. A strong signal was detected in thefollicle cells using an antisense RNA probe (Figure1a). In contrast, hybridization with a sense control
probe produced no significant signal (Figure 1b). In
marine bivalves, autosynthesis of yolk proteins in
the ovary has been postulated, based on the morphological evidence (Pipe 1987, Dorange and Le Pennec
1989, Eckelbarger and Davis 1996). In oyster andother bivalve species, the ovarian acinus is a simple

Figure 1. Localization of putative Vg mRNA in the ovary by insitu hybridization. A) Strong expression of putative Vg wasseen in the follicle cells with an antisense probe in the ovary. B)
Hybridization with a sense probe produced no significant signal
in the ovary. Scale bar = 50 µm.

21

structure containing only developing oocytes andassociated follicle cells within a thin germinal epithelium. The function(s) of follicle cells in the bivalve
ovary are not well understood, although they aresuspected of playing some role in oocyte nutrition.
In the present study, Vg mRNA expression was in
the follicle cells in the ovary of Pacific oysters. Thissuggests that yolk synthesis in Pacific oysters may
take place in the follicle cells.

Oyster Estrogen Receptor (cgER)

Using the degenerate primers derived fromconserved regions within human ERs, we cloned a
PCR fragment from oyster ovary. The amino acidsequence of the fragment showed high similarity
with those of human ERs. Subsequently the 5' and 3'
ends of this cDNA were obtained by 5' and 3' RACE.
The cDNA sequence of cgER is deposited as Accession number AB259818 (Matsumoto et al. 2007). It
contained an ORF of 1,455 bp encoding a polypeptide of 485 amino acids. The molecular mass of themature receptor protein, based on its deduced aminoacid sequence, is 53.6 kDa. The deduced protein
contains eight cysteine residues that constitute twozinc finger structures and P-box (CEGCKA) in the C
domain. The position of cysteine residues and otherresidues around them are conserved in mollusk ERs
including cgER.
The amino acid sequence of cgER protein revealed a high identity to mollusk ERs. The phylogenetic analysis indicated that there are three groupsof ER: ER-a, ER-b, and mollusk ERs and cgER is
most closely related to the other mollusk ERs (Figure2). This indicates that the cgER we isolated is anortholog of Aplysia ER, tcER1, and octopus ER. We
showed that cgER protein did not activate luciferase
expression in the presence of E2 in the mammalian cell-culture system. This is consistent with theresults on other mollusk ERs (Thornton et al. 2003,
Kajiwara et al. 2006, Keay et al. 2006). The cgER’s insensitivity to E2 is unlikely to be due to a lack of ER
protein, because the expressions of receptor proteinsin U2OS cells were confirmed by immunoblotting
analyses.
The cgER that we have isolated was not active
in mammalian cells in our reporter assay. However,the cgER mRNA was detected in all tissues tested,with the higher expression in the ovary. In addition,
The immunohistochemical study indicated that cgER
was mainly localized in the nuclei of follicle cells in
the oyster ovary (Figure 3a). The negative control
using preimmune rabbit serum instead of antiserum

22

Figure 2. Phylogenetic tree based on amino acid sequences ofERs. The unrooted phylogenetic tree was constructed by theneighbor-joining method after alignment. The sequence data
have been submitted to the GenBank database under the following accession numbers: Octopus (DQ533956), Thais (AB077032),
Aplysia (AY327135), Human-α (M12674), Chicken-α (X03805),
Mouse-α (M38651), Human-ß (AB006590), and Mouse-ß(U81451). Bootstrap values are calculated from 1,000 replicatesand expressed as percentages. Scale bar = 0.1 substitutions per
site.

showed no immunoreactivity (Figure 3b). In oviparous species, E2 mainly controls vitellogenesis mediated by ER (Wallace 1985). These results imply that
cgER could regulate the transactivation of the Vg
gene in the nuclei of follicle cells. Moreover, cgER
immunoreactivity was also detected in the nuclei ofoocytes (Figure 3a), suggesting that estrogens playvarious roles in regulating reproductive events. Our
results will facilitate further functional research tounderstand the roles of estrogens in the oyster.

MatsumotoDorange, G., and M. Le Pennec. 1989. Ultrastructural study of oogenesis and oocytic degeneration in
Pecten maximus from the Bay of St. Brieuc. Mar. Biol.
103:339–348.
Eckelbarger, K. J., and C. V. Davis. 1996. Ultrastructure of the gonad and gametogenesis in the easternoyster, Crassostrea virginica. I. Ovary and oogenesis.
Mar. Biol. 127:79–87.

Figure 3. Immunohistochemical staining for ER in ovary of oyster. A) The cgER immunoreactivity was visualized using diaminobenzidine. The cgER immunostaining was detected in the nuclei
of follicle cells (arrows) and oocytes (o). B) Preimmune rabbit
serum was used as a negative control. Scale bar = 25 µm.

Gauthier-Clerc, S., J. Pellerin, and J. C. Amiard. 2006.
Estradiol-17beta and testosterone concentrations inmale and female Mya arenaria (Mollusca bivalvia)
during the reproductive cycle. Gen. Comp. Endocrinol. 145:133–139.
Kajiwara, M., S. Kuraku, T. Kurokawa, K. Kato, S.
Toda, H. Hirose, S. Takahashi, Y. Shibata, T. Iguchi,
T. Matsumoto, T. Miyata, T. Miura, and Y. Takahashi.
2006. Tissue preferential expression of estrogen receptor gene in the marine snail, Thais clavigera. Gen.
Comp. Endocrinol. 148:315–326.
Keay, J., J. T. Bridgham, and J. W. Thornton. 2006.
The Octopus vulgaris estrogen receptor is a constitutive transcriptional activator: Evolutionary and functional implications. Endocrinology 147:3861–3869.
Krust, A., S. Green, P. Argos, V. Kumar, P. Walter,
J. M. Bornert, and P. Chambon. 1986. The chickenoestrogen receptor sequence: Homology with v-erbA
and the human oestrogen and glucocorticoid receptors. EMBO (Eur. Mol. Biol. Organ.) J. 5:891–897.

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66:1–3.
Botticelli, C. R., F. L. Hisaw Jr., and H. H. Wotiz.
1961. Estrogens and progesterone in the sea urchin(Strongylocentrotus franciscanus) and pecten (Pecten
hericius). Proc. Soc. Exp. Biol. Med. 106:887–889.
Chen, J. S., W. L. Cho, and A. S. Raikhel. 1994. Analysis of mosquito vitellogenin cDNA. Similarity withvertebrate phosvitins and arthropod serum proteins.
J. Mol. Biol. 237:641–647.

MatsumotoLi, Q., M. Osada, T. Suzuki, and K. Mori. 1998.
Changes in vitellin during oogenesis and effect of
estradiol-17ß on vitellogenesis in the Pacific oyster
Crassostrea gigas. Invertebr. Reprod. Dev. 33:87–93.
Mangelsdorf, D. J., C. Thummel, M. Beato, P.
Herrlich, G. Schutz, K. Umesono, B. Blumberg, P.
Kastner, M. Mark, and P. Chambon. 1995. The
nuclear receptor superfamily: The second decade.
Cell 83:835–839.
Matsumoto, T., A. M. Nakamura, K. Mori, I. Akiyama, H. Hirose, and Y. Takahashi. 2007. Oyster
estrogen receptor: cDNA cloning and immunolocalization. Gen. Comp. Endocrinol. 151:195–201.

23

Matsumoto, T., A. M. Nakamura, K. Mori, and
T. Kayano. 2003. Molecular characterization of
a cDNA encoding putative vitellogenin from the
Pacific oyster Crassostrea gigas. Zool. Sci. (Tokyo)
20:37–42.

Schwabe, J. W., L. Chapman, J. T. Finch, and D.
Rhodes. 1993. The crystal structure of the estrogen
receptor DNA-binding domain bound to DNA: How
receptors discriminate between their response elements. Cell 75:567–578.

Matsumoto, T., M. Osada, Y. Osawa, and K. Mori.
1997. Gonadal estrogen profile and immunohistochemical localization of steroidogenic enzymes in
the oyster and scallop during sexual maturation.
Comp. Biochem. Physiol. B 118:811–817.

Stefano, G. B., P. Cadet, K. Mantione, J. J. Cho, D.
Jones, and W. Zhu. 2003. Estrogen signaling at thecell surface coupled to nitric oxide release in Mytilus
edulis nervous system. Endocrinology 144:1234–
1240.

Okuno, A., W. J. Yang, V. Jayasankar, H. SaidoSakanaka, Do T. T. Huong, S. Jasmani, M. Atmomarsono, T. Subramoniam, N. Tsutsui, T. Ohira, I.
Kawazoe, K. Aida, and M. N. Wilder. 2002. Deduced primary structure of vitellogenin in the giant
freshwater prawn, Macrobrachium rosenbergii, andyolk processing during ovarian maturation. J. Exp.
Zool. 292:417–429.

Suzuki, T., A. Hara, K. Yamaguchi, and K. Mori.
1992. Purification and immunolocalization of a vitellin-like protein from the Pacific oyster Crassostrea
gigas. Mar. Biol. 113:239–245.

Osada, M., M. Harata, M. Kishida, and A. Kijima.
2004. Molecular cloning and expression analysis ofvitellogenin in scallop, Patinopecten yessoensis (Bivalvia, Mollusca). Mol. Reprod. Dev. 67:273–281.

Tsutsui, N., I. Kawazoe, T. Ohira, S. Jasmani, W. J.
Yang, M. N. Wilder, and K. Aida. 2000. Molecularcharacterization of a cDNA encoding vitellogenin
and its expression in the hepatopancreas and ovary
during vitellogenesis in the kuruma prawn, Penaeus
japonicus. Zool. Sci. (Tokyo) 17:651–660.

Osada, M., T. Takamura, H. Sato, and K. Mori. 2003.
Vitellogenin synthesis in the ovary of scallop, Patinopecten yessoensis: Control by estradiol-17 beta and thecentral nervous system. J. Exp. Zool. 299:172–179.
Osada, M., T. Unuma, and K. Mori. 1992. Purification and characterization of a yolk protein from thescallop ovary. Nippon Suisan Gakkaisha 58:2283–
2289.
Pakdel, F., S. Feon, F. Le Gac, F. Le Menn, and Y.
Valotaire. 1991. In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation withvitellogenin mRNA in rainbow trout. Mol. Cell.
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Thornton, J. W., E. Need, and D. Crews. 2003.
Resurrecting the ancestral steroid receptor: Ancientorigin of estrogen signaling. Science 301:1714–1717.

Umesono, K., and R. M. Evans. 1989. Determinantsof target gene specificity for steroid/thyroid hormone receptors. Cell 57:1139–1146.
Wallace, R. A. 1985. Vitellogenesis and oocyte
growth in non-mammalian vertebrates. In L. W.
Browder (ed.), Developmental biology, Vol. 1, Oogenesis, p. 127–177. Plenum Press, New York.
Yano, K., M. T. Sakurai, S. Watabe, S. Izumi, and S.
Tomino. 1994. Structure and expression of mRNAfor vitellogenin in Bombyx mori. Biochem. Biophys
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Pipe, R. K. 1987. Oogenesis in the marine mussel
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24

MatsumotoDisease Diagnostics and Treatment of Hemic Neoplasia in the Softshell Clam(Mya arenaria)
S. Anne Böttger and Charles W. Walker
University of New Hampshire
Department of Zoology
46 College Road
Durham, New Hampshire 03824
boettger at cisunix.unh.edu
Keywords: hemic neoplasia, leukemia, softshell clam
Abstract

The softshell clam (Mya arenaria) has been harvested commercially in the United States and wasa reliable source of income for local fisherman. Since the 1980s, the annual harvest has drastically declined, and the loss of seasonal and full-time jobs has been significant in New England and the Chesapeake Bay area. The situation is particularly severe in New Hampshire, where commercial clam digging
has been banned since 1951, though recreational harvesting still occurs.
Softshell clam hemic neoplasia (leukemia), one of the six most devastating bivalve diseases, is one ofvery few marine diseases that has been characterized at the molecular level. Yet almost nothing is known
about the environmental triggers of this disease. Leukemic clam hemocytes express a highly conserved
homolog for human p53 protein that is rendered nonfunctional by sequestration in the cytoplasm bymortalin when the latter protein is overexpressed.
Treatment of leukemic clam hemocytes with etoposide overcomes mortalin-based cytoplasmic
sequestration and promotes translocation of clam p53 protein from the cytoplasm to the nucleus. Cytotoxicity, DNA damage, and apoptosis of leukemic clam hemocytes follow. Since disease diagnosis,
treatment, and prevention are among the most significant variables in aquaculture, these results will aid
in generating and distributing information and finally developing treatments to potentially cure hemic
neoplasia.

Böttger and Walker

25

The Japanese Monitoring System on Shellfish Toxins and Development of Liquid
Chromatography–Mass Spectrometry of Lipophilic Toxins Associated with Diarrhetic
Shellfish Poisoning for Improvement of the Monitoring System
Toshiyuki Suzuki, Yutaka Okumura, and Takashi Kamiyama
Fisheries Research Agency
Tohoku National Fisheries Research Institute3-27-5 Shinhama
Shiogama, Miyagi 985-0001, Japantsuzuki at affrc.go.jp
Keywords: shellfish, diarrhetic shellfish poisoning, toxins
Abstract

Diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP) cause serious quality
assurance problems for bivalve industries in Japan. The toxicities of cultured bivalves are periodicallymonitored by mouse bioassay (MBA) at selected monitoring stations. When the toxicity of the bivalves
exceeds the quarantine levels (0.05 MU/g wet weight for DSP, 4.0 MU/g wet weight for PSP), harvestingceases. Shellfish harvesting is not resumed until testing indicates that the toxicity of the bivalves is below the quarantine levels on three successive weeks. Since establishment of the monitoring system forshellfish toxins, no human poisonings due to contaminated bivalves distributed on commercial markets
have been reported. In addition to the direct monitoring of bivalve toxicity, the occurrence and abundance of the planktonic dinoflagellates which cause DSP (Dinophysis spp.) and PSP (Alexandrium spp.)
are also monitored. This provides an early warning and risk assessment of the chances of toxicity developing in the bivalves.
Recently, we developed liquid chromatography–mass spectrometry (LC-MS) of lipophilic toxins inbivalves associated with DSP. Using a C8-silica reversed phase column and a mobile phase of aqueous
acetonitrile containing 2 mM ammonium formate and 50 mM formic acid, okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoyldinophysistoxin-1 (DTX3), pectenotoxin-1 (PTX1), pectenotoxin-2 (PTX2),
pectenotoxin-6 (PTX6), pectenotoxin-2 seco-acid (PTX2sa), yessotoxin (YTX), and 45-hydroxyyessotoxin(45-OHYTX) in bivalves were quantified by LC-MS. Approximately 200 bivalve samples collected fromvarious production areas in Japan were analyzed by LC-MS. Comparison of the quantitative results
obtained for bivalve samples between LC-MS and MBA showed that many samples assayed as being
below the quarantine level (0.05 MU/g whole tissues) by MBA were quantified as exceeding the quarantine level by LC-MS. This difference was due to YTXs, which are poorly detected by MBA. The results
indicate that LC-MS is a better method than MBA in terms of sensitivity and accuracy to quantify known
lipophilic toxins including YTXs.
Introduction
Diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP) cause serious quality
assurance problems for the bivalve industries in Japan; however, no human poisonings due to contaminated bivalves with DSP or PSP toxins distributedon commercial markets have been reported since
establishment of the monitoring system for shellfish
toxins in 1979. This indicates that the monitoring
system is successful. The present Japanese shellfish
toxin monitoring system is described in this report.
DSP is a condition resulting from ingestion of

26

shellfish contaminated with lipophilic toxins (Yasumoto et al. 1978). Three lipophilic toxin groups (Figure 1) differing in basic structures have been associated with DSP (Yasumoto et al 1985, Yasumoto and
Murata 1993). The most important toxins responsible for diarrhea are okadaic acid (OA) analogues.
These toxins have been shown to be potent phosphatase inhibitors (Bialojan and Takai 1988), a property
which can cause inflammation of the intestinal tractand diarrhea (Terao et al. 1986). OA analogues havealso been shown to have tumor-promoting activity

Suzuki et al.

bivalves were analyzed by our recently developed
LC-MS method (Suzuki et al. 2005). A comparisonof the quantitative results obtained for these bivalve
samples between LC-MS and mouse bioassay (MBA)was carried out to investigate the applicability of thisLC-MS method for routine monitoring of lipophilic
toxins in Japanese bivalves.

Materials and Methods

Figure 1. Structure of typical lipophilic toxins found in Japanesebivalves. Okadaic acid is a rare toxin in Japanese bivalves.

(Fujiki et al. 1988). In contrast to OA analogues, it
has been suggested that the risk assessment of theother two toxin groups for mammals may be relatively low compared to OA analogues when they are
injected orally (Ogino et al. 1997, Aune et al. 2002,
Miles et al. 2004).
European authorities have set a higher control
limit for the yessotoxins (YTXs) than OA analogues.
The different functional toxicities and control limits for the three toxin groups require a method forshellfish testing that can quantify each toxin groupseparately. Recently, we developed liquid chromatography–mass spectrometry (LC-MS) of lipophilic
toxins in bivalves associated with DSP (Suzuki et al.
2005).
In 2003 in Japan, harvesting of scallops (Patinopecten yessoensis) and mussels (Mytilus galloprovincialis and M. coruscus) ceased due to contamination with lipophilic toxins. During that event,approximately 200 bivalve samples were collectedfrom production areas and lipophilic toxins in the

Suzuki et al.

Samples. Approximately 200 bivalve samples werecollected from six production areas during a DSP
outbreak from May to September 2003 (Figure 2).
Scallops were collected in several production sites in
Hokkaido (5 sites), Aomori (10 sites), Iwate (8 sites),and Miyagi (12 sites). Mediterranean mussels (M.
galloprovincialis) were collected in Aomori (2 sites),
Iwate (1 site), and Miyagi (3 sites). Mussels (M. coruscus) were collected in Akita (1 site), Yamagata(2 sites), and Niigata (1 site).
Hepatopancreas (HP) samples from more than
10 individuals collected from the same monitoringsite on the same date were combined and homogenized. An aliquot of the homogenate was immediately analyzed by the official Japanese testing
method for DSP by MBA (Japanese Ministry of
Health and Welfare 1981). The remainder of thehomogenate was kept frozen at –30°C until used forextraction of toxins for LC-MS.

Figure 2. Production area of bivalves where harvesting wasprohibited due to DSP events in 2003.

27

Extraction of toxins for LC-MS. The remainder (ca.
5 g) of the homogenates tested by MBA was extracted with nine times volume of 90% methanol and theextracts were centrifuged at 3,000 rpm for 5 min. An
aliquot of each supernatant was passed through a 0.5µm filter (Millipore Corp., Billerica, Massachusetts)for direct injection into the LC-MS.
LC-MS. A Hewlett-Packard Model 1050 series liquid
chromatograph was coupled to a Finnigan MAT
SSQ-7000 instrument (San Jose, California) equippedwith an atmospheric pressure electrospray ionization (ESI) interface. Separations were performedon Quicksilver cartridge columns (50 mm x 2.1 mm
internal diameter) packed with 3 µm Hypersil-BDSC8 (Keystone Scientific, Bellefonte, Pennsylvania)
maintained at 35°C. Eluent A was water and B was
acetonitrile/water (95:5), both containing 2 mM ammonium formate and 50 mM formic acid (Suzuki etal. 2005). Gradient elution from 20% to 100% B was
performed over 10 min, then held at 100% B for 15
min for LC separation. Re-equilibration was takenfor 13 min with 20% B. The flow rate was 0.2 mL/
min and the injection volume was 5 µL.
Using a divert valve, LC eluent between 9 and
15 min and between 19 and 22 min were introduced
into an ESI interface. The other eluent fractions
were discarded to reduce contamination of the ion
source and MS detector by bivalve matrices. Toxins
were detected using selected ion monitoring (SIM)of negatively charged ions for [M-H]- of OA (m/z803.5), DTX1 (m/z 817.5), DTX3 (m/z 1055.7), PTX2
seco acid (m/z 875.5), and for [M+HCOOH-H]- of
PTX1 (m/z 919.5), PTX2 (m/z 903.5), PTX6 (m/z933.5), and for [M-2Na+H]- of YTX (m/z 1141.5),
45-OHYTX (m/z 1157.5). The SIM scan width foreach toxin was 0.3 U (unit mass). The voltage on
the ESI interface was maintained at approximately
4.5 kV. The temperature of the heated capillary was
200°C. High-purity nitrogen was used as a sheathgas (nebulizer gas) at an operating pressure of 480
kPa and auxiliary gas flow at 5 units.
MBA. MBA was carried out according to the official testing method in Japan (Japanese Ministryof Health and Welfare 1981). Whole tissues andseparated HP of each sample were weighed and theweight percentage (w/w%) of the HP was calculated. The homogenate of the HP was extracted withacetone three times. After removal of the acetone byevaporation, the aqueous suspension was extractedwith diethyl ether. The ether solution was back-28

washed with distilled water and evaporated. The
residue was suspended in 1.0 mL of 1% Tween-60
saline solution for each 20 g whole tissue equivalent.
An aliquot (1.0 mL) of this solution corresponding to
20 g of the whole tissues was injected intraperitoneally into each of three mice. When more than two
mice die within the 24 hour observation period, thetoxicity of the extracts is defined as greater than 1
mouse unit (MU) and the whole contents are definedas more than 0.05 MU/g whole tissues, the current
Japanese quarantine level.
Comparison of mouse toxicities obtained by LCMS and MBA. Total mouse toxicities of samples
quantified by LC-MS were compared to the MBA
results. The conversion factors to the mouse toxicities of OA, DTX1, DTX3 (7-O-palDTX1), PTX1, PTX2,
PTX6, YTX, and 45-OHYTX are 4.0, 3.2, 5.0, 5.0, 4.6,
10, 2.0, and 10 µg/MU, respectively (Yasumoto et al.
1995, Satyake et al. 1996). Mouse toxicities obtainedby MBA are shown between minimum and maximum toxicities according to the dilution factors of bivalve extracts (e.g., 0.05–0.1 MU/g). Average values
obtained by the MBA were used for the comparison
to the LC-MS results.

Results and Discussion

Figure 3 shows the SIM LC-MS chromatogram
obtained from a mixture of standard toxins. A cleanchromatogram was obtained, showing discrete
peaks for all toxins. Although YTX, PTX1, and PTX6
coeluted, this was not problematic for the quantification of these toxins due to the SIM separation.
Figure 4 shows a comparison of mouse toxicities
quantified by LC-MS and MBA for the 196 scallopand mussel samples. Ninety-six samples were quantified by both LC-MS and MBA as being below thequarantine level. Thirty-three samples were above
the quarantine level by both LC-MS and MBA. Six
samples tested as exceeding the quarantine level
by MBA were quantified by LC-MS as being below
the quarantine level. As the toxicities of these six
samples were between 0.05 and 0.1 MU/g by MBA,which is the lowest level measurable by the MBA,
false positives due to free fatty acids in the samples
are suspected (Tagaki et al. 1984, Lawrence et al.
1994, Suzuki et al. 1996). Sixty-one samples as exceeding the quarantine level by LC-MS were quantified by MBA as being below the quarantine level.
The Japanese MBA protocol includes a partition of bivalve extracts between water and diethyl
ether to remove water soluble saxitoxin analogues.

Suzuki et al.

Figure 3. SIM LC-MS chromatogram for a standard mixture (100ng/mL) of lipophilic toxins. Toxins were detected using ESIand SIM for [M-H]- of OA (m/z 803.5), DTX1 (m/z 817.5), DTX3
(m/z 1055.7), and for [M+HCOOH-H]- of PTX1 (m/z 919.5), PTX2
(m/z 903.5), PTX6 (m/z 933.5), and for [M-2Na+H]- of YTX (m/z1141.5).

It is reported that a major portion of the YTXs are
partitioned into the aqueous fraction (Ramstad et al.
2001) and are therefore not measured by MBA. The
discrepancies are probably caused by YTXs that arepoorly detected by MBA. These results indicate that
the LC-MS is definitely a better method than MBA,in terms of the sensitivity and the accuracy, to quantify known lipophilic toxins including YTX. It isnoteworthy that the numbers of samples exceeding
the quarantine level increase if MBA is replaced with
LC-MS in routine monitoring of toxins in Japanesebivalves (Figure 4). This impact will be huge to scallop industries because a dominant toxin in scallops
is YTX.
The risk assessment of YTX to mammals isthought to be lower than the other toxin groups(Ogino et al. 1997, Aune et al. 2002), and the European Union (EU) recently set a higher control limit for
the YTXs than OA analogues. When the LC-MS isapplied to the toxin monitoring in Japanese bivalves,an adequate control limit for the YTXs as proposedby the EU should be considered to decrease economic impacts to scallop industries.
A simple and rapid LC-MS method without
time-consuming cleanup of samples for the quantification of lipophilic toxins in Japanese bivalves
was proposed in the present study. Approximately
200 bivalve samples collected in various production areas in Japan have been tested by the method.
Comparisons between LC-MS and MBA results forthese samples demonstrate that the present LC-MS
method is applicable to routine monitoring of lipophilic toxins in Japanese bivalves. When the different control limit for each toxin group is required as
proposed by EU, the LC-MS method is a suitable
alternative to MBA.

Acknowledgments

Figure 4. Comparison of total mouse toxicities obtained by
LC-MS and MBA for the same scallop and mussel samples (n =sample numbers in the square).

Suzuki et al.

The research was funded through a research
project of Ministry of Agriculture, Forestry, andFisheries of Japan: Research Project for Utilizing
Advanced Technologies in Agriculture, Forestry and
Fisheries (No. 1504). We express our gratitude to thecollectors of the bivalve samples.

29References

Aune, T., R. Sorby, T. Yasumoto, H. Ramstad, and T.
Landsverk. 2002. Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon
40:7782.
Bialojan, C., and T. Takai. 1988. Inhibitory effectof a marine-sponge toxin, okadaic acid, on protein
phosphatases. Specificity and kinetics. Biochem. J.
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Fujiki, H., M. Suganuma, H. Suguri, S. Yoshizawa,
K. Takagi, N. Uda, K. Wakamatsu, K. Yamada, M.
Murata, T. Yasumoto, and T. Sugimura. 1988. Diarrhetic shellfish toxin, dinophysistoxin-1, is a potent
tumor promoter on mouse skin. Jpn. J. Cancer Res.
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Food Sanitat. Res. 7:60.
Lawrence, J. F., R. K. Chadha, W. M. Nimal Ratnayake, and J. F. Truelove. 1994. An incident of elevatedlevels of unsaturated free fatty acids in mussels from
Nova Scotia and their toxic effect in mice after intraperitoneal injection. Nat. Toxins 2:318–321.
Miles, C. O., A. L. Wilkins, R. Munday, M. H. Dines,
A. D. Hawkes, L. R. Briggs, M. Sandvik, D. J. Jensen,
J. M. Cooney, P. T. Holland, M. A. Quilliam, A. L.
MacKenzie, V. Beuzenberg, and N. R. Towers. 2004.
Isolation of pectenotoxin-2 from Dinophysis acuta
and its conversion to pectenotoxin-2 seco acid, andpreliminary assessment of their acute toxicities.
Toxicon 43:1–9.
Ogino, H., M. Kumagai, and T. Yasumoto. 1997.
Toxicologic evaluation of yessotoxin. Nat. Toxins
5:255–259.

Suzuki, T., T. Jin, Y. Shirota, T. Mitsuya, Y. Okumura,and T. Kamiyama. 2005. Quantification of lipophilic
toxins associated with diarrhetic shellfish poisoningin Japanese bivalves by liquid chromatography-mass
spectrometry and comparison with mouse bioassay.
Fish. Sci. 71:1370–1378.
Suzuki, T., R. Yoshizawa, T. Kawamura, and M.
Yamasaki. 1996. Interference of free fatty acids fromthe hepatopancreas of mussels with the mouse bioassay for shellfish toxins. Lipids 31:641–645.
Takagi, T., K. Hayashi, and Y. Itabashi. 1984. Toxic
effect of free unsaturated fatty acids in the mouseassay of diarrhetic shellfish toxin by intraperitoneal
injection. Bull. Jpn. Soc. Sci. Fish. 50:1413–1418.
Terao, K., E. Ito, T. Yanagi, and T. Yasumoto. 1986.
Histopathological studies on experimental marine
toxin poisoning I: Ultrastructural changes in thesmall intestine and liver of suckling mice induced
by dinophysistoxin-1 and pectenotoxin-1. Toxicon
24:1141–1151.
Yasumoto, T., M. Fukui, K. Sasaki, and K. Sugiyama.
1995. Determinations of marine toxins in foods. J.
Am. Organ. Agr. Chem. 78:574–582.
Yasumoto, T., and M. Murata. 1993. Marine toxins.
Chem. Rev. 93:1897–1909.
Yasumoto, T., M. Murata, Y. Oshima, M. Sano, G. K.
Matsumoto, and J. Clardy. 1985. Diarrhetic shellfish
toxins. Tetrahedron 41:1019–1025.
Yasumoto, T., Y. Oshima, and M. Yamaguchi. 1978.
Occurrence of a new type of shellfish poisoning
in the Tohoku district. Nippon Suisan Gakkaishi
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Ramstad, H., S. Larsen, and T. Aune. 2001. Repeatability and validity of a fluorimetric HPLC methodin the quantification of yessotoxin in blue mussels(Mytilus edulis) related to the mouse bioassay. Toxicon 39:1393–1397.
Satake, M., K. Terasawa, Y. Kadowaki, and T. Yasumoto. 1996. Relative configuration of yessotoxin
and isolation of two new analogs from toxic scallops.
Tetrahedron Lett. 37:5955–5958.

30

Suzuki et al.

Bivalves as Biofilter: Efficient, Profitable, and Tasty as Well!
Muki Shpigel
Israel Oceanographic and Limnological Research
National Center for Mariculture
P.O. Box 1212
Eilat, Israel
shpigelm at agri.huji.ac.il
Keywords: bivalves, commercial culture, polytrophic
Abstract

Bivalve production has increased dramatically worldwide in the last three decades. According to the
Food and Agriculture Organization of the United Nations, fishery and aquaculture production increased
fourfold, reaching almost 12 million metric tons in 2000.
The choice of a particular bivalve species for commercial culture involves considerations such as fast
growth rate, low food conversion ratio, resistance to pests, and tolerance to a wide range of environmental conditions. The technology for its reproduction and culture should be straightforward and userfriendly for the growers, and the mollusk should meet market demands with respect to appearance, taste,smell, texture, processing considerations, and market behavior. Profitability depends on yield per unit ofarea, grow-out time, harvest frequency, farm-gate price, and the cost of waste treatment.
Bivalves can be cultured by sea ranching or in land-based facilities. Sea ranching usually involves
bottom culture, rack culture, or suspended culture. In the open sea, the bivalves are vulnerable to weather conditions, predation, red tide, and poaching. Because they are generally cultured close to shore, they
are also subjected to urban pollution. As filter feeders, bivalves can accumulate high concentrations of
toxic and pathogenic material, which can affect the economics of bivalve culture. Land-based facilities,
wherein bivalves are cultured in ponds, tanks, or indoor hatcheries and nurseries, are safer because thequality of the incoming water can be controlled. Bivalve monoculture in such systems is of doubtful
profitability due to the high costs of construction, the need for highly trained technicians, water pumping, food (microalgae) supply, and waste control.
Polytrophic culture in integrated systems holds much greater economic promise because it saves
resources such as feed and water purification, diversifies the farm’s market products, allows intensification and optimization, and is environmentally friendly. In this system, fishpond effluent, rich in dissolved nutrients, drains through an earthen sedimentation pond. The dissolved nutrients, coupled withthe high incidence of solar radiation, generate an extremely high phytoplankton production, mainly of
diatoms, that supports the growth of bivalves on the bottom of the sedimentation pond.
This presentation summarized state-of-the-art research and development in the use of bivalves as
biofilters and as safe, valuable by-products in land-based integrated aquaculture systems, and thus as avalid alternative to open sea monoculture.

Shpigel31Considerations on the Fine-scale Topography in Sand Flats, Habitat Heterogeneity,
and Refugia for Clams
Hajime Saito, Hideyuki Takahashi, Akihiko Matsuda, and Hisami Kuwahara
Fisheries Research Agency
National Research Institute of Fisheries Engineering
Ibaraki 314-0408, Japan
theora at affrc.go.jp
Yuka Ishihi, Tomoko Sakami*, and Junya Higano
Fisheries Research Agency
National Research Institute of Aquaculture
Mie 516-0193, Japan*Present address:
Fisheries Research Agency
Tohoku National Fisheries InstituteMiyogi 985-0001, Japan
Keywords: clams, topography, habitat, refugia
Abstract

We discuss the use of the fine-scale topography on sand flats as an explanatory variable for spatial
patterns of clams. Topography on a sand flat is a fingerprint of sediment-water interactions, although
its interpretation has not yet been systematically confirmed by fluid mechanics. We attempted to derivesome implications about the effect of hydrodynamics on clam populations from the correspondence between topography and spatial patterns of clams. Individual numbers of two common clam species, Manila clams (Ruditapes philippinarum) and surf clams (Mactra veneriformis) were compared. For the meanlog transformed individual numbers of Manila clams, effects of position between sandbars and within asandbar were significant, while the effect of ripples and the interaction term were not. Within the middle
sandbar, the mean individual number in the trough was greater than in other positions. Between threecrests of sandbars, the mean decreased seaward.
Individual numbers of surf clams did not show significant variations within the middle sandbar.
In the test for the effects of position between sandbars and ripples, the interaction term was significant.
Vertical profiles of ripples showed that ripples on the trough of the middle sandbar were concave upwards having rounded tops. Ripples on other positions in the same sandbar were convex and slightly
skewed seaward. On crests of sandbars, ripples in the landward sandbar were concave upwards while
ripples in other sandbars were convex and slightly skewed. The small abundance of Manila clams corresponded with occurrence of convex ripples that suggest unidirectional currents mixed with wave actions.

Introduction

Any element of habitat structure that diminishes
the likelihood that an organism on or near it will die
is a spatial refuge for that organism, and is crucial tounderstanding the interactions between the mortality source and the distribution and abundance ofrefugia (Woodin 1978). Spatial patterns of benthic
organisms on sand flats are a result of physical andbiotic processes. Recruitment of benthic organisms and their larvae through bedload transport

32

has crucial roles in recovery processes of disturbed
communities. Hydrodynamic processes have asignificant effect on larval settlement (Jonsson et al.
1991, Butman and Grassle 1992, Grassle et al. 1992a,
1992b, Snelgrove et al. 1993, Roegner et al. 1995,
Snelgrove et al. 1998). Adults and juveniles are also
moved through passive transport and active drifting(Sigurdsson et al. 1976, Beukema and Devlas 1989,
Hiddink et al. 2002).

Saito et al.

To establish sound strategies for managementof aquaculture and fishing grounds on tidal flats, it
is important to understand the relationship between
spatial heterogeneity of hydrodynamic conditions
and the distribution of refugia that have a significant
effect on growth and degradation of clam aggregations. In some cases, refugia emerge after alternationof the sediment surface structure. For example, it
was suggested that shell debris has a role in increasing and maintaining biodiversity by reducing disturbance regimes (Hewitt et al. 2005). Dense tube bedsof polychaetes persist for several years and offer a
refuge wherein infaunal diversities are greater thanthose of more hydrodynamically rigorous areas(Bolam and Fernandes 2002). Local aggregations ofManila clams (Ruditapes philippinarum) were formed
on a tidal flat in Kumamoto, Japan, after artificial
capping with coarse sand.
Indices that identify location and scale of refugia
on sand flats, if developed, would be helpful in the
management of fishing grounds on tidal flats. Grainsize distribution can be used as a marker for changes
in the physical sedimentary conditions for benthic
organisms on the sand flat because grain size is an
important factor influencing benthic communities
and is often correlated with other sediment properties (Zajac and Whitlatch 2003). On the other hand,
there are diverse topographic patterns with various
spatial scales on sand flats. Roughness of the bottom
produced by grain size distribution of the substrateis the smallest structure, on the order of 0.1–1 mm.
Ripples and dunes on the order of 1–10 cm areoften seen on the sandy bottom. Multiple sandbars,
ridges, and runnels are large structures on the orderof 10 m developing on sand flats exposed to strong
wave forces.
Besides spatial structures formed by physicalprocesses, biological and human activities modify
the surface structure of sand flats (e.g., mounds,
tubes, mats, pits by predation, digging, and humanfootprints). These structures produce shaded andexposed sides to wave and current forces, and thisdifferentiation in the strength of physical disturbance may be an important factor that creates habitat
heterogeneity on sand flats. Multiple sandbars areapparent and their shapes are easily captured using
topographical survey techniques.
Roughness of the sand surface can be estimatedfrom the grain size distribution of the sediment. It
is difficult to capture the real shape of ripples in thefield. Wave length and amplitude of ripples can beobtained with replicate measurements in the field,

Saito et al.

but their detailed shapes cannot be included in thedata.
Sand flat topography provides a fingerprint of
sediment-water interactions, although its interpretation has not yet been systematically confirmed by
fluid mechanics. In tentative bed state definitions,wave ripples have concave-upwards two-sided (ormore) slip faces, and there is vortex shedding fromthe tops when sharp crested with top angles approximating the angle of repose, whereas current dunes
have approximately triangular cross sections but
often convex-upwards sides, lee sides commonly at
the angle of repose and vortex shedding (Kleinhans
2005). There are also mixed-flow ripples; bedforms
with characteristics of both wave and current bedforms. Thus bedforms provide important information on the physical state in the benthic boundary
layer, relative contribution of waves and currents,
and disturbance for the benthic community.
In this study, we discuss the use of the fine-scale
topography on sand flats as an explanatory variable
for spatial patterns of clams. Previous literature
has correlated benthic assemblages with physicochemical factors. Many of them included sedimentcharacteristics and water qualities, but less attention
has been paid to hydrodynamics, whereas they are
critical in the settlement and survival of young benthic organisms. We tried to derive some implications
about the effect of hydrodynamics on clam populations from the correspondence between the topography and the spatial pattern of two common clams,
Manila clams and surf clams (Mactra veneriformis).

Materials and methods

Matsunase Beach is an intertidal sand flat at theeastern margin in the mouth of the Kushida River(lat 34°36’N, long 13°35’E), Matsusaka City, Japan(Figure 1). The catchment area of the river is 436
km2. The sand flat is covered with various sizesof substrate material from fine sand to cobbles. In
winter, the shallow subtidal area is filled with thousands of bamboo rods with nets extended between
them for mariculture. Seaweeds grow densely in thesubtidal zone and in deep troughs of sandbars thatemerge at low water during spring tides. Bivalvespredominate the macrofauna on the sand flat. Other
taxonomic groups are less abundant than bivalves.
Major bivalve species are Manila clams, surf clams,and horse mussel (Musculista senhousia).
We established sampling stations along a linetransect from the coastal sand dune to the pointof low water during spring tide. Location of the33

line transect was determined to make elevationalchanges over sandbars along it as contrasting as
possible. On 6 September 2006, land elevations at
haphazardly chosen points scattered along the linetransect were measured with a VRS-RTK receiver(Trimble 5800, Trimble Navigation Ltd., Sunnyvale,
California). From the results of this survey, the land
shape of the sand flat was described in detail andit was shown that the upper intertidal zone was a
gradual slope while multiple sandbars developedin the lower intertidal zone (Figure 2). Wave lengthof the sandbar was equal to 30–40 m, and amplitude
was from 20 to 60 cm. Sampling was conducted on 7
and 8 September 2006 at stations established on the
crests of three discrete sandbars (landward, middle,
and seaward sandbar) and three other locations(trough, onshore side slope, and offshore side slope)on the middle sandbar. Two additional stations (5and 6) were established on the foreshore gradualslope (Figure 2).
At each station, photographs of the sand surface enclosed by a 60x60 cm2 unit made from plastic
pipes placed on the sand surface were taken using
two digital cameras (Coolpix 7900, Nikon Co.). At
the same time, a compass and six circular markers were included in the pictures to be used in thecalibration process. The cameras were mounted inparallel and looked downward under a tripod. The
distance between the centers of the camera lenses
was 23 cm. The height of the lenses from the sandsurface was approximately 85 cm. After taking
pictures, 6 sediment samples were collected using
96x46x46 mm rectangular stainless quadrats haphazardly placed in the square unit. Three replicateswere obtained from the crests of sand ripples andanother three replicates from troughs. Sediment
samples were sieved through a 1.0 mm mesh screen,and materials retained on it were fixed in a 5% formaldehyde solution.
Fixed specimens were rinsed with water inthe laboratory and organisms were sorted under abinocular microscope. Shell lengths of specimens ofManila clams and surf clams were determined, andthe individuals were placed in 0.25 mm intervals
and counted. Shell length distribution was evaluated using the Solver function in Microsoft Excel 2000
to fit the summation of normal distribution curves
using a published cohort-analysis program (Aizawa
and Takiguchi 1999). In this program, fitness of themodel was evaluated employing the maximum likelihood method. Intersections of normal distribution
curves were recognized as the border of different

34

Figure 1. Locality of Matsunase Beach tidal flat and the positionof the line transect for this study.

Figure 2. Vertical profile of the line transect and positions ofsampling stations.

Saito et al.

size groups, and the individual number of separated
size groups was counted. In order to focus on therelationship between the spatial pattern of young
clams and the latest topography, older individuals
were eliminated from further analyses.
The numbers of clams in each replicate unit
were log-transformed. Two-way analysis of variance(ANOVA) was calculated using the “aov” functionin R, a high level language and an environment fordata analysis and graphics distributed by the Comprehensive R Archive Network. The null hypotheses
tested were:• There was no difference in individual numbers
		 between troughs and crests of sand ripples• There was no variation in individual numbers 				 between locations of sandbars including the		 two extra stations in the foreshore slope• There was no variation in individual numbers 				 between positions in the middle sandbar.
To test these hypotheses, we employed several
two-way ANOVA models. One model was composed of two orthogonal fixed factors, the effect ofripple (R, n = 2) crossed with the effect of locationof sandbars (B, n = 5). The other model was composed of two orthogonal fixed factors, the effect ofripple (R, n = 2) crossed with the effect of position
within the middle sandbar (P, n = 4). Homogeneityof variance between sampling stations was tested
with Cochran’s test for variance outliers (Kanji 2006).
Post hoc multiple comparisons were performed todetect subgroups between sampling stations using
Tukey HSD.
The three-dimensional shape of the sand surface enclosed with the square unit in the field wasvisualized from several stereo pictures (a stereo pair)with image analysis software (PI-3000 version 3.0,
Topcon Co., Tokyo, Japan). This procedure involved
selecting six circular targets manually in each of theimages to determine the initial estimates of locations,and the process was then completed by the automatic estimation of all remaining locations. Locations
were estimated from the similarity of pixel patterns,
parallax in a stereo pair, and the calibration file foradjusting the lens distortion. The calibration file
was prepared using PI-Calib software (Topcon Co.).
The shape of the sand surface was visualized as atriangulated irregular network (TIN) produced in
5 mm grids. Inclination of the sand surface against
the camera angle was removed by subtracting theheight of the regressed plain from the corresponding
apex of TIN. Then the vertical profile of ripples was

Saito et al.

sectioned along an assigned line rectangular to thecrest line of ripples.

Results

The cohort analyses revealed that there wereseveral size groups in Manila clams and surf clams(Figure 3). Individual numbers of Manila clams
smaller than 17 mm and surf clams smaller than 5
mm in shell length were enumerated. The border for
Manila clams, 17 mm in shell length, was adopted
to eliminate a few large individuals. Although it
is likely that there are two cohorts in Manila clams
smaller than 17 mm in shell length (shown as S and
M in Figure 3), they were pooled in further analyses
because the border between them was very narrow
and the distinction was not clear. The distinction
between the smaller two cohorts of surf clam was
not clear (shown as S and M in Figure 3), but we estimated that there were at least two cohorts because
the size distribution would be strongly skewed to

Figure 3. Shell length distributions of Manila clams (Ruditapes
philippinarum), above, and surf clams (Mactra veneriformis), below. Curves fitted to the shell length histograms are the resultsof cohort analyses.

35

Table 1. Results of two-way ANOVAs on log-transformed individual numbers of Manila clams (Ruditapes philippinarum), shell length< 17 mm, and surf clams (Mactra veneriformis), shell length < 5 mm. Effects of spatial positions within the middle sandbar (P), locations between crests of three discrete sandbars and two stations in the foreshore slope (B) and the difference between troughs andcrest of sand ripples (R) were included in the model as fixed factors.

Df

Sum sqMean sq

F value

Pr(>F)

Significance

3
1
3
16

3.188
0.003
0.016
0.724

1.063
0.003
0.005
0.045

23.479
0.071
0.120

0.000
0.793
0.947

***		
ns
ns

3
1
3
16

0.186
0.016
0.078
0.764

0.062
0.016
0.026
0.048

1.298
0.332
0.542

0.309
0.572
0.660

ns
ns
ns

4
1
4
20

3.328
0.000
0.016
0.486

0.832
0.000
0.020
0.024

34.208
0.004
0.822

0.000
0.949
0.527

***
ns
ns

4
1
4
20

1.327
0.000
0.562
0.845

0.332
0.000
0.141
0.042

7.855
0.000
3.327

0.001
0.991
0.030

***
ns
*

R. Philippinarum
P
R
PxR
Residuals

M. veneriformis
P
R
PxR
Residuals

R. philippinarum
B
R
BxR
Residuals

M. veneriformis
B
R
BxR
Residuals

the right if regarded as single cohort. In surf clams,
there was another size group of large individuals,
larger than 22 mm in shell length, that was eliminated in the following analyses.
Two-way ANOVA to test the effects of ripples(R) and positions within the middle sandbar (P)showed that, for Manila clams, the effect of positions was strongly significant (F = 23.479, p < 0.001)
whereas the effect of ripples was not significant(Table 1). Neither effect was significant in the caseof surf clams. Tests on the effects of ripples andsandbars (including the two extra stations in theforeshore slope) revealed that the effect of sandbars
was strongly significant for Manila clams (F = 34.208,
p < 0.001) and surf clams (F = 7.855, p < 0.001), andinteraction between ripples and sandbars was significant in the case of surf clams (F = 3.327, p < 0.05).
Cochran’s test for variance outliers found that the36

null hypothesis about homogeneity of variance presupposed in ANOVA was retained in all tests above(p > 0.05).
In multiple comparisons, replicate samples fromtroughs and crests of ripples were not separated if
the effect of ripples and the interaction term were
not significant in ANOVAs. Tukey HSD revealed
that the mean log-transformed individual numberof Manila clams in the trough of the middle sandbar
was significantly greater than those of other positions (Figure 4). Subgroups detected were shownalphabetically. Surf clams did not show significant
variations in the mean log-transformed individual
numbers among four positions in the middle sandbar. Multiple comparisons between sandbar crests,
including the two extra stations in the foreshore
slope, revealed that the mean log-transformed individual number of Manila clams in the landward

Saito et al.

sandbar was prominently greater than those in otherstations. Mean log-transformed individual numbersof surf clams were overlapped among five stations.
The interaction term B×R was significant in
ANOVA though the difference between troughs andcrests of ripples in the same unit was not significant
for all stations. As an example, a three-dimensional
image and the contour plot of sand ripples on the
crest of the middle sandbar were superimposed in
Figure 5. Vertical profiles of ripples showed thatripples on the trough of the middle sandbar wereconcave upward having rounded tops (Figure 6).
Ripples of this type have wide and relatively flat
troughs. Ripples at other positions on the same
sandbar were convex upward and slightly skewed
seaward having flat tops. Those had narrow troughs
compared to ripples that concave upward. Referring to the tentative bed state definition in Kleinhans(2005), the former ripples were classified as wave
ripples while the latter were classified as mixed-flow
ripples.
Wave length of ripples in the trough, onshore
slope, crest, and offshore slope of the middle sandbar were approximately 12, 10, 8, and 8 cm, respectively, and heights of the ripples were approximately
2.0, 1.0, 1.0, and 0.7 cm, respectively. There wereoverlaps of ripples having different phases on theonshore and the offshore slopes. Along the crestsof sandbars, ripples on the landward sandbar wererather concave upward while ripples on other sandbars were convex upward (Figure 7). Ripples wereslightly skewed seaward.
Wave lengths of ripples on the foreshore slope
and the crest of the landward sandbar were approximately 11, 12, and 9 cm, respectively, and heights ofripples were approximately 1.0, 1.3, and 1.3, respectively. Ripples on the crest of the seaward sandbar
had very flat shapes with height of only about 0.7
cm, and wave lengths were irregular, ranging from
7 to 9 cm. From these data, steepness of ripples as
defined by height/wave length could be calculated.
Steepness of ripples in the trough, onshore slope,
crest, and offshore slope of the middle sandbar were0.17, 0.10, 0.13, and 0.09, respectively. Steepness ofripples in the foreshore slope and the crest of thelandward sandbar were 0.09, 0.11 and 0.14, respectively. Steepness of ripples on the crest of the seaward sandbar ranged from 0.08 to 0.10.

Discussion

In bottom culture systems, large clams survive
better than small clams (Gosling 2003). Retention

Saito et al.

Within the middle sandbar

Between sandbars

Figure 4. Mean log-transformed individual numbers of Manila
clams (Ruditapes philippinarum), shell length < 17 mm, above,and surf clams (Mactra veneriformis), shell length < 5 mm, below.
Individual numbers were compared at four positions in the
middle sandbar (left), and the crests of three sandbars and two
stations on the foreshore slope. Troughs and crests of ripples
were compared separately for surf clams because the interaction
term was significant in ANOVA.

37

rate of Manila clams of 26–28 mm shell length in thefield experiment decreased when bottom friction
velocity was greater than 4 cm/s (Kakino 2000). It is
likely that disturbance caused by waves and currents has critical effects on clam survival. Thread
secretion by young Manila clams to attach to large
objects such as stones and shells is an adaptation toturbulent conditions on the bottom. However, clams
attached to the objects would be passively transported under strong wave conditions that remove theobjects or break the threads.
Recovery rates of benthic communities from
impacts of disturbances on sand flats are dependent on spatial scales of disturbed sites (Thrush etal. 1996). Generally, a larger disturbed site needs
a longer period to recover its community structure
than ambient areas. Community recovery occurred
after 4.5 months in 1 m2 disturbed plots on a sandflat (Zajac and Whitlatch 2003). Recovery times for
disturbances of larger areas, 100 m2 to 1 km2 in size,
ranged from about 1 to 2 years (Zajac 1999). Wave
length of multiple sandbars in this study was 30–40
m, and crests of sandbars may be exposed to strong
Figure 5. An example of three-dimensional analysis on the finescale topography of the sand surface. A photograph of the sandsurface was pasted on the TIN, above. A contour plot in 1 mm
intervals was automatically drawn, below.

Figure 6. Vertical profiles of sand ripples in four stations established in the middle sandbar.

38

Figure 7. Vertical profiles of sand ripples at two stations established in the foreshore slope and on the crests of three discrete
sandbars.

Saito et al.

wave and current forces. If sandbar crests were
exposed to yearly or more frequent strong wave
actions, the benthic community on them would beseverely disturbed before it recovers from the previous disturbance. In such situations, a sandbar
trough, shaded by a sandbar crest, would work as a
refuge against wave exposure for many days until
extreme waves change the morphology and locationof the sandbars.
In a study on the morphology of intertidal
multiple sandbars of Okoshiki Beach in Ariake Bay,
Japan, the sand bar morphology appears to be a permanent feature in both form and position (Yamada
and Kobayashi 2007). On the other hand, it wasreported that the morphology of multiple sandbars,
referred as ridges and runnels, on Mablethorpe
Beach, North Lincolnshire, England, responded tochanging wave conditions (Masselink 2004).
In this study, abundance of Manila clams in
the trough on the middle sandbar was greater thanat other positions on the same sandbar. When the
crests of three sandbars were compared, the abundance of this clam was greatest on the landward
sandbar where the ripple shape was rather concave
upward and slightly steep, suggesting that the effectof unidirectional current was milder than those at
the other two stations. On the onshore parts of thesandbar zone, the bottom surface may be more protected from exposure than that on the offshore partthrough a shading effect by offshore sandbars. The
abundance of Manila clams on the foreshore slope
was small, especially at the station located at higher
part of the slope (Figure 4).
Steepness of ripples on the foreshore slope wassmall, and the ripple shape there was similar to
those on the crest of the middle and seaward sandbar where the clam was not abundant. This impliedthat the hydrodynamic condition on the bottom of
the foreshore slope was different from that on the
crest of the landward sandbar where the clam wasabundant. Further, extremely shallow parts may beharsh environments for the clam because the submerged period is limited. The low abundance ofManila clams corresponded with the occurrence of
convex ripples that suggested the effect of unidirectional currents mixed with wave actions. In contrast,
this clam was abundant in the trough of the middle
sand bar where the ripple shape was concave upward. This type of ripples—wave ripples—is seen
where unidirectional currents are weak relative tooscillatory waves. It has been reported that Manila
clam abundance was greater in the trough of the

Saito et al.

sandbar than in other positions (Kakino 1996).
Abundance of Manila clams was not significantly different between troughs and crests of sandripples in this study. However, a contrasting phenomenon was that the difference in the community
structure between troughs and crests of ripples
was observed in the subtidal soft bottom (Barros etal. 2004) though the height of the ripples was approximately 5–10 cm, which is much greater thanobserved in this study. A technical problem insampling may be another reason why the difference
was not significant in the present study. The widthof quadrats used in this study, 5 cm, was not smallenough to separate troughs and crests of ripples
when the wave length of the ripples was less than
10 cm. The wave length of ripples observed in thisstudy was sometimes smaller than 10 cm. In thiscase, troughs and crests could not be clearly separated because the area sampled partly overlapped.
The spatial pattern of surf clams was different
from that of Manila clams, which were evenly distributed within the middle sandbar (Figure 4). Comparing the abundances on three sandbar crests, surf
clams increased landward. That pattern was similar
to that of Manila clams, however the difference inabundance between stations was more contrasting in
Manila clams. To explain the contrast between thetwo species, behavioral responses to wave exposure
should be compared in future studies. From theskew of sand ripples, it was estimated that landward
motion is stronger than seaward motion. Small
clams may experience a high risk to be passivelytransported landward.
In studies on benthic communities, the literature referring to ripple shapes on sand flats is limited (Shull 1997, Barros et al. 2004). However, fromthe patterns of ripples, we may be able to deduce
the similarity or the dissimilarity in hydrodynamic
conditions on the sea bottom among sites and localities. Hydrodynamic processes on the sea bottom are
crucial environmental factors that control movement
and settlement of young benthic organisms. However, generalization and theorization of nonsteadyhydrodynamics in turbulent conditions is difficult.
A modular approach characterizing the ecologicalrole of benthos in large coastal seas and estuaries
identified types of modules that comprise coastal
systems and depends on the overall complexity ofgeomorphology and coastlines (Tenore et al. 2006).
The approach aimed at providing insights as to how
the ecology of the benthos may be related to physicalvariables that are used in classification schemes for39

coastal systems. Fine-scale topographies like sandripples may be used as indices of physical conditionsin the modular approach.
Three-dimensional analyses on ripple shape
under water were developed in a previous study
using video cameras in waterproof housings, thoughmeasurement of ripple profiles was not successful
because it was not possible to identify distinct points
on the ripple profiles in each of the images comprising the stereo pairs (Doucette et al. 2002). Our application method is able to obtain stereo pairs using
portable cameras, although the process is limited tointertidal zones. Not being used underwater, ourmethod was successful, although improvement of
the process so it can be used under submerged conditions should be a future task. A detailed explanation of our method and examination of its ability isin preparation.
In previous literature on movable bed roughness
in unsteady oscillatory flow that employed a model
wherein the roughness was divided into contributions due to the form drag over wave-formed ripples
and the near-bed sediment transport, authors demonstrated that when ripples having steepness greaterthan 0.1 are present, they determine the majority ofthe magnitude of the roughness under the waves,
while the roughness depends to a large extent on
the intensity of the near-bed sediment transport for
ripple steepness less than about 0.1 under high sediment transport conditions (Grant and Madsen 1982).
In another study conducted on reef-associated sediments of an exposed coast that used the mean ripple
wave length as an index of exposure to wave energy,
significant negative relationships were detected
between relative wave exposure (as estimated fromripple wave length) and taxonomic richness, numberof individuals, and diversity (DeFelice and Parrish2001).
In the present study, ripple steepness less than
0.1 was observed on the offshore slope on the middle
sandbar, the crest of seaward sandbar, and the upper
part of the foreshore slope. At those stations, theabundance of Manila clams was small compared tothat in other stations (Figure 4). On the other hand,ripple steepness was no less than 0.14 in the troughon the middle sandbar and the crest of the landward
sandbar. The abundance of Manila clams there wasmuch greater than at other stations. From this, it
was suggested that juvenile Manila clams survived
better in places where most of the roughness magnitude is determined by the presence of steep ripples.
Such places may serve as spatial refugia against40

wave and current exposure. Contrasting with theresult of Manila clams, abundance to surf clams
did not show any clear correspondence with ripple
steepness.
In conclusion, common clams in Matsunase
Beach, especially Manila clams, showed a spatial
pattern related to the sandbar topography. This spatial pattern corresponded to some extent with ripple
shapes on the sand surface. Presence of steep ripples
concave upward may be used as an index of refugia
against wave and current exposure, although susceptibility to the exposure would be different among
species. TIN images and vertical profiles of ripples
made from stereo pictures taken in the field will help
us share information on the fine-scale topography ofsand surfaces that is difficult to describe and needs
to be quantified in the field. Statistical tests on clamabundance performed here are not unambiguous.
Random spatial variations within each sampling station were not included in the tests because the scaleof square units equivalent to 60x60 cm adjusted tothe size of stereo pictures was too small to represent
the whole area considered. To keep the robustnessof the tests, the square units should have been replicated within each station. An extensive sampling
design is necessary to solve this problem.

Acknowledgments

We deeply thank Dr. Tomomi Mizuno andTakuya Maruyama (Mie Prefecture Fisheries Research Institute) for their kind assistance, and wegreatly appreciate devotion to duty by the editors
who kindly revised this manuscript. This study
was funded by the Special Coordination Funds for
Promoting Science and Technology, Ministry of
Education, Culture, Sports, Science, and Technologyof Japan.

Saito et al.

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Methods, Economics, and Commercialization of Open Ocean Mussel Culture in the
Northeast United States
Richard Langan
University of New Hampshire
Atlantic Marine Aquaculture Center, Open Ocean Aquaculture Program
Durham, New Hampshire 03824
rlangan at unh.edu
Keywords: open ocean, culture, mussel, protocols
Abstract

Constraints on expansion of culture operations in protected, nearshore embayments where sea
conditions are favorable for raft and surface-referenced longline culture are forcing mussel industries to
explore the potential for developing farms in the open ocean.
A project at the University of New Hampshire has taken this exploratory approach and establishedan open ocean aquaculture demonstration site located 10 km from shore in the open waters of the U.S.
Gulf of Maine. Water depth is 52 m at the site, which is fully exposed to wind and waves from all directions and can experience significant wave heights of 9 m during severe storms. Two longlines, each approximately 120 m in length and submerged 12 m below the surface, were installed in 1999.
The project was designed to identify and demonstrate offshore commercial aquaculture opportunities for local and regional capture fishing communities; therefore, a fishing vessel typical of those usedin nearshore ocean fisheries was equipped to tend submerged longlines. Gear and technology used insurface referenced longline culture was modified for use in the open ocean environment, and several different types of buoys, growout ropes, and socking materials and methods were evaluated to determine
optimal materials and practices for use in offshore environments.
The project has been successful in developing operational protocols and production strategies andhas demonstrated that excellent growth and production can be achieved in the open sea. Since 1999 eight
seed cohorts of blue mussels (Mytilus edulis) have been grown to market size with an average production
cycle of 13 months from spat settlement to 55 mm shell height. Yield at market size has ranged from 7.5
to 12 kg/m of mussel rope, depending on the initial seeding density. The product quality and meat yieldhas been consistently excellent, with meat yields ranging from 42% to greater than 55% depending on
density and season.
An economic analysis that examined optimal farm size, ownership options, and capital costs concluded that high quality mussels could be produced at a cost of $0.53 USD per kilogram, indicating excellent potential for profitability. Project personnel have worked with the regional fishing industry in New
England to transfer technology and a commercial farm was established in 2005. Project personnel continue to provide technical support for commercial start-ups. This presentation highlighted system design,
production strategies, economics, and the process of moving from applied research to commercialization.

Langan43

Bivalve Mollusk Culture on the U.S. West Coast
Bill Dewey
Taylor Shellfish Company
130 SE Lynch Road
Shelton, Washington 98584
billd at taylorshellfish.com
Keywords: oysters, clams, mussels, shellfish farms
Abstract

Shellfish farms on the U.S. West Coast annually produce roughly 48,000 mt of oysters, clams, andmussels valued at approximately $111 million. Oysters dominate production with an estimated 42,731 mt
valued at $84.8 million. The bulk of the oyster production is Pacific oysters (Crassostrea gigas) with edible oyster (Ostrea edulis), Olympia oyster (O. lurida), Kumamoto oyster (C. sikamea), and eastern oyster(C. virginica) being produced in smaller quantities for the live half shell oyster market.
Manila clams (Venerupis philippinarum) are the next most significant species farmed with an estimated 3,880 mt produced annually valued at $17 million. Mediterranean mussels (Mytilus galoprovincialis) and foolish mussels (M. trossulus) are two species of mussels farmed on the U.S. West Coast with anestimated annual production of 1,238 mt valued at $3.5 million. Pacific geoduck clams (Panopea abrupta)
are relatively new to the suite of bivalves cultured. With an estimated 850,000 pounds annual production
valued at more than $5 million, they are the most valuable species per pound.
Washington dominates West Coast production. This is largely attributable to laws passed in the late
1800s allowing the sale of tidelands into private ownership specifically for the purpose of culturing shellfish. Subsequently, Washington has encouraged the development of a robust shellfish industry. Pacificand Grays Harbor counties in southwest Washington rely on the shellfish culture industry to support asignificant portion of their economy.
Oysters are cultured predominantly in the intertidal zone, planted directly on the bottom. The
market in recent years has shifted from fresh shucked oyster meats to live oysters on the half shell trade.
With these shifting markets have come improvements in nursery systems for rearing single oyster seed
and culture and processing systems for producing quality single oysters. In recent years, there have also
been advancements in the mechanization of Manila clam culture and harvest.

44

Dewey

Relationship Between Gametogenesis and Food Quality in Sea Urchin Gonads
Tatsuya Unuma
Fisheries Research Agency
Japan Sea National Fisheries Research InstituteSuido-cho, Niigata 951-8121, Japan
unuma at fra.affrc.go.jp
Charles W. Walker
University of New Hampshire
Department of Zoology
Center for Marine Biology and Marine Biomedical Research GroupDurham, New Hampshire 03824
Keywords: aquaculture, food quality, gametogenesis, ovary, testis, sea urchin
Abstract

The sea urchin gonad contains two main types of the cells: germinal cells (GCs) and somatic nutrient storage cells called nutritive phagocytes (NPs). The proportion of GCs and NPs varies during theyear. Before gametogenesis, NPs fill the gonadal lumina and increase in size by accumulating nutrients
derived from food. As gametogenesis proceeds, NPs decrease in size as nutrients are mobilized andtransferred to GCs. In fully mature gonads, ova or spermatozoa fill the gonadal lumina and NPs shrink
to their smallest size. The best season for eating gonads is restricted to a few months around the initiation of gametogenesis when NPs begin to mobilize their nutrients. Before that period, the size of the
gonad is too small. After that period, as gametogenesis proceeds, the quality of gonads as food products
gradually decreases. The tissues of the gonads become fragile as they mature. After the ripe gonads are
removed from the testis, ova or spermatozoa ooze from the gonoduct, reducing their commercial value.
In some species, strong bitterness develops in the ovary as oogenesis proceeds. To prolong the period
during which commercially valuable sea urchin gonads can be harvested and to improve the quality of
the gonad, two strategies are proposed. One is to accelerate the growth of NPs by feeding sea urchinswith an artificial feed instead of macroalgae. The other is to suppress gametogenesis by manipulating
environmental cues that trigger gametogenesis or by generating infertile, triploid sea urchins.

Introduction

The sea urchin is a popular seafood in Japan,
where market demand for this delicacy has been
growing for decades. Japan is annually importing
more than 6,000 tons of sea urchin gonads (FAO
1996), which means that the Japanese consume about
80% of the total world catch. As wild stocks of sea
urchins gradually decline as a result of overfishing,aquaculturists have become interested in culturing
several species (Keesing and Hall 1998). However,sea urchin aquaculture is still in its infancy in Japan,
the United States, and some other countries. As
only the gonad is edible in sea urchins, knowledgeof unique characteristics of the sea urchin gonad
as food products would be useful for successful aquaculture. Here we describe the features ofgametogenesis, describe the relationship between

Unuma and Walker

gametogenesis and quality of gonads as food products, and propose some possible strategies to improve the food quality. We use the northern green
sea urchin (Strongylocentrotus droebachiensis) and red
sea urchin (Pseudocentrotus depressus) as models, as
they are popular edible sea urchins in the northeastern United States and in the southern part of Japan,
respectively.

Structure of the Gonad

The sea urchin has five gonads, each of which is
attached internally to the test (shell) and can releasegametes to the outside world by a separate gonoduct. Each gonad consists of hundreds of acini andresembles a bunch of grapes. Two sacs (Figure 1b)of tissues (outer and inner) compose the wall of each

45

Figure 1. a) Diagrammatic representation of the sea urchin
reproductive system in aboral side view, b) Diagrammatic representation of the tissues in the sea urchin gonadal wall, afterStrenger (1973), c) Lobe of a sea urchin gonad stained with phalloidin to show muscles on the exterior surface of the GHS of theinner sac (white strips). A = anus, CTL = connective tissue layer,
GL = gonadal lumen, M = madreporite, NP = nutritive phagocytes, GCS = genital coelomic sinus, GHS = genital hemal sinus,
TF = tube feet, and VP = visceral peritoneum. Reprinted withpermission from Walker et al. 2006.

acinus and each sac consists of several characteristic
layers (Figure 1a) (Walker et al. 2006). Throughout
the gonad, the genital coelomic sinus (GCS) separates the outer sac from the inner sac. The outer sac
includes a visceral peritoneum that faces the perivisceral coelom and is attached to a connective tissue
layer (CTL). Nonmuscular epithelial cells also line
the CTL on its opposite surface toward the GCS. The
inner sac is a genital hemal sinus (GHS) that bears
ciliated myoepithelial cells on its outer face (Figure
1b,c,) (Walker 1982). These muscles contract rhythmically during gamete release (Okada et al. 1984).
On its luminal face, the GHS supports the germinal
epithelium. The principal functions of the inner sac
are gametogenesis and nutrient storage.
The germinal epithelium consists of the two major populations of cells: germinal cells (GCs, ranging

46

from oogonia to ova in the ovary and from spermatogonia to fully differentiated spermatozoa in thetestis) and somatic cells called nutritive phagocytes(NPs) that are present in both sexes (Walker 1982).
NPs are versatile cells that provide a structural andnutritional microenvironment for GCs throughout
gametogenesis (Figure 2) (Walker et al. 2005). NPs
have multiple functions. NPs may contain amitotic
oogonia or spermatogonia and growing primaryoocytes or spermatogenic cells basally and simultaneously phagocytize residual ova and spermatozoa
luminally (Walker et al. 1998, 2005). At full maturity,each ovarian NP encloses a single, growing vitellogenic oocyte in a basal incubation chamber where
it may be enveloped by dissolved or particulate
nutrients (Figure 2a) (Walker et al. 2005). The sameNPs may also simultaneously contain additional
GCs at several earlier stages of oogenesis withinsmaller, discrete basal incubation chambers. Testes
also contain numerous NPs that cooperate to provide large basal incubation chambers that ultimately
become continuous and together supply nutrients toenormous numbers of spermatogenic cells at diversestages (Figure 2b) (Walker et al. 2005).

Stages in Gametogenesis

During the annual reproductive cycle, gonadsof both sea urchin sexes pass through a predictable
series of structural changes. Nutrient accumulation in NPs and its use for gametogenesis are linked
processes in sea urchin reproduction (Walker et al.
2006). The proportion of GCs and NPs in the gonadal acini varies with the progress of gametogenesis.
Figure 3 shows structural changes inside the gonadof red sea urchins during gametogenesis as classified
into five stages by Fuji (1960) with modifications forthat species (Unuma et al. 1996, Unuma 2002).
Stage 1. Before gametogenesis: In both sexes, eachacinus is filled with NPs (eosinophilic cells). In
ovaries, a few young oocytes are present at the periphery of the acini. Hematoxylin-stained roundspots, residue from phagocytized ova (Masuda
and Dan 1977, Tominaga and Takashima 1987), areoccasionally present centrally in the ovarian lumen. In testes, detection of spermatogenic cells isdifficult at this stage in paraffin sections. Instead,many hematoxylin-stained speckles, residue fromphagocytized spermatozoa (Kato and Ishikawa
1982, Reunov et al. 2004), may be present in NPs.
Unlike the round spots observed in the immature
ovary, these speckles are amorphous, and are a

Unuma and Walker

useful feature to distinguish testes from ovaries.
Stage 2. Beginning of
gametogenesis: Many
developing oocytes orclusters of spermatogonia
are present at the periphery of the acini, and the
gonadal lumina are stillfilled with NPs.

Figure 2. Nutritive phagocytes (NPs) in a) ovaries and b) testes of northern green sea urchins at
each gametogenic stage with germ cells removed from NP incubation chambers (not to scale). For
ovaries, the shapes and dimensions of representative, individual NPs are shown, as well as the positions of various germ cell types within discrete NP incubation chambers. For testes, the positionsof germ cells relative to groups of NPs are shown, especially the continuous incubation chamberthat eventually forms. AO = amitotic oogonia, AS = amitotic spermatogonia, NVPO = new vitellogenic primary oocyte, O = ovum, LSS = later spermatogenic stages, RVPO = residual vitellogenic
primary oocyte, NP = nutritive phagocyte, NS = new spermatozoa, and MS = mitotic spermatogonia. Modified with permission from Walker et al. 2005.

Figure 3. Representative structure of the ovary (upper panels) and testis (lower panels) of red sea
urchins at different gametogenic stages. Paraffin-embedded sections are stained with hematoxylin and eosin. At stage 1, the gonadal lumina are filled with nutritive phagocytes. At stage 2, many
developing oocytes or clusters of spermatogonia or spermatocytes are present at the periphery.
At stage 3, nutritive phagocytes are replaced with ripe ova or spermatozoa in the center of thelumina. At stage 4, the lumina are filled with ripe ova or spermatozoa. At stage 5, the lumina contain a few residual ova or spermatozoa. NP = nutritive phagocyte, OC = oocyte, OV = ripe ovum, SG= spermatogonium, SC = spermatocyte, and SZ = spermatozoon. Inset a, round spot representinga phagocytized residual ovum. Inset b, amorphous speckles representing phagocytized residual
spermatozoa. Scale bar represents 100 µm. Modified with permission from Unuma 2002.

Unuma and Walker

Stage 3. Middle ofgametogenesis: NPs are
replaced with ripe ova or
spermatozoa in the centerof the gonadal lumina.
Numerous developing
oocytes or clusters of spermatogonia and spermatocytes (Ward and Nishioka
1993, Walker et al. 2005)
are present at the periphery of the acini. NPs aregradually decreasing
in size and are present
between the GCs.
Stage 4. End of gametogenesis: The gonadal lumina are filled with ova or
spermatozoa. Shrunken
NPs, which have alreadylost nutrients, are present
only at the periphery of
the acini.
Stage 5. After spawning:
The gonadal lumina havenumerous empty spaces
and a few residual ova or
spermatozoa. NPs gradually phagocytize residual
gametes and begin togrow as they store nutrients. After this stage,
gonads return to Stage 1
and a new cycle starts.

47

Seasonal Changes in Gonadal Size and GCs/
NPs Proportion

Sea urchin gonads grow in size not only because
gametogenesis increases the size or numbers of
GCs but also because NPs store extensive nutrient
reserves before gametogenesis (Walker et al. 2006).
Figure 4 shows the seasonal changes in the gonad index and in the proportion of GCs and NPs in red sea
urchins (Unuma 2002). This species spawns around
November and gonad indices rapidly decrease.
After spawning, the gonad index gradually increases
until the next spawning. The increase before gametogenesis is attributable to the growth of NPs. NPs
accumulate various nutrients, such as proteins, lipids, and carbohydrates, derived from ingested foodand increase in size. About 80% of the total protein
contained in NPs is a glycoprotein with a molecularmass of about 170 kDa (Unuma et al. 2003). This
protein was originally identified as the predominantcomponent of yolk granules in sea urchin eggs andtermed the major yolk protein (MYP) (Yokota andSappington 2002). Unlike other oviparous animals
where the yolk protein is female-specific, both maleand female sea urchins produce MYP, store it in NPs
prior to gametogenesis (Unuma et al. 1998), and utilize it as nutrient source for gametogenesis (Unuma
et al. 2003, Unuma and Walker in press).
Proliferation and development of GCs begin twomonths before spawning in red sea urchins (Unuma
2002). After gametogenesis begins, the size of the
gonad continues to increase but the proportion of
NPs in the gonad rapidly decreases. The periodof gametogenesis of red sea urchins is shorter thanthose of other species, where gametogenesis usuallytakes more than three months.

Relationship Between Gametogenesis andFood Quality

In most countries that consume sea urchins,
gonads containing fewer GCs than NPs (unripe) are
preferred as food, although there may be some exceptions (Walker et al. 2006). In red sea urchins, the best
season to eat gonads is from June to August, when
NPs have grown sufficiently but gametogenesis has
not yet begun (Unuma 2002). Before that period,
the size of the gonad is too small and the color of the
gonad is not attractive (brownish). After the period,
as gametogenesis proceeds, the quality of gonads as
food products gradually deteriorates. This is due
to adverse events, the most serious of which is theoozing of gametes (Unuma 2002). The tissues of the
gonads become fragile as gametogenesis proceeds.

48

Figure 4. Diagrammatic representation of the seasonal changes
in the gonad index and in the proportion of nutritive phagocytes (NPs) and germ cells (GCs) in red sea urchins. Stages ofgametogenesis are indicated below the graph. Spawning occurs
around November. The long-term increase in the gonad index
before gametogenesis is mostly attributable to the growth ofNPs. Modified with permission from Unuma 2002.

After ripe gonads are removed from the testis,they appear to be melting because of the oozing of
gametes (Figure 5). Eggs or sperm ooze from thegonoduct, and the gonads cannot maintain theirconsistency. This phenomenon, common among
all the edible sea urchins, considerably reduces thecommercial value of the gonads. To suppress thisgamete ooze, alum, an aluminum compound, is usually used in Japan. After removing the gonads fromthe testis, they are lightly washed in salt water andthen immersed in a 0.5–1.5% alum solution for 5–20
minutes. In this way, the mature gonads can maintain their shape for a longer time. The alum cannot,
however, prevent gamete ooze completely; besides,
it alters the taste of gonads, which become astringent
and bitter.
Another reduction in quality results from thebitterness of mature ovaries in some species. In the
red sea urchin and the green sea urchin (Hemicentrotus pulcherrimus, another popular species in thesouthern part of Japan), mature ovaries have an extremely bitter taste, unlike the mature testis. Recently, pulcherrimine, a novel sulfur-containing aminoacid, has been isolated as the bitter component fromthe mature ovary of the green sea urchin (Murata
and Sata 2000, Murata et al. 2001, 2002). The bitter
component in the mature ovary of red sea urchinshas not been identified. The bitterness of mature

Unuma and Walker

in GCs/NPs proportions, the food quality of the gonad could be improved and the season during which
commercial quality gonads can be harvested would
be prolonged. For this purpose, acceleration of NP
growth or suppression of gametogenesis should be
effective as shown in Figure 6 (Unuma 2002). Bothmethods prolong the season during which large
gonads that contain fewer GCs can be harvested.
However, the acceleration of NP growth is easierthan the suppression of gametogenesis and is now
being put to practical use. In contrast, the suppression of gametogenesis, which is more effective atyielding large gonads with fewer GCs, is technicallydifficult and requires much additional research before it can be put to practical use.

Figure 5. Deterioration of commercial quality in the gonads of
red sea urchins. Mature testes were taken out of the testis andplaced in a petri dish for 30 minutes. The testes appeared to bemelting because of oozing sperm. The same process also occurs
in the mature ovaries. Reprinted with permission from Unuma
2002.

Use of Formulated Feed to Accelerate NP
GrowthMost edible sea urchins are herbivorous and eat

macroalgae (seaweed or kelp). However, food that
contains higher amounts of protein than macroalgae
can enhance the gonadal growth more efficiently
than diets of algae, for example, formulated feeds(de Jong-Westman et al. 1995, Lawrence et al. 1997,
Barker et al. 1998, Walker and Lesser 1998, Akiyama

ovaries in these two species results in a shortenedcommercial season compared to that of other species
where ovaries at the end of gametogenesis are notbitter. Usually, red sea urchins and green sea urchins
are harvested only before gametogenesis begins(Stage 1), while other species in which the mature
ovary has no bitterness have commercial value until
mid gametogenesis (Stage 3).
In addition to the deterioration in quality withthe advance of gametogenesis, subsequent spawning causes a large year-to-year decrease in the sizeof the gonad (Unuma 2002). Following spawning,it takes time for the gonads to grow to marketablesize again. The best season for harvesting sea urchin
gonads is rather short because of these problems.
Low-quality or small gonads can be harvested for alonger period of time, but large gonads of commercial quality can be harvested for only about one or
two months. Therefore, gametogenesis and subsequent spawning are disadvantageous for culturing
sea urchins.

Methods to Improve the Food Quality

For successful sea urchin aquaculture, it isimportant to produce large gonads that contain few
GCs. If the gonad can be enlarged without increase

Unuma and Walker

Figure 6. Two strategies for extending the season of harvesting high-quality gonads. If growth of NPs is accelerated (left) or
gametogenesis is suppressed (right), the season during which
quality gonads containing fewer GC can be harvested is dramatically prolonged. Modified with permission from Unuma 2002.

49

et al. 2001, Pearce et al. 2002, Hammer et al. 2006)
and fish meat (Agatsuma and Nishikiori 1991). Thisimplies that protein ingestion and assimilation arelimiting factors for gonadal growth. Figure 7 shows
the gonad indices of young red sea urchins fed formulated feeds of various protein levels and brown
alga Eisenia bicyclis for 8 weeks, one of the main
sources of food for natural populations of red sea
urchins (Akiyama et al. 2001). The gonad indices ofthe sea urchins fed the formulated feeds are higher
than those fed the brown alga and highest in feeds
containing 20% and 30% protein, suggesting that the
optimum protein level for gonadal growth of young
red sea urchins is about 25%.
The protein level of formulated feeds is correlated to the taste of gonads, which becomes increasingly bitter in sea urchins fed higher protein feeds(Hoshikawa 1993). Considering the taste, the appropriate protein level for red sea urchins should belower than 25%. Besides the bitter taste, formulated
feeds have some disadvantages compared to algae:
the color of the gonad becomes unattractive (whitish), formulated feeds are often more costly, and they
decay more quickly than algae (Unuma 2002). These
negative aspects must be overcome before formulated feeds will be practical in sea urchin aquaculture.
Indeed, the performance of formulated feeds has
improved dramatically year by year.

Control of Environmental Conditions to Suppress Gametogenesis

Land-based aquaculture systems for sea urchin
are rapidly developing (Devin 2002, Böttger et al.
2004). In such systems, suppression of gametogenesis is advantageous for more efficient production ofcommercial quality gonads (Unuma 2002). Control

Figure 7. Gonad indicesof red sea urchins fed different diets. Formulated
feeds of 10–50% protein
level and the brown alga
Eisenia bicyclis were provided for 8 weeks to theyoung red sea urchins of
15 mm testis diameter.
Casein was used as theprotein source for theformulated feed. Basedon data from Akiyama etal. 2001.

50

of environmental conditions can be utilized to suppress gametogenesis in land-based systems (Böttger
et al. 2006, Walker et al 2006). In purple sea urchins(Strongylocentrotus purpuratus), northern green sea
urchins, and pencil urchins (Euchidaris tribuloides),the environmental cue to initiating gametogenesis issuggested to be the photoperiod (Pearse et al. 1986,
Bay-Schmith and Pearse 1987, Walker and Lesser
1998, Böttger et al. 2006, Dumont et al. 2006), whereas in red sea urchins, green sea urchins, and violet
sea urchins (Anthocidaris crassispina), it is suggested
to be the water temperature (Yamamoto et al. 1988,
Sakairi et al. 1989).
In some hatcheries in Japan, which produce
seedlings for restocking of fish and shellfish including sea urchins, out-of-season maturation of broodstock of red sea urchins and green sea urchins is
induced by manipulating water temperature (Ito etal. 1989, Masaki and Kawahara 1995, Noguchi et al.
1995). Alternatively, for culturing adult sea urchins,
suppression of gametogenesis is required. In orderto suppress gametogenesis by manipulating photoperiod or water temperature, the control regime
needs to be further investigated. However, controlling water temperature by use of cooling or heating
may not be financially practical for commercial-scaleaquaculture because of high energy costs. Economical methods, such as use of warm effluent from anelectric power station or cold seawater from the deepsea, are essential to control water temperature inaquaculture facilities.
Böttger et al. (2006) investigated the effects ofinvariant summer photoperiod and progressing
ambient (fall/winter) photoperiod on the gonads ofnorthern green sea urchins. Maintenance under invariant photoperiod yielded gonads containing fewer GCs than NPs (unripe), while maintenance underambient photoperiod yielded ripe gonads (Figure 8).
There was no significant difference in the gonad size
between the two photoperiod regimes. These results
imply that rearing northern green sea urchins underinvariant summer photoperiod may improve thefood quality and prolong the harvest season of thespecies in a land-based aquaculture system.

Production of Infertile Sea Urchins

Production of infertile sea urchins may also be
utilized to suppress gametogenesis. To generateinfertile adults, triploidy is often induced in otherorganisms, such as fish and bivalves (Arai 2001, Nell
2002). In sea urchins, however, there are very few
reports concerning triploidy. One of the difficulties

Unuma and Walker

ing the first cleavage after fertilization and its subsequent crossing with a normal sea urchin may be
an efficient method to generate triploid sea urchins(Unuma 2002).
Recently, Böttger et al. (in press) succeeded ingenerating triploid embryos of northern green sea
urchins by fusing two eggs followed by fertilization
with normal sperm (Figure 9), although development of the triploid embryos was not observed past
the gastrula stage. If this method can be improved,
it may become a useful technique to generate triploid urchins.
If production of infertile sea urchins is accomplished, it would solve all the drawbacks associated with gametogenesis, loss of gonad size resulting from spawning, oozing of gametes, bitterness
caused by oogenesis, and astringency caused by theuse of alum. As a result, production of infertile sea
urchins can dramatically improve the food quality
and prolong the season during which commercial
quality gonads can be harvested. Needless to say, it
is crucial to confirm that infertile triploid sea urchinsaccumulate nutrients in NPs as normal diploid sea
urchins do.

Conclusions
Figure 8. Plastic sections of A) a representative northern green
sea urchin ovary and B) testis maintained for 5 months underinvariant photoperiod showing predominance of NPs and few
gametes. Limited numbers of residual (RO) primary oocytes(no new ones) are present near the ovarian wall and limited
numbers of new spermatozoa (arrows) are evident between
the expanded NPs as are spermatogonial mitoses among thespermatogenic cells (SC). C) Plastic section of a representative
northern sea urchin ovary and D) testis maintained for 5 months
under ambient photoperiod showing growing residual (RO) andnew (NO) primary oocytes in NP incubation chambers. The testicular lumen is filled with new spermatozoa and the NPs (darkgranule containing cells) are reduced in size with only a slenderstrand of cytoplasm connecting them to the testicular wall; circle
points out a spermatogonial mitosis (SC); C = coelom. Scale bar
represents 50 µm. Reprinted with permission from Böttger et al.
2006.

Intragonadal nutrient storage and its utilization
for gametogenesis are linked processes in sea urchin
reproduction. This situation, which is not foundin other echinoderms including sea stars and sea
cucumbers, is the basis for using sea urchin gonads
as food products. A thorough understanding of sea
urchin reproduction should permit some methods
to improve gonad quality and prolong the season
during which quality gonads can be harvested in sea
urchin aquaculture.

Acknowledgments

Grant support was provided by Sea Grant, U.S.
Department of Agriculture, and NRAC to CWW.

in generating triploid sea urchins is that sea urchin
eggs complete both meiotic divisions before spawning (Walker et al. 2006). Therefore, retention of thepolar body, a popular method of generating triploidfish and shellfish, cannot be applied to sea urchins.
Production of a tetraploid sea urchin by suppress-

Unuma and Walker

51

A

B

C

D Diploid = 43

E Triploid = 63

F Diploid = 43

Figure 9. Triploidy in northern
green sea urchins. A) two ova
with the jelly coat and vitelline
membranes removed, B) several ova just prior to fusion, C)
three successful fusions with
two other ova beginning tofuse, D) normal diploid chromosomal spread, E) triploid
chromosomal spread, F) normal diploid blastula, and G)
triploid blastula (notice larger
blastomeres). Courtesy of S.
A. Böttger et al., from Böttger
et al. in press.

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An Overview of the Culture of Marine Invertebrates in Maine
Dana L. Morse
University of Maine Cooperative Extension
Maine Sea Grant College Program
193 Clarks Cove Road
Walpole, Maine 04573
dana.morse at maine.edu
Keywords: Maine, aquaculture, invertebrates, polyculture
Abstract

Maine’s aquaculture industry encompasses a growing variety of producer sectors and an increasingly differentiated set of products and culture methods. Once solely recognized as a salmon-producing
state, Maine enjoys a growing awareness for the quality and value of its other products and the care withwhich producers grow their crops. Marine invertebrates are principal examples, with the premier species
being eastern oyster (Crassostrea virginica) and blue mussel (Mytilus edulis). Other marine invertebrates
raised in Maine include hard clam (Mercenaria mercenaria), softshell clam (Mya arenaria), sand worm
(Nereis virens), sea scallop (Placopecten magellanicus), and green sea urchin (Strongylocentrotus droebachiensis). Species are raised for the food market, for stock enhancement, and for other uses, employing
a broad variety of culture techniques. Macroalgae are also being investigated as companion species inpolyculture arrangements.
The annual value of marine invertebrates raised in Maine is approximately $10 million, though precise figures are difficult to obtain. Approximately 50 producing companies are distributed through every
coastal county, with many industry members having history in traditional capture fisheries. Several academic institutions are engaged in education and applied research, most frequently in collaboration withindustry partners, and both organizational and physical infrastructure in the state is improving. Funding
for research and development within the state is nonetheless fairly limited, though applicants have beensuccessful at attracting outside funding. The industry association is strongly engaged in a variety of issues.
A review of the species and methods of production was given, along with details about regulatory
structure, market conditions, and current opportunities and challenges.

Morse

55

Polyculture of Red Abalone (Haliotis rufescens) and Pacific Dulse (Palmaria mollis)
in a Land-based Recirculation System
Chris Langdon and Ford Evans
Oregon State University
Department of Fisheries and Wildlife
Coastal Oregon Marine Experiment Station
Hatfield Marine Science Center
Newport, Oregon 97365
chris.langdon at oregonstate.edu
Keywords: polyculture, red abalone, Pacific dulse, protein
Abstract

Integrated, intensive polyculture usually depends on maintaining a balanced system made up of different, complementary biological units that share a common water source. Exchanges of water, nutrients,and organic material among the linked components are often designed to enhance production, reduce
nutrient loss, and enhance economic gains.
We studied a simple marine integrated polyculture system in which red macroalga Pacific dulse(Palmaria mollis) was cocultured with red abalone (Haliotis rufescens) in the same culture system. Dulse
provided abalone with a high quality diet. Ammonia and carbon dioxide produced by abalone were efficiently removed by dulse and converted into feed for abalone. Light, nutrients, and inorganic carbon
were added to the system.
Under optimal summer conditions in Oregon, (lat 44°37’N, long 124°02’W), we measured dulse
specific growth rates (SGR, % increase in dry weight d-1) of 17%, dulse productivities of 67 g dry weightm-2 d-1 (equivalent to 413 g wet weight m-2 d-1), and light utilization efficiencies as high as 7.2% for dulse.
These production rates and efficiencies were comparable to those reported for high-yielding terrestrial
agricultural crops, such as maize, rice, and sugar cane.
Dulse was an excellent food for abalone and had a high average protein content of 28% dry weight.
Juvenile red abalone (shell length 25 mm) fed on dulse at 18°C showed growth rates as high as 198 μm
shell length increase d-1, exceeding previously reported growth rates for this species. Food conversion
efficiencies were as high as 20%, resulting in an overall energy conversion efficiency of light energy into
abalone biomass of up to 1.41% for this system.
In this integrated polyculture system, abalone production was much more limited by dulse production rates than the capacity of dulse to maintain water quality by removing excreted ammonia. Dulse
production rates and efficiencies may be improved by optimizing conditions for gas exchange, lightavailability, and by selecting fast-growing dulse strains suited to intensive culture conditions. However,
the economic benefits of these improvements should be balanced against costs in a commercial facility.

56

Langdon and Evans

Wave Induced Flow in Seawater Exchange Structures for Improving Seawater Quality
Yoshihiro Ohmura
Fisheries Research Agency
National Research Institute of Fisheries Engineering
7620-7 Hasaki, Kamisu
Ibaraki, 314-0408, Japanohmura at fra.affrc.go.jp
Keywords: seawater exchange, coastal structures, physical model, theoretical considerations
Abstract

In order to improve and preserve the quality of seawater and the seabed in dead water regions suchas ports and harbors—including aquaculture grounds—seawater circulation and exchange needs to beenhanced. It may be possible to use a seawater exchange structure driven by wave motion. Among themany kinds of seawater exchange structures that have been developed, seawater exchange structureswith blockwork mounds may be one of the most effective. This paper presents the results obtained
from physical model tests and theoretical considerations performed on modified structures. Two models aimed at flexible applicability were used in the experiment; both of the modified structures proved
effective. The research affirms that the volume rate of discharge for inlet flow through a conduit can be
estimated quantitatively by the present discharge model.
Introduction
Seawater circulation and exchange in ports
and harbors are inevitably restrained by surrounding facilities. If effective countermeasures against
stagnation are not adopted, there may be pollution
problems in ports and harbors, including aquaculture grounds, which can sometimes result in
ecological damage. Enhanced seawater circulationand exchange are needed, in order to improve andpreserve the quality of the seawater and seabed insuch regions.
A large amount of work has been conducted todevelop countermeasures against seawater pollution
and various systems have been adopted in stagnant
regions (Table 1). Some designs enhance the advection-diffusion of pollutant loads through seawaterexchange, while others directly improve seawaterand sediment conditions. Use of tidal and ocean
currents or wave induced currents may be one ofthe possible solutions to the problem. However, thedriving forces of such currents are generally weak in
ports and harbors and consequently ineffective. It
may be possible to use a seawater exchange structure driven by wave motions.
In Japan, many kinds of seawater exchange
breakwaters that conduct seawater into basinsthrough the use of wave power have been constructed for fisheries in ports and harbors. We havedeveloped a seawater exchange breakwater in which

Ohmurathe unidirectional current is excited by vortex flows(Ohmura et al. 2005). I have also developed anotherseawater exchange structure, in which one-way flow is
excited by mass transport due to waves (Ohmura 2002).
Among the many kinds of seawater exchange
breakwaters, seawater exchange structures withblockwork mounds may be one of the most effective(Figure 1). The structure was originally developedby Yamamoto et al. (1992) as a breakwater in which
one-way flow is excited in a wave chamber due towave breaking or wave overtopping. When waterlevel exceeds the crest of mound, wave setup occurs
by wave breaking. When water level is below the
crest, wave setup occurs by wave overtopping. The
structures not only have an ability to conduct seawater seaward through a conduit into the basin, but

Figure 1. Mechanism of wave setup induced flow in seawaterexchange structures with blockwork mounds.

57

Table 1. Representative works for improvement and preservation of seawater and sediment conditions.

Function

Works (energy)

Enhancement ofseawater exchange

Seawater exchange structure (wave, tide, current)		
Waterway (tide, current, internal tide)
Current training wall (tide, current, internal tide)
Bay mouth improvement (tide, current, internal tide)
Water gate (tide, electric power)
Seawater training pump (wave, electric power)
Air bubble curtain (electric power)

Supply oxygento seawater

Seawater exchange structure (wave)
Tideland (wave, tide)
Sand beach (wave, tide)
Seaweed (solar)
Armor blocks (wave)
Rubble mound type gentle sloping breakwater (wave)
Revetment with aeration function (wave)
Air bubble curtain (electric power)

Organic digestion
by benthos andplanktonTideland (solar, wave, tide)
Sand beach (solar, wave, tide)
Facilities for oxidation digestion by rocks (wave, tide, current, electric power)
Rubble mound type gentle sloping breakwater (wave, tide, current)

Keeping nutrient salts

Tideland (solar, wave, tide)
Sand Beach (solar, wave, tide)
Seaweed (solar)

Preventing nutrient
salts from sediment

Sediment dredge
Sand capping
Supply oxygen

also help supply oxygen to the seawater. It is known
that when the distance from seawater level to the topof the crest becomes more than the values of incident
wave height, the structures do not produce one way
flow at all.
This paper presents information on the effectiveness of modified seawater exchange structures withblockwork mounds for a wide range of applicability
such as wide tidal change and long conduits. Physical model tests and theoretical considerations were
employed to investigate hydraulic characteristics ofthe modified structures (Ohmura 2007).

58

Physical Model Tests

Experimental setup. Experiments were conducted in a wave basin at the National ResearchInstitute of Fisheries Engineering as shown in Figure 2. The wave basin was 40 m long, 22 m wide,
and 0.8 m deep. It was equipped with a 10-m wide
piston-type wave generator. Two modified models
not only for wide tidal exchange but also for longconduits were used in the experiment. Model A had
different blockwork mounds consisting of low crest,high crest, and intermediate crest as shown in Figure
3. We theorized that different crest heights would
play an important role to induce wave setup in wave
chambers over a wide range of water levels. The

OhmuraFigure 2. Plane view of a wave basin.

outer face of each mound had a 1:2 slope in orderto enhance more wave setup in the wave chamber.
Each conduit was made of PVC pipe with a diameterof 0.1 m and a length of 4.3 m. Model B used a rearchamber as shown in Figure 4. The specificationsof model B were the same as model A except for therear chamber. The conduit was made of PVC pipewith a diameter of 0.1 m and a length of 6.7 m. Figure 5 is a photograph of model B used in the experiment.
Waves acting on the models were the regularwaves under the following test conditions:• Deepwater wave height: H0 = 2, 4, 6, 8 cm
• Wave period: T = 1.0, 1.5 s• Water depth: h = 25.0, 28.1, 31.3, 34.4, 37.5, 		
40.6, 43.8 cm
The relationships among seven water levels andeach crest height are shown in Figure 3 and Figure 4.
The geometric scale was assumed about 1/10 to 1/20
according to Froude similitude. Measurements taken
were surface elevation in the wave chambers in frontof the wave paddle and at the exit of the pipe by capacitance-type wave gauges, and velocity in the center ofthe pipe at the exit by electromagnetic velocity meters.

Calculation methods of spatially averaged
velocity

In order to calculate the volume rate of discharge through the pipe, adequate methods shouldbe employed to estimate the spatially averaged
velocity from the center velocity at the exit. The
procedures described below were adopted.
Log-law equation for turbulent flow in pipes
may be expressed as:								
(1)
where y is the polar coordinate, y0 is the length
representative, u(y) is the velocity at y, u is the shear*
velocity, and k = 0.4 is the von Karman constant.
Integrating with respect to r, the relationshipbetween the spatially averaged velocity U and thecenter velocity ,uc is given by:						
Figure 3. Plane view and cross section of model A with threedifferent crests.

Ohmura(2)where Q is the volume rate of discharge, A is thearea of pipe, and r is the radius of pipe.

59

Shear velocity u and the friction loss factor f are*defined as:														
(3)						
														
(4)
where t0 is the shear stress, r is the water density, gis the gravitational acceleration, D is the diameter ofpipe, and n is the Manning’s roughness coefficient.
Consequently, relation between the spatially averaged velocity U and the center velocity uc is givenby:														
(5)
In the experiments, D, n, and g in Equation 5
should be taken as 0.10 m, 0.010 m-1/3s, and 9.8 m/s2,
respectively, and finally U=0.82uc is obtained.

Methodology for Prediction of Discharge

Figure 4. Plane view and cross section of model B with three different crests and rear chamber.

Volume rate of discharge from the mound. The discharge model may not only be necessary for predicting the volume rate of discharge, but is also helpful
to investigate hydraulic characteristics on the seawater exchange structures with blockwork mounds.
The discharge model on the structures for regularwaves were newly developed and calculated.
The relationship between the time-averaged
surface elevation in the wave chamber without discharge and the deepwater wave height for regularwaves may be given by:											
						

Figure 5. Photograph of seawater exchange structures withblockwork mounds in the experiment.

60

(6)

where hc is the time-averaged surface elevationin wave chamber without discharge, hc is the crest
elevation from still water level, and H0 is the deepwater wave height.
Considering both of the past experimental
results on blockwork mounds and the results fromthis study, formulas in Equation 6 are determined to
be a concise expression excluding the effect of wave
period. Yamamoto et al. (1988) examined the relationship between the time-averaged surface eleva-

Ohmuration in the wave chamber without discharge and the
deepwater wave height for regular waves, and determined that h = 0.49H0 when hc = 0 was obtained.
We assumed that the phenomena in the wave
chamber are steady as shown in Figure 6. Based on
Bernoulli’s theorem, the volume rate of discharge forincident wave Q1 and the discharge for return flowfrom wave chamber QR can be expressed as:														
(7)														
(8)
where C1 is the coefficient of discharge for incident
wave, CR is the coefficient of discharge for returnflow, h is the time-averaged surface elevation inwave chamber with discharge, and B is the crest
length.
The volume rate of discharge for inlet flow QT
may be written as:						
						
(9)													

(10)

where hout is the time averaged surface elevation atexit, Cin is the coefficient of discharge for inlet flow,
fe is the entrance loss factor, fo is the outlet loss factor,
fb is the bending loss factor, and l is the pipe length.
The continuity of the volume rate of discharge
in the wave chamber gives:						
						
(11)
Surface elevation with discharge h can be calculated easily from these equations mentioned above
with the Newton-Raphson method. It should consider the conditions which are 0 ≤ h ≤ hc and hc = 0
for hc < 0.
The volume rate of discharge for inlet flow QT
can be calculated by the present discharge model.
Volume rate of discharge from rear chamber. The
volume rate of discharge for inlet flow from eachmound can be calculated by the discharge model
described above. The volume rate of discharge fromthe rear water chamber may also be expressed asgiven by Equation 9 and Equation 10 by substituting the time-averaged surface elevation at the rearchamber hwc into h. It is also necessary to take into

Ohmuraaccount the continuity of the volume rate of discharge in the rear chamber. The equation of continuity is written as:														
(12)
where QT,k is the volume rate of discharge for the
inlet flow from each mound and QT,4 is the volumerate of discharge from the rear water chamber.
Equation 12 can be arranged as:											

(13)

Then hwc can be calculated by solving Equation
13. Thus QT,k and QT,4 can be obtained. The conditions are 0 ≤ hwc ≤ max (h1, h2, h3), hk = hwc for hk +hc,k < 0 and hwc + hc,k < 0, hk = hc,k for hk + hc,k < 0, andhwc + hc,k ≥ 0.

Results and Discussion

Figure 7 shows the results on the mass of watertransport from each mound Q for model A. From
the figures, it is easily seen that the discharge fromeach mound appears effective at a different waterlevel. When water level is comparatively low, thedischarge from low crest mounds shows high performance. On the other hand, when water level iscomparatively high, the discharge from high crest
mounds shows high ability. The reason is that thewave setup in the wave chamber that induces thedischarge for inlet flow from each mound is influenced by the crest elevation from the still waterlevel. The volume rate of discharge becomes gradually larger for longer and larger waves.
Figure 8 shows the dimensionless volume rateof discharge from each mound Q* for model A as afunction of crest elevation by deepwater wave heighthc/H0. The volume of water transport for unit width
Q/B is normalized by the amount HL/2π, which isequivalent to the water volume set in motion during a half wave period where H is the incident wave
height and L is the wave length. From the figure, it
can be seen that the value of the dimensionless volume rate of discharge becomes maximum, say about
0.25 when hc/H0 is around –0.2 for each mound. It
affirms that the water transport function is effective
when –1.2 < hc/H0 < 1.2.
Figure 9 shows the time-averaged surface elevation in wave chamber for model A as a function of61

Figure 6. Time-averaged phenomena in wave chamber.

Figure 8. Dimensionless volume rate of discharge for inlet flowfrom each mound for model A.

Figure 9. Time-averaged surface elevation in wave chamber formodel A.

Figure 7. Volume rate of discharge for inlet flow from eachmound for model A.

62

hc/H0. From the figure, it can be seen that the valueof the dimensionless surface elevation becomesmaximum at about 0.5 when hc/H0 is around –0.2
for each mound. It is also confirmed that the surface elevation occurs when –1.2 < hc/H0 <1.2. The
relationship between surface elevation and hc/H0 islinear over the range of –1.2 < hc/H0 < 1.2, which isthe same tendency as shown in Figure 8.
Figure 10 shows the comparison of measured
and calculated time-averaged surface elevation inwave chamber for model A. In the calculation, CI,
CR, ffe, ffo, ffb, and hout were chosen as 1.0, 1.0, 1.0, 1.0,
0.29, and 0.0, respectively. The calculated surface
elevations show fairly good agreement.
Figure 11 shows the comparison of measured
and calculated volume rates of discharge for inlet
flow from each mound for model A. The calculated

OhmuraFigure 10 (at right).
Comparison of
measured andcalculated timeaveraged surface
elevation in wave
chamber for modelA.

Figure 12. Comparison of measured and calculated volume rateof discharge from rear chamber for model B.
Figure 11 (at right).
Comparison of
measured andcalculated volumerate of discharge
for inlet flow fromeach mound formodel A.

values also show fairly good agreement. The data
show that the volume rate of discharge for inlet flow
through the conduit can be estimated quantitatively
by the present discharge model. However, thecalculated values are overestimates compared withthe value from the experiment, so there needs to be acorrection to increase accuracy.
Figure 12 shows the comparison of measured andcalculated volume rates of discharge from the rearchamber for model B. In the calculation, fe, fo, fb, andhout for pipes from the rear chamber were chosen as
1.0, 1.0, 0.256, 0.0, respectively, and CI, CR, ffe, ffo, andffb for pipes from each mound were 1.0, 1.0, 1.0, 1.0,
and 0.0, respectively. From the figure, we see thatboth measured and calculated volume rates of discharge from the rear chamber appear reasonable overa range of water levels. However, when the waterlevel is relatively high, say levels 6 and 7, the volumerate of discharge decreases. It should be noted thatthe calculated values show fairly good agreement

OhmuraFigure 13. Calculated volume rate of discharge for inlet flowfrom each mound for model B.

compared to the values from the experiment.
Figure 13 shows the calculated volume rate ofdischarge for inlet flow from each mound in model
B. We see that the volume rate of discharge for inlet
flow from low mounds show negative values when
the water level is relatively high. It may be necessary to change the specifications of the structuresin order to prevent return flow from the rear waterchamber. The present discharge model may be useful in predicting the performance and specificationsof the structures.

63

Conclusions

Our results confirm that both of the models are
effective not only for wide tidal change but also forlong conduits because the different crest heights play
an important role to induce the wave setup in wave
chambers over a wide range of water levels. We
also affirmed that the volume rate of discharge for
inlet flow through conduits can be estimated quantitatively by the present discharge model, althoughcorrection may be needed for more accuracy. In addition, the present discharge model may be helpfulin predicting the effective specifications for modified seawater exchange structures using blockwork
mounds. It is hoped that the technology of seawaterexchange structures can play a significant role on
aquaculture grounds in the near future.

References

Ohmura, Y. 2002. Development and testing of theseawater intake works with cylindrical holes. Fish.
Eng. 39:131–138 [in Japanese].
Ohmura, Y. 2007. Wave set-up induced flow in seawater exchange structures with blockwork mounds.
In Coastal Engineering 2006, Proceedings of the 30th
International Conference, p. 4374–4385. World Scientific, Hackensack, NJ.
Ohmura, Y., T. Nakamura, and K. Ohi. 2005. Excitation of vortex induced currents by piston mode
wave resonance in double-curtain walled breakwaters. In Coastal Engineering 2004, Proceedings of the
29th International Conference, p. 3787–3799. World
Scientific, Hackensack, NJ.
Yamamoto, M., M. Nakaizumi, and M. Manabe.
1988. Development of the breakwater with remotesubmerged rubble mound for the purpose of interchanging sea water I: Investigation on its function
and the form of the submerged rubble mound. Technical Report of the National Research Institute ofFisheries Engineering, Aquaculture and Fishing Port
Engineering 10:39–55 [in Japanese].
Yamamoto, M., M. Nakaizumi, M. Manabe, and A.
Moriguchi. 1992. Development of the breakwaterwith submerged mound for interchanging sea water.
Bulletin of National Research Institute of Fisheries
Engineering 13:11–30 [in Japanese].

64

OhmuraNovel Culture Feed for Short-neck Clam Using Single-cell Material from Porphyra
Takao Yoshimatsu
Fisheries Research Agency
National Research Institute of Aquaculture
Mie 516-0193, Japan
takaoyos at fra.affrc.go.jp
Alok Kalla, Nakib Dad Kahn, and Toshiyoshi Araki
Mie University
Graduate School of Bioresources
Mie 514-8507, Japan
Shuichi Sakamoto
Oriental Yeast Industry Co. Ltd.
Tokyo 174-8505, Japan
Keywords: short-neck clam, Porphyra, food supplement
Abstract

The short-neck clam (Manila clam [Ruditapes philippinarum]) is an important Japanese seafood andsupports profitable coastal fishing. Short-neck clam culture is a key component and rapidly increasingarea of Japanese aquatic production. Mainstream production is from natural populations while, increasingly, stocks are close to or have exceeded utmost sustainable yields. Stock improvement through thecapture and imparting of natural seed in both extensive and intensive forms of culture is a frequent
practice.
Nevertheless, the consistency of natural recruitment can never be guaranteed. A method to understand how to meet the seed requirements of the short-neck clam is the hatchery culture trial. The production of seed through hatchery propagation accounts at present for only a small percentage and may
be attributed to the unavailability of artificial feeds. Although shortneck clam larvae and spats havebeen reared successfully for algal foods (i.e., mainly diatoms), these are expensive to produce and do notalways coordinate with producer’s requirements; researchers have paid insufficient attention to investigating substitutes for algal foods. Expensive algal concentrates, universally used throughout the world,
have overcome some constraints associated with shelf life of algal concentrate, including storage of algal
foods.
Red algae (Porphyra spp.) are widely cultured on the coastal lines of Japan, are rich in nutrients,
and are known as functional seafoods. Generally, Porphyra has a high percentage of protein and also
contains an outstandingly high amount of taurine, which is an important amino acid for larval marine
animals in addition to various kinds of minerals and vitamins.
Recent innovations in biotechnology including protoplast/spheroplast isolation techniques using
polysaccharide-degrading enzymes have shown some promise to use these nutritious algae as a foodsupplement without cell walls, because these single-cell materials are easily digestible when ingested.
We therefore attempted to develop a novel feed for culturing shellfish by using single-cell materials obtained by enzymatic means and to determine the dietary effect of this feed for growth and production ofshort-neck clam. Many successful results have been obtained in the series of experiments so far.
This research was supported by a grant from the Agriculture, Forestry, and Fisheries Research Council in Japan: Research Project for Utilizing Advanced Technologies in Agriculture, Forestry, and Fisheries(No. 1681, 2004–2006).

Yoshimatsu et al.

65

Culture Systems and Habitat Evaluation Branch Overview–
Molluscan Aquaculture
Ronald Goldberg
Northeast Fisheries Science Center
Aquaculture and Enhancement Division, Milford Laboratory
212 Rogers Avenue
Milford, Connecticut 06460
ronald.goldberg at noaa.gov
Keywords: aquaculture, bivalves, bay scallops
Abstract

The Culture Systems and Habitat Evaluation Branch of the Aquaculture and Enhancement Division can trace its scientific roots to the history of Milford Laboratory. Early research led by Dr. Victor
Loosanoff succeeded in devising techniques for larval rearing of bivalves, enabling the development of
the aquaculture industry as we know it today. Milford research was extended to nutrition, immunology,
disease control, field grow out, and predator control.
While early Milford research focused on the eastern oyster (Crassostrea virginica), rearing methods
were applied to many bivalve species. Research during the 1970s developed culture methods for the bay
scallop (Argopecten irradians irradians), the Atlantic surf clam (Spisula solidissima), and the hard clam
(Mercenaria mercenaria).
Current branch research has concentrated on investigating recirculating aquaculture seawater systems to grow large (25 mm) bay scallop seed under controlled conditions. The ideal system would behighly automated, economic to operate, and generate little or no waste discharge. Producing large seedbay scallops allows for single season grow out to market size in the northeastern United States and also
provides release animals that would likely spawn naturally in stock enhancement efforts. Research
topics include understanding the chemical ecology within closed systems, measuring the physiologicalresponses of scallops to different culture parameters, overcoming unexplained winter mortalities, anddefining the ecology of natural populations.
The branch has recently incorporated research themes that reflect the National Oceanic and Atmospheric Administration’s priorities to further expand the U.S. aquaculture industry. These include refining sustainable high-volume seed production methods, determining interactions between aquaculture
operations and the environment, and exploring stock enhancement strategies for shellfish. Research on
aquaculture and stock enhancement of bay scallops is particularly pertinent because of marked declines
in natural bay scallop populations over the past decades.

66

Goldberg

Bay Scallop (Argopecten irradians irradians) Husbandry Program
James C. Widman Jr.
Northeast Fisheries Science Center, Milford Laboratory
212 Rogers Avenue
Milford, Connecticut 06460
jwidman at mi.nmfs.gov
Keywords: bay scallop, husbandry, adductor muscle, seed
Abstract

The bay scallop (Argopecten irradians irradians) is a recreationally and commercially harvested
bivalve in the eastern United States. Imported to China in the early 1980s, it has become a major partof that country’s aquaculture production. It has many characteristics that make it a prime candidate foraquaculture: rapid growth rate, achieving market size in less than 18 months, well-known husbandry
methods, amenability to culture in nets or pens, high consumer acceptance, and a primary consumer.
Unfortunately, the bay scallop has variable survival throughout the winter in New England, whichcurrently restricts bay scallop aquaculture to a single-season endeavor. When scallops were grown inlantern nets in a single season in Long Island Sound, Connecticut, they reached a mean shell height of
50 mm. In the United States, only the scallops’ adductor muscle is consumed. Raising scallops to 50 mm
yields a three gram adductor muscle, which is not economically profitable in the United States at thistime.
It is possible to produce a larger adductor muscle in a single season by relying on shellfish hatcheries
to produce scallop seed early in the season. If a 25 mm scallop could be deployed by May in the northeast United States, it should grow to a minimum size of 62 mm, which would double the adductor muscle
yield to six grams per scallop.
Any method to produce larger seed in the northeast United States must rely on heated sea waterand economically producing large amounts of phytoplankton. One method to reduce seawater heating demands would be to rely on partially recirculated bay scallop culture of post-set (dissoconchs). In
recirculating shellfish culture systems at the Milford laboratory, we need to monitor the accumulationof toxic nitrogenous compounds. Recently we found that scallops were most susceptible to ammonia incomparison to nitrite or nitrate. We have developed automated demand feeding systems to minimize thenitrogen load, in particular ammonia, in recirculating scallop culture systems.
Our current research indicates that low cell concentrations, 1,000 cells/ml of the phytoplankton Tetraselmis chuii, yields faster growth of scallops than those cultured at higher cell concentrations. Research
is now focusing on augmenting the single-algal species to produce a superior diet that will yield faster
growth rates.

Widman67

Growth and Stress Response of Bivalve Mollusks in Response
to Different Microalgal Diets and Arachidonic Acid Supplementation
Lisa Milke, Shannon Meseck, and Gary Wikfors
Northeast Fisheries Science Center, Milford Laboratory
212 Rogers Avenue
Milford, Connecticut 06460
lisa.milke at noaa.gov
Monica Bricelj
Institute for Marine Biosciences
Halifax, Nova Scotia, Canada
Christopher Parrish
Memorial University of Newfoundland
St. John’s, Newfoundland, Canada
Keywords: scallop, diets, fatty acids
Abstract

The bay scallop (Argopecten irradians irradians) and sea scallop (Placopecten magellanicus) are twocommercially important scallop species in the United States. Few data exist concerning dietary requirements during the early postlarval stages of these species, and therefore costly multispecies algal diets areoften used in hatcheries to ensure high growth and survival. Thus there is an interest in identifying costeffective, high-performance algal diets for implementation in commercial hatcheries, and in identifying
specific dietary compounds which will not only enhance growth but also reduce stress response, thereby
improving health and survival.
Bay and sea scallops were grown in recirculating systems during five separate trials, each lasting 3–4
weeks. Scallops were offered unialgal and binary diets consisting of one of three diatoms and one of fiveflagellates. Two binary diet combinations, CHGRA (Chaetoceros muelleri) in combination with either
Pavlova spp. (CCMP strain 459, Pav 459) or P. pinguis, consistently ranked highest among the diets tested
for both scallop species.
While previous work has established a requirement for n-3 fatty acids in bivalves, our work strongly
suggests that scallop growth rate is influenced by two n-6 polyunsaturated fatty acids (PUFA): arachidonic acid (AA) found in CHGRA and docosapentaenoic acid provided by Pav 459 and P. pinguis. Enrichment in tissues (relative to diet) of these individual fatty acids, as well as total n-6 fatty acids, wereobserved in tissues of both scallop species regardless of dietary treatment, suggesting a requirement for
n-6 fatty acids in pectinids that has been largely overlooked.
The specific role of AA was further examined by offering AA supplemented algal diets to bay scallop
larvae and juveniles. Changes in hemocyte morphology were associated with AA supplementation as
well as with stress, imposed by centrifugation. Future work will determine whether cortisol concentrations can be used as a measure of stress in bivalves, as previously shown in vertebrate systems. To thisend, an enzyme-linked immunosorbent assay, also known as an ELISA, is currently under development
to measure cortisol concentrations in bivalve hemolymph.

68

Milke et al.

Biotechnology Branch Overview on Probiotic Bacteriaand Harmful Algal Bloom Research
Gary Wikfors
Northeast Fisheries Science Center, Milford Laboratory
Aquaculture and Enhancement Division, Biotechnology Branch
212 Rogers Avenue
Milford, Connecticut 06460
Gary.Wikfors at noaa.gov
Keywords: biotechnology, HAB, probiotic bacteria, bivalves
Abstract

The research of the Northeast Fisheries Science Center’s Biotechnology Branch applies the contemporary tools of several scientific disciplines, including genetics, proteomics, microbiology, immunology,
chemistry, and ecology to research relevant to marine aquaculture and its ecosystem interactions. Examples of several specific projects are presented by other branch personnel; this presentation focuses on
two additional areas of research: probiotic bacteria for use in bivalve hatcheries and harmful algal bloom(HAB) interactions with aquacultured mollusks.
The project on probiotic bacteria was established by a postdoctorate from the National Fisheries
Research and Development Institute in Korea, Dr. Hyun-Jeong Lim. Working with branch staff, sheisolated a number of bacterial strains from within the shells of healthy mollusks and screened these forbiological effects on oyster larvae and on bacterial pathogens of oyster larvae. Promising strains havebeen used in challenge experiments with oyster larvae. General findings are that the probiotic strains
can support improved survival and growth of larval oysters, alone and in the presence of pathogens, butthat the effectiveness depends on the dose administered. Experiments are continuing to refine effective
administration of probiotic strains.
The branch’s HAB research has focused primarily on trophic interactions between bivalves and HABtaxa, including dinoflagellates, prymnesiophytes, and raphidophytes. Our main accomplishments in thisarea are: 1) demonstrated variable expression of toxic effects by the dinoflagellate Prorocentrum minimum
on several shellfish species, including pathologies, immunomodulation, and mortality; 2) developed a
new method to produce large numbers of Alexandrium resting cysts, allowing experiments demonstrating accumulation of saxitoxins in oysters feeding on the resting cysts; 3) revealed a widespread risk that
HABs can be introduced into receiving waters when bivalves are transplanted, but 4) found a cost-effective means to mitigate this risk by holding shellfish 24 hours out of water between harvest and transplant; and 5) showed that bivalves infested with parasitic diseases are more susceptible to immunomodulation and development of pathologies by HABs than are nonparasitized individuals.
In addition to research, the branch provides direct support to the shellfish industry by convening the
Milford Aquaculture Seminar and Milford Microalgal Culture Workshop, by providing microalgal seed
cultures and advice on their use in commercial hatcheries, and by troubleshooting microalgal culture anddisease problems in hatcheries, either on-site or remotely. These activities serve industry throughout theUnited States, not just in the northeast region.

Wikfors

69

The Importance of Nutrients, Light Duration, Light Intensity, and pH
when Growing Large Volumes of Phytoplankton
Shannon L. Meseck
Northeast Fisheries Science Center, Milford Laboratory
Aquaculture and Enhancement Division, Biotechnology Branch
212 Rogers Avenue
Milford, Connecticut 06460
Shannon.L.Meseck at noaa.gov
Keywords: mass cultures, microalgae
Abstract

A number of variables, including pH, light intensity, day length, nutrient availability, and temperature, are important in mass culturing microalgae. In aquaculture, large volumes of phytoplankton foodare often grown outside to reduce culturing costs. However, mass cultures of phytoplankton outdoors
are complex because light intensity, day length, and temperature are not as easy to control as in the laboratory. Furthermore, outdoor cultures often become contaminated with bacteria and other algal species.
This presentation focused on how different light intensity, day length, pH, and contaminates that areoften seen when growing cultures outdoors can affect the growth of a phytoplankton feed.

70

MeseckPhytoplankton Ecology and Aquaculture
Judy Yaqin Li
Northeast Fisheries Science Center, Milford Laboratory
Aquaculture and Enhancement Division, Biotechnology Branch
212 Rogers Avenue
Milford, Connecticut 06460
Judy.Yaqin.Li at noaa.gov
Keywords: phytoplankton, shellfish, toxicity, scallops
Abstract

Phytoplankton provide not only food for shellfish, but also can include species harmful or toxic
to shellfish. Shellfish, on the other hand, as one major group of consumers of phytoplankton in somecoastal marine environments, do not affect phytoplankton by simply reducing their biomass, but also by
selectively consuming some groups of phytoplankton and by providing recycled nutrients. Thus the interactions between phytoplankton and shellfish can be complex. My research is aimed at studying suchinteractions in laboratory, seminatural, and in the near future, natural aquaculture settings.
In the laboratory, we examined the possible relationship between the toxicity of the dinoflagellate
Prorocentrum minimum to bay scallops (Argopecten irradians irradians) and proteins that this harmful
alga excretes into the culture medium. Although P. minimum is generally considered a harmful species,
its toxicity varies between strains and according to different physiological status.
In this study, a gradient of toxicity of this particular strain was achieved by reducing the supply ofphosphate or carbon. Using state-of-the-art protein-profiling technology—the ProteinChip and surface
enhanced laser disorption ionization, time of flight mass spectrometry (SELDI-TOF-MS)—a number ofproteins were detected and some patterns of protein expression associated with toxicity were revealed.
This study can help with the identification of the toxin or toxins responsible and may lead to the development of tools for detecting P. minimum toxicity and providing an early warning in a natural environment.
The impact of scallops on their surroundings was examined in a close-to-natural, yet controlledenvironment by using 10 m–long raceway tanks with constant flow of seawater. In this seminatural setting, scallops were exposed to natural seawater with mixed particles, and the net uptake of particles wasquantified by analyzing inflows, outflows, and settling. The removal of phytoplankton by scallops wasquantified, but the other effects of scallops upon phytoplankton community structure, such as those attributable to the change in nutrient supply, could not be examined as the residence time in the tank was
too short.
Thus our next step will be examining the interaction of shellfish with the environment in a natural
aquaculture setting.

Li

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Cross-field Technologies in Aquaculture
Barry C. Smith
Northeast Fisheries Science Center, Milford Laboratory
212 Rogers Avenue
Milford, Connecticut 06460
barry.smith at noaa.gov
Keywords: software, cross applications, aquaculture
Abstract

When determining how to do something new, it makes sense to consider whether there are tools already available that can be used. Often tools and techniques, collectively referred to as technologies, aredeveloped within specific fields or industries for specific purposes. These techniques often can be transferred, with or without modification, to other seemingly unrelated applications.
Adaptation, scale-up, and scale-down of existing technologies for aquaculture applications will havespecial considerations and conditions under any given circumstance. Requirements for precision may
constrain the level of technology to apply. For example, there may be no reason to invest in industrial
process control software and programming if a light timer can accomplish the task needed. However,process control software and requisite interfaces are desirable for research and development applications where requirements evolve and change. One of the most important aspects of these considerations
is a thorough knowledge of not only the engineering aspects of a project but also how an aquacultured
organism can perform in the given conditions.
Hollow fiber filters, as well as tangential flow and membrane plate filters, were developed for the
food processing and blood dialysis industries. These filters are readily employable in the aquacultureindustry. However, prefiltration is absolutely essential in aquaculture applications. Industrial processcontrol software and circuit systems are steadily being applied to aquaculture processes as the aquaculture industry grows and evolves. Cross applications such as these are only a few examples of what has
been accomplished and what is still to come as aquaculture evolves.

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Smith

An Overview of Genetic Studies on Commercial Species of Bivalves
Sheila Stiles, Joseph Choromanski, and Dorothy Jeffress
Northeast Fisheries Science Center, Milford Laboratory
212 Rogers Avenue
Milford, Connecticut 06460
sheila.stiles at noaa.gov
Keywords: genetics, inbreeding depression, markers
Abstract

With significant advances in the culture of marine organisms has come increased interest in improving genetic traits—especially those of economic importance, such as growth, survival, and disease resistance—for increased productivity.
The overall goal of breeding or management of a species is to maximize productivity. An understanding of what it takes to maximize production enables the breeder or culturist to recognize signs ofinbreeding depression which could be manifested as slower growth, decreased viability, disease susceptibility, or overall decreased production. An example of inadequate attention to genetic consequences ofculture with bay scallops (Argopecten irradians irradians) occurred when some of the industry collapsed
which was attributed to inbreeding depression from a narrow gene pool.
Genetics of commercial oysters, clams, and scallops were reviewed from such perspectives encompassing three aspects: breeding or quantitative genetics, chromosomal or cytogenetics, and molecular(DNA) genetics. These areas of genetics have been applied to commercial bivalves separately and incombination with varying degrees of success as measured by different responses for hatchery culture andin field programs.
Conventional approaches consist of breeding methodology similar to that applied historically in agricultural genetics with the domestication of farm animals and crops. Selective breeding and heritability
values have indicated positive responses for growth in bivalves. Chromosome manipulation to induce
polyploidy and cloning also has produced some favorable results. Alternatively, biotechnological techniques can be employed to facilitate progress in improvements.
Various types of molecular markers are being used to supplement conventional approaches of breeding to improve characteristics with quantitative trait loci in marker-assisted selection. In addition, genetic markers are being used to identify stocks and estimate genetic diversity of wild populations. DNA
markers have been observed to result in variation with many alleles that could be useful for applications
such as species, stock, and population identification. If environmental and habitat qualities are not suitable, however, genetic improvement in desired traits may not find expression, an important consideration
for future developments in increasing the commercial production of bivalves.

Stiles et al.

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