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

and

Milford, Connecticut

November 2, 2007

Aquaculture Technologies

for Invertebrates

Proceedings of the Thirty-sixth

U.S.-Japan Aquaculture Panel

Symposium

Durham, New Hampshire

October 29-30, 2007

and

Milford, 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

*
ii

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.
iii

Table of

Contents 1

2

3

7

8

15

17

18

19

20

Aquaculture Overview

The Basic Plan for Fisheries and Current Status of Aquaculture 

Research in Japan

Kazumasa Ikuta

Overview of NOAA’s Aquaculture Program

Michael Rubino

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 

Mushiake

Marine Ornamental Aquaculture

Junda Lin

Nutritional Significance of n-3 Highly Unsaturated Fatty Acids 

for Larval Survival and Development in Mass Production of 

Brachyuran Crab Larvae

Shigeki Dan, Katsuyuki Hamasaki, Takayuki Kogane, Tadao 

Jinbo, and Takashi Ichikawa

The Chesapeake Bay Blue Crab (Callinectes sapidus): 

A Multidisciplinary Approach to Responsible Stock 

Enhancement

Oded Zmora, Yonathan Zohar, Anson H. Hines, and

Eric G. Johnson

An Outline of the Research Project, Development of Seed 

Production Technology in Japanese Spiny Lobster

Hideaki Aono, Keisuke Murakami, and Masahiko Awaji

Mollusk Culture

Shellfish Aquaculture Research on Oysters and Clams at 

Haskin Shellfish Research Laboratory

John Kraeuter

Improvements in Yield of the Pacific Oyster (Crassostrea 

gigas) by Selective Breeding

Chris Langdon, Ford Evans, and Alan Barton

Synthesis of Vitellogenin in the Pacific Oyster (Crassostrea gigas)

Toshie Matsumoto
iv

Disease Diagnostics and Treatment of Hemic Neoplasia in the Softshell 

Clam (Mya arenaria)

S. Anne Böttger and Charles W. Walker

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

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

Muki Shpigel

Considerations on the Fine-scale Topography in Sand Flats, Habitat 

Heterogeneity, and Refugia for Clams

Hajime Saito, Hideyuki Takahashi, Akihiko Matsuda, Hisami Kuwahara, Yuka Ishihi, 

Tomoko Sakami, and Junya Higano

Methods, Economics, and Commercialization of Open Ocean Mussel 

Culture in the Northeast United States

Richard Langan

Bivalve Mollusk Culture on the U.S. West Coast

Bill Dewey

Echinoderms and Other Species

Relationship between Gametogenesis and Food Quality in Sea Urchin 

Gonads

Tatsuya Unuma and Charles W. Walker

An Overview of the Culture of Marine Invertebrates in Maine

Dana L. Morse

Polyculture of Red Abalone (Haliotis rufescens) and Pacific Dulse

(Palmaria mollis) in a Land-based Recirculation System

Chris Langdon and Ford Evans

Wave Induced Flow in Seawater Exchange Structures for Improving 

Seawater Quality

Yoshihiro Ohmura

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

Porphyra

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

Sakamoto

25

26

31

32

43

44

45

55

56

57

65
v

66

67

68

69

70

71

72

73

Milford Laboratory Minisymposium Presentations

Culture Systems and Habitat Evaluation Branch Overview—Molluscan 

Aquaculture

Ronald Goldberg

Bay Scallop (Argopecten irradians irradians) Husbandry Program

James C. Widman Jr.

Growth and Stress Response of Bivalve Mollusks in Response to Different 

Microalgal Diets and Arachidonic Acid Supplementation

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

Biotechnology Branch Overview on Probiotic Bacteria and Harmful Algal 

Bloom Research

Gary Wikfors

The Importance of Nutrients, Light Duration, Light Intensity, and pH when 

Growing Large Volumes of Phytoplankton

Shannon Meseck

Phytoplankton Ecology and Aquaculture

Judy Yaqin Li

Cross-field Technologies in Aquaculture

Barry C. Smith

An Overview of Genetic Studies on Commercial Species of Bivalves

Sheila Stiles, Joseph Choromanski, and Dorothy Jeffress
vi
vii

Introduction The U.S.-Japan Cooperative Program in Natural Resources (UJNR) was established 

on January 29, 1964. Since its creation, the UJNR has evolved to become one of 

the oldest and most effective cooperative agreements between Japan and the United 

States. In 1969, the UJNR Aquaculture Panel was created as a vehicle for scientists 

of both countries to meet and discuss aquaculture research accomplishments, needs, 

and priorities, as well as provide opportunities for cooperative research and scientific 

exchange.

Under UJNR Aquaculture Panel auspices, U.S. and Japanese scientists have met 

annually since 1971 without interruption. The venue for these meetings has alternated 

between 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 of 

brief papers or abstracts.

Hiroshi Nakano, Japan Panel Chairman

Robert Iwamoto, United States Panel Chairman

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, Japan

ikutak 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 based 

on 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
2

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 research 

and the importance of nurturing important international partnerships, such as the U.S.-Japan Natural 

Resources Panel on Aquaculture.

Rubino
3

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, Japan

tsugaya 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) and 

mitochondrial 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 related 

groups 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 the 

temperate 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 establishment 

of stock enhancement programs resulting in the 

annual 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 of 

suitable tags to distinguish introduced shrimp from 

wild ones at all life history stages. In addition, the 

massive 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 et 

al. 1979). In particular, the control region that does 

not code for proteins or RNA shows high variability 

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 with 

unknown pedigree (Finch et al. 1996, Cronin et al. 

1999). Therefore, DNA markers should be useful for 

the 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 seven 

experimentally 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 calcuSugaya et al.
4

lated from multilocus 

genotypes of five MSDNA loci. The averages 

of the distances were 

0.392 ±0.161 within 

siblings and 0.783 ±0.138 

between siblings. Moreover, each sibling was 

clearly distinct on an 

unweighted pair group 

method with arithmetic 

mean (UPGMA)–based 

dendrogram constructed 

from the identities 

among individuals 

(Figure 1). These results 

demonstrate the availability of the MS-DNA 

markers for pedigree 

analysis estimating genetic relatedness among 

individuals in shrimp 

populations of unknown pedigree.

The Detection of Released Shrimp

in a Stocking Area

The feasibility of the DNA markers as tags for 

released shrimp was examined with a mark-recapture experiment in Saiki Bay along the eastern coast 

of Kyusyu island in southern Japan (Figure 2). Two 

hundred 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 by 

experimental trawling and gill net fishing by commercial fishermen. The pedigree analysis by the 

MS-DNA and mtDNA markers detected 51 offspring 

from the six females in the samples. The contributions of the offspring ranged from 2 to 17.2% on a 

monthly basis (Figure 3). From that data we can 

conclude that the microsatellite and mitochondrial 

DNA markers are useful as tags for released kuruma

prawns in stocking programs. In addition, the relatedness analysis of the samples revealed the existence 

of many related shrimp in the bay. Although mean 

genetic relatedness (rxy) of Queller and Goodnight 

(1989) estimated by the five MS-DNA markers was 

almost zero, the relatedness among individuals sharing common haplotypes of mtDNA markers were 

from 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 fishery 

union. The relatedness analysis is considered to 

have detected those released prawns. This may 

show the possibility of the analysis of large-scale 

stocking effectiveness estimation.

Figure 1. Genetic relationship 

among seven full-sib groups: 

Group 1 = l

Group 2 = p

Group 3 = i

Group 4 = r

Group 5 = o

Group 6 = o, and

Group 7 = n.

Sugaya et al.
5

Analysis of Genetic Relatedness Structure

of 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 from 

the information of the five MS-DNA loci ranged 

from 0.059 to 0.063. The values from Kagoshima and 

Kumamoto were significantly higher than simulated 

values estimated on the assumption of random 

mating (permutation test, p < 0.05). The frequencies 

of rxy in Kumamoto and Kagoshima were high 

(permutation test, p < 0.05, Figure 6). Furthermore, 

the relationship between rxy and the distribution 

of the haplotypes of mtDNA markers examined 

by 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 theoretical 

values of rxy were approximately 0.5 among the full 

sibs. In the East China Sea, including Kumamoto 

and Kagoshima, 40–90 million kuruma prawns have 

been released annually. Since those shrimp are generally reared from a limited number of parents, the 

release might explain the closely related individuals detected in those two localities. However, these 

results do not necessarily show the gene flow among 

wild and released populations, as we cannot deterSugaya et al.

Figure 2.

Location of

Saiki Bay.

Figure 4. Relatedness distributions among the individuals with 

common haplotypes (dotted line) and among those with different haplotypes (solid line) each month.

Figure 3.

Distribution of 

released prawns 

detected by 

pedigree 

analysis in each 

month.

Figure 5.

Sampling location of 

wild kuruma prawn.

mine whether the closely related individuals were 

born in the wild or in a hatchery. Hence the magnitude of gene flow should be examined to reveal the 

genetic effects of release in the future.
6

References

Brown, W. M. 1985. The mitochondrial genome of 

animals. 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 mitochondrial 

DNA. Proc. Natl. Acad. Sci. 76:1967–1971.

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 the 

Penaeidae. Adv. Mar. Biol. 27:1–489.

Finch, M. O., and D. M. Lambert. 1996. Kinship and 

genetic 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 in 

Japan. 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.

Tauz, D. 1989. Hypervariability of simple sequences 

as a general source for polymorphic DNA markers. 

Nucleic Acids Res. 17:6463-6471.

Sugaya et al.

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.

Figure 6. Distribution of relatedness among individuals calculated from the data of MS-DNA in localities of Kumamoto and 

Kagoshima. Relatedness less than zero was regarded as zero. 

Observed value = o, simulated value ±95% confidence interval 

(CI) = l, and significant difference = *.
7

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 and 

toxins (e.g., cyanides), have caused grave concerns. In recent years, efforts have been made to understand 

the 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 or 

without mating) again. The larvae are composed of many (9–13) zoea stages. Artemia nauplii can be 

used 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 caretakers 

of 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 demand 

higher 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
8 Dan et al.

Nutritional Significance of n-3 Highly Unsaturated Fatty Acids for Larval Survival and 

Development in Mass Production of Brachyuran Crab Larvae

Shigeki Dan

Fisheries Research Agency

Seikai National Fisheries Research Institute

Ishigaki Tropical Station

Ishigaki, Okinawa 907-0451, Japan

sdan 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 large 

tanks, 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. Horsehair 

crab 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-
9

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.

Dan et al.

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 been 

studied 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 are 

warm water species, snow crab and horsehair crab 

inhabit cold water.

Between 1993 and 2005, approximately 50 million juveniles and several million juveniles of the 

first 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 mass 

mortality 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 the 

other hand, large-scale larval production techniques 

remain undeveloped for snow crab and horsehair 

crab (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) and 

docosahexaenoic 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 well 

known 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 been 

studied in detail. Since the early 1990s, we have 

investigated this issue to improve and develop mass 

production technologies for brachyuran crabs.

We conducted larval rearing experiments for 

swimming crab, mud crabs, horsehair crab, and 

snow crab using small vessels and large larval 

production tanks with live foods containing various 

levels of n-3 HUFA and examined the significance 

of dietary n-3 HUFA for larval development and 

survival of the species of interest. In this paper, we 

summarize the results of our experiments and discuss future prospects for the development of mass 

seed production techniques of brachyuran species 

from the point of view of n-3 HUFA requirement by 

larvae.

Materials and Methods

Rearing experiments in small vessels. Larvae of 

each 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 beaker 

with seawater and prey (rotifers, Brachionus plicatilis

sp. complex, or Artemia sp.). The larval developmental stages were determined when they were 

transferred 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 first 

zoeal stages for mud and snow crabs, respectively. 

Phytoplankton (Chlorella vulgaris, Nannochloropsis

sp.) were also added to the tanks as food for the 

rotifers and Artemia. The rotifers were enriched with 

brewer’s yeast or phytoplankton containing cellular 

n-3 HUFA before being fed to the larvae. Larval 

survival rates were estimated by the volumetric 

method. The length of chelae and carapace of fifth 

stage zoea of mud crabs were measured under a 

microscope 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.
10

Fatty acid analysis. The n-3 HUFA contents of 

larvae 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 and 

analyzed with a gas chromatograph equipped with 

a silica capillary column. Fatty acid methyl esters 

were identified by comparing retention times against 

standards.

Results and Discussion

Effect of dietary n-3 HUFA on larvae of swimming 

and mud crabs. Larvae of the swimming crab and 

the two mud crab species fed rotifers and Artemia

enriched with n-3 HUFA showed higher survival 

rates than larvae fed unenriched live foods in the 

rearing experiments using small vessels (Takeuchi et 

al. 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 larvae 

of 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 fail 

to 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 during 

molting); type B, partial abnormality (larva has shed 

its exuvia but was unable to shed the integument 

of 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, percentages 

of larvae showing abnormal molting were plotted 

against the relative chelae lengths as an index of the 

degree of morphogenesis of fifth stage zoea (Figure 

4). Almost all larvae with a relative chela length 

exceeding 50% failed to molt successfully and the 

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 

Dan et al.

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 production 

of 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.)
11

tanks with phytoplankton that contained different 

supplementary levels n-3 HUFA in their cells and 

relative chela length of last stage zoeas (Figure 5). 

The relative chela lengths increased with increasing 

n-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 was 

around 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) (see 

Figure 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).

Dan et al.

Figure 3. Photographs show the molting patterns during the 

metamorphosis to megalops in the production of S. serrata. Type 

A, 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 the 

chelipeds 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.)

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 et 

al. 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.)
12 Dan et al.

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 lengths 

of 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 larval 

morphogenesis should be examined to evaluate the 

rearing experiments with swimming crab and mud 

crabs.

Effect of dietary n-3 HUFA on larvae of horsehair 

and snow crabs. Horsehair crab larvae were reared 

in 2 L beakers with Artemia unenriched and enriched 

with n-3 HUFA (Jinbo unpubl. data). Horsehair 

crab larvae showed high survival rates to the first 

crab 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 the 

larval rearing tanks and snow crab larvae reared in 

large 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 growth 

through second stage zoea to megalops stages and 

larval mass mortality occurred (Figure 7) during metamorphosis to megalops (Kogane et al. 2007b). This 

was 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 to 

improve the mass production of snow crab.

Our recent research demonstrated that dietary 

n-3 HUFA is essential to improve larval survival and 

development in the production of swimming crab, mud 

crabs, and snow crab. However, the requirement by 

larvae of brachyuran crabs for n-3 HUFA varied with 

species. So, strategies for n-3HUFA enrichment in live 

food require individual verification for each species in 

future research.

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 on 

data from Kogane et al. 2007b.)

Figure 7. Survival rates of larvae (o) and percentage composition of megalops (p) of the snow crab (Chionocetes opilio) reared 

in 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, especially 

K. Murakami and M. Ashidate for their kind hospitality and cooperation throughout the experiment.
13

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Zmora et al. 15

The Chesapeake Bay Blue Crab (Callinectes sapidus):

A Multidisciplinary Approach to Responsible Stock Enhancement

Oded Zmora and Yonathan Zohar

University of Maryland Biotechnology Institute

Center 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 (more 

than $100 million in value), dropped over 70% from record highs in the early 1990s. Overfishing and 

environmental 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 under 

carrying capacity. This situation makes the Chesapeake Bay blue crab an excellent candidate for stock 

enhancement 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 the 

potential 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 monitoring 

of 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 some 

minor differences in morphology and behavior.

Enhancement doubled to tripled the wild population in release sites, and survival from release to 

sexual 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 the 

spawning sanctuary in the lower bay, suggesting that hatchery crabs can contribute to the spawning 
16 Zmora et al.

stock as soon as several months postrelease. This, together with evidence showing the effectiveness of 

the 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.
Aono et al. 17

An Outline of the Research Project, Development of Seed Production Technology in 

Japanese 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 of 

the 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 hindered 

significant 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 and 

develop 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.
18 Kraeuter

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 eastern 

oysters (Crassostrea virginica) using our 50-year breeding program that has developed disease resistant 

lines. 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) and 

Dermo (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 of 

oysters in Delaware Bay using classic shelling techniques. This restoration program is being coupled 

with 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 to 

model 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 hard 

clam 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 disease 

on growth and reproduction in France.

In other studies, we have just begun a small project investigating the potential to use aquaculture to 

enhance 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).
Langdon et al. 19

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 broodstock 

oysters 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 the 

selection 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.
20

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 nematode 

species, especially in the N-terminal region. Vitellogenin mRNA expression was detected only in the 

ovary. To determine the distribution of oyster vitellogenin mRNA expression in the ovary, we performed 

in 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 other 

mollusk 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 et 

al. 1992). In the oyster, vitellin has been isolated and 

characterized (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 Pacific 

oyster (Crassostrea gigas) Vg cDNA and examined the 

expression of its mRNA.

Estrogen receptors (ERs) are members of the 

nuclear receptor superfamily, which have a number of common features, and their proteins can be 

divided into several distinct domains (Krust et al. 

1986). The DNA-binding domain (C domain) and 

the 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 is 

clearly established that the Vg gene expression is 

regulated 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
21

Estrogens have been detected in blue mussels (Mytilus edulis) and softshell clams (Mya arenaria) and are 

considered to have various physiological functions 

in bivalves (Reis-Henriques et al. 1990, Stefano et al. 

2003, Gauthier-Clerc et al. 2006). In the oyster and 

scallop, 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 control 

of 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 or 

other steroid hormone receptors have yet been 

cloned 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 the 

ovary, 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 a 

cDNA 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 cDNA 

is 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 acid 

residues with a predicted molecular mass of 179,191 

Da. The deduced amino acid sequence contained a 

consensus cleavage site, R-X-R-R (Arg828 to Arg831), 

capable of undergoing processing by endoproteases of the subtilisin family (Barr 1991) that have 

also 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%) and 

Matsumoto

the 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 the 

polyserine 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 compared 

with Vg of the other known species.

The levels of Vg mRNA in various tissues from 

female 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 the 

follicle cells using an antisense RNA probe (Figure 

1a). 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 and 

other bivalve species, the ovarian acinus is a simple 

Figure 1. Localization of putative Vg mRNA in the ovary by in 

situ hybridization. A) Strong expression of putative Vg was 

seen 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.
22 Matsumoto

structure containing only developing oocytes and 

associated follicle cells within a thin germinal epithelium. The function(s) of follicle cells in the bivalve 

ovary are not well understood, although they are 

suspected 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. This 

suggests that yolk synthesis in Pacific oysters may 

take place in the follicle cells.

Oyster Estrogen Receptor (cgER)

Using the degenerate primers derived from 

conserved regions within human ERs, we cloned a 

PCR fragment from oyster ovary. The amino acid 

sequence 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 the 

mature receptor protein, based on its deduced amino 

acid sequence, is 53.6 kDa. The deduced protein 

contains eight cysteine residues that constitute two 

zinc finger structures and P-box (CEGCKA) in the C 

domain. The position of cysteine residues and other 

residues 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 groups 

of ER: ER-a, ER-b, and mollusk ERs and cgER is 

most closely related to the other mollusk ERs (Figure 

2). This indicates that the cgER we isolated is an 

ortholog 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 the 

results 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 proteins 

in 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 

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 of 

oocytes (Figure 3a), suggesting that estrogens play 

various roles in regulating reproductive events. Our 

results will facilitate further functional research to 

understand the roles of estrogens in the oyster.

Figure 2. Phylogenetic tree based on amino acid sequences of 

ERs. The unrooted phylogenetic tree was constructed by the 

neighbor-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 replicates 

and expressed as percentages. Scale bar = 0.1 substitutions per 

site.
Matsumoto 23

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and its expression in the hepatopancreas and ovary 

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of target gene specificity for steroid/thyroid hormone receptors. Cell 57:1139–1146.

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Böttger and Walker 25

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 was 

a 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 of 

very 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 by 

mortalin 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.
26

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 Institute

3-27-5 Shinhama

Shiogama, Miyagi 985-0001, Japan

tsuzuki 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 periodically 

monitored 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), harvesting 

ceases. 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 for 

shellfish 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 in 

bivalves 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 from 

various 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 distributed 

on 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 

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 tract 

and diarrhea (Terao et al. 1986). OA analogues have 

also been shown to have tumor-promoting activity 

Suzuki et al.
Suzuki et al. 27

(Fujiki et al. 1988). In contrast to OA analogues, it 

has been suggested that the risk assessment of the 

other 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 for 

shellfish testing that can quantify each toxin group 

separately. 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 collected 

from production areas and lipophilic toxins in the 

bivalves were analyzed by our recently developed 

LC-MS method (Suzuki et al. 2005). A comparison 

of the quantitative results obtained for these bivalve 

samples between LC-MS and mouse bioassay (MBA) 

was carried out to investigate the applicability of this 

LC-MS method for routine monitoring of lipophilic 

toxins in Japanese bivalves.

Materials and Methods

Samples. Approximately 200 bivalve samples were 

collected 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 monitoring 

site 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 the 

homogenate was kept frozen at –30°C until used for 

extraction of toxins for LC-MS.

Figure 1. Structure of typical lipophilic toxins found in Japanese 

bivalves. Okadaic acid is a rare toxin in Japanese bivalves.

Figure 2. Production area of bivalves where harvesting was 

prohibited due to DSP events in 2003.
28 Suzuki et al.

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, the 

toxicity of the extracts is defined as greater than 1 

mouse unit (MU) and the whole contents are defined 

as 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 obtained 

by 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 clean 

chromatogram 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 scallop 

and mussel samples. Ninety-six samples were quantified by both LC-MS and MBA as being below the 

quarantine 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. 

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 the 

extracts were centrifuged at 3,000 rpm for 5 min. An 

aliquot of each supernatant was passed through a 0.5 

μm 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) equipped 

with an atmospheric pressure electrospray ionization (ESI) interface. Separations were performed 

on 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 et 

al. 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 taken 

for 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/z 

803.5), 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/z 

933.5), and for [M-2Na+H]- of YTX (m/z 1141.5), 

45-OHYTX (m/z 1157.5). The SIM scan width for 

each 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 sheath 

gas (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 Ministry 

of Health and Welfare 1981). Whole tissues and 

separated HP of each sample were weighed and the 

weight percentage (w/w%) of the HP was calculated. The homogenate of the HP was extracted with 

acetone three times. After removal of the acetone by 

evaporation, the aqueous suspension was extracted 

with diethyl ether. The ether solution was back-
Suzuki et al. 29

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 are 

poorly 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 is 

noteworthy that the numbers of samples exceeding 

the quarantine level increase if MBA is replaced with 

LC-MS in routine monitoring of toxins in Japanese 

bivalves (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 is 

thought 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 is 

applied to the toxin monitoring in Japanese bivalves, 

an adequate control limit for the YTXs as proposed 

by 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 for 

these 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

The research was funded through a research 

project of Ministry of Agriculture, Forestry, and 

Fisheries of Japan: Research Project for Utilizing 

Advanced Technologies in Agriculture, Forestry and 

Fisheries (No. 1504). We express our gratitude to the 

collectors of the bivalve samples.

Figure 3. SIM LC-MS chromatogram for a standard mixture (100 

ng/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/z 

1141.5).

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).
30 Suzuki et al.

Suzuki, T., T. Jin, Y. Shirota, T. Mitsuya, Y. Okumura, 

and T. Kamiyama. 2005. Quantification of lipophilic 

toxins associated with diarrhetic shellfish poisoning 

in 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 from 

the 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 mouse 

assay 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 the 

small 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 

44:1249–1255.

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Landsverk. 2002. Comparison of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon 

40:7782.

Bialojan, C., and T. Takai. 1988. Inhibitory effect 

of a marine-sponge toxin, okadaic acid, on protein 

phosphatases. Specificity and kinetics. Biochem. J. 

256:283–290.

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. 

79:1089–1093. 

Japanese Ministry of Health and Welfare. 1981. 

Food Sanitat. Res. 7:60.

Lawrence, J. F., R. K. Chadha, W. M. Nimal Ratnayake, and J. F. Truelove. 1994. An incident of elevated 

levels 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, and 

preliminary 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.

Ramstad, H., S. Larsen, and T. Aune. 2001. Repeatability and validity of a fluorimetric HPLC method 

in 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.
Shpigel 31

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 of 

area, 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 the 

quality 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 with 

the 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 a 

valid alternative to open sea monoculture.
32 Saito et al.

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 Institute

Miyogi 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 derive 

some 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 mean 

log transformed individual numbers of Manila clams, effects of position between sandbars and within a 

sandbar 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 three 

crests 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 to 

understanding the interactions between the mortality source and the distribution and abundance of 

refugia (Woodin 1978). Spatial patterns of benthic 

organisms on sand flats are a result of physical and 

biotic processes. Recruitment of benthic organisms and their larvae through bedload transport 

has crucial roles in recovery processes of disturbed 

communities. Hydrodynamic processes have a 

significant 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. 33

To establish sound strategies for management 

of 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 alternation 

of 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 beds 

of polychaetes persist for several years and offer a 

refuge wherein infaunal diversities are greater than 

those of more hydrodynamically rigorous areas 

(Bolam and Fernandes 2002). Local aggregations of 

Manila 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. Grain 

size 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 substrate 

is the smallest structure, on the order of 0.1–1 mm. 

Ripples and dunes on the order of 1–10 cm are 

often seen on the sandy bottom. Multiple sandbars, 

ridges, and runnels are large structures on the order 

of 10 m developing on sand flats exposed to strong 

wave forces.

Besides spatial structures formed by physical 

processes, biological and human activities modify 

the surface structure of sand flats (e.g., mounds, 

tubes, mats, pits by predation, digging, and human 

footprints). These structures produce shaded and 

exposed sides to wave and current forces, and this 

differentiation in the strength of physical disturbance may be an important factor that creates habitat 

heterogeneity on sand flats. Multiple sandbars are 

apparent and their shapes are easily captured using 

topographical survey techniques.

Roughness of the sand surface can be estimated 

from the grain size distribution of the sediment. It 

is difficult to capture the real shape of ripples in the 

field. Wave length and amplitude of ripples can be 

obtained with replicate measurements in the field, 

but their detailed shapes cannot be included in the 

data.

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 (or 

more) slip faces, and there is vortex shedding from 

the 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 sediment 

characteristics 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 the 

eastern 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 sizes 

of 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 the 

subtidal zone and in deep troughs of sandbars that 

emerge at low water during spring tides. Bivalves 

predominate 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 line 

transect from the coastal sand dune to the point 

of low water during spring tide. Location of the 
34 Saito et al.

line transect was determined to make elevational 

changes over sandbars along it as contrasting as 

possible. On 6 September 2006, land elevations at 

haphazardly chosen points scattered along the line 

transect 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 and 

it was shown that the upper intertidal zone was a 

gradual slope while multiple sandbars developed 

in the lower intertidal zone (Figure 2). Wave length 

of 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 (5 

and 6) were established on the foreshore gradual 

slope (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 the 

calibration process. The cameras were mounted in 

parallel 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 sand 

surface 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 replicates 

were obtained from the crests of sand ripples and 

another 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 in 

the laboratory and organisms were sorted under a 

binocular microscope. Shell lengths of specimens of 

Manila clams and surf clams were determined, and 

the 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 the 

model was evaluated employing the maximum likelihood method. Intersections of normal distribution 

curves were recognized as the border of different 

Figure 1. Locality of Matsunase Beach tidal flat and the position 

of the line transect for this study.

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

sampling stations.
Saito et al. 35

size groups, and the individual number of separated 

size groups was counted. In order to focus on the 

relationship 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” function 

in R, a high level language and an environment for 

data 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 of 

ripple (R, n = 2) crossed with the effect of location 

of sandbars (B, n = 5). The other model was composed of two orthogonal fixed factors, the effect of 

ripple (R, n = 2) crossed with the effect of position 

within the middle sandbar (P, n = 4). Homogeneity 

of variance between sampling stations was tested 

with Cochran’s test for variance outliers (Kanji 2006). 

Post hoc multiple comparisons were performed to 

detect subgroups between sampling stations using 

Tukey HSD.

The three-dimensional shape of the sand surface enclosed with the square unit in the field was 

visualized 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 the 

images 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 for 

adjusting the lens distortion. The calibration file 

was prepared using PI-Calib software (Topcon Co.). 

The shape of the sand surface was visualized as a 

triangulated irregular network (TIN) produced in 

5 mm grids. Inclination of the sand surface against 

the camera angle was removed by subtracting the 

height of the regressed plain from the corresponding 

apex of TIN. Then the vertical profile of ripples was 

sectioned along an assigned line rectangular to the 

crest line of ripples.

Results

The cohort analyses revealed that there were 

several 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 results 

of cohort analyses.
36

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 case 

of surf clams. Tests on the effects of ripples and 

sandbars (including the two extra stations in the 

foreshore 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), and 

interaction 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 the 

Saito et al.

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 and 

crest of sand ripples (R) were included in the model as fixed factors.

Df Sum sq Mean sq F value Pr(>F) Significance

R. Philippinarum

P 3 3.188 1.063 23.479 0.000 *** 

R 1 0.003 0.003 0.071 0.793 ns

P x R 3 0.016 0.005 0.120 0.947 ns

Residuals 16 0.724 0.045

M. veneriformis

P 3 0.186 0.062 1.298 0.309 ns

R 1 0.016 0.016 0.332 0.572 ns

P x R 3 0.078 0.026 0.542 0.660 ns

Residuals 16 0.764 0.048

R. philippinarum

B 4 3.328 0.832 34.208 0.000 ***

R 1 0.000 0.000 0.004 0.949 ns

B x R 4 0.016 0.020 0.822 0.527 ns

Residuals 20 0.486 0.024

M. veneriformis

B 4 1.327 0.332 7.855 0.001 ***

R 1 0.000 0.000 0.000 0.991 ns

B x R 4 0.562 0.141 3.327 0.030 *

Residuals 20 0.845 0.042

null hypothesis about homogeneity of variance presupposed in ANOVA was retained in all tests above 

(p > 0.05).

In multiple comparisons, replicate samples from 

troughs 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 number 

of Manila clams in the trough of the middle sandbar 

was significantly greater than those of other positions (Figure 4). Subgroups detected were shown 

alphabetically. 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 
37

Within the middle sandbar

Between sandbars

Saito et al.

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.

sandbar was prominently greater than those in other 

stations. Mean log-transformed individual numbers 

of surf clams were overlapped among five stations.

The interaction term B×R was significant in 

ANOVA though the difference between troughs and 

crests 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 that 

ripples on the trough of the middle sandbar were 

concave 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 were 

overlaps of ripples having different phases on the 

onshore and the offshore slopes. Along the crests 

of sandbars, ripples on the landward sandbar were 

rather concave upward while ripples on other sandbars were convex upward (Figure 7). Ripples were 

slightly 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 of 

ripples 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 were 

0.17, 0.10, 0.13, and 0.09, respectively. Steepness of 

ripples in the foreshore slope and the crest of the 

landward 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 
38 Saito et al.

rate of Manila clams of 26–28 mm shell length in the 

field 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 to 

turbulent conditions on the bottom. However, clams 

attached to the objects would be passively transported under strong wave conditions that remove the 

objects 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 et 

al. 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 sand 

flat (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 sand 

surface 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.

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. 39

wave and current forces. If sandbar crests were 

exposed to yearly or more frequent strong wave 

actions, the benthic community on them would be 

severely 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 location 

of 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 was 

reported that the morphology of multiple sandbars, 

referred as ridges and runnels, on Mablethorpe 

Beach, North Lincolnshire, England, responded to 

changing wave conditions (Masselink 2004).

In this study, abundance of Manila clams in 

the trough on the middle sandbar was greater than 

at 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 effect 

of unidirectional current was milder than those at 

the other two stations. On the onshore parts of the 

sandbar zone, the bottom surface may be more protected from exposure than that on the offshore part 

through 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 was 

small, 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 implied 

that the hydrodynamic condition on the bottom of 

the foreshore slope was different from that on the 

crest of the landward sandbar where the clam was 

abundant. Further, extremely shallow parts may be 

harsh environments for the clam because the submerged period is limited. The low abundance of 

Manila 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 to 

oscillatory waves. It has been reported that Manila 

clam abundance was greater in the trough of the 

sandbar than in other positions (Kakino 1996).

Abundance of Manila clams was not significantly different between troughs and crests of sand 

ripples 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 et 

al. 2004) though the height of the ripples was approximately 5–10 cm, which is much greater than 

observed in this study. A technical problem in 

sampling may be another reason why the difference 

was not significant in the present study. The width 

of quadrats used in this study, 5 cm, was not small 

enough 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 this 

study was sometimes smaller than 10 cm. In this 

case, 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 in 

abundance between stations was more contrasting in 

Manila clams. To explain the contrast between the 

two species, behavioral responses to wave exposure 

should be compared in future studies. From the 

skew of sand ripples, it was estimated that landward 

motion is stronger than seaward motion. Small 

clams may experience a high risk to be passively 

transported landward.

In studies on benthic communities, the literature referring to ripple shapes on sand flats is limited (Shull 1997, Barros et al. 2004). However, from 

the 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 nonsteady 

hydrodynamics in turbulent conditions is difficult. 

A modular approach characterizing the ecological 

role of benthos in large coastal seas and estuaries 

identified types of modules that comprise coastal 

systems and depends on the overall complexity of 

geomorphology and coastlines (Tenore et al. 2006). 

The approach aimed at providing insights as to how 

the ecology of the benthos may be related to physical 

variables that are used in classification schemes for 
40 Saito et al.

wave and current exposure. Contrasting with the 

result 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 of 

sand surfaces that is difficult to describe and needs 

to be quantified in the field. Statistical tests on clam 

abundance performed here are not unambiguous. 

Random spatial variations within each sampling station were not included in the tests because the scale 

of square units equivalent to 60x60 cm adjusted to 

the size of stereo pictures was too small to represent 

the whole area considered. To keep the robustness 

of 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 and 

Takuya Maruyama (Mie Prefecture Fisheries Research Institute) for their kind assistance, and we 

greatly 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 Technology 

of Japan.

coastal systems. Fine-scale topographies like sand 

ripples may be used as indices of physical conditions 

in the modular approach.

Three-dimensional analyses on ripple shape 

under water were developed in a previous study 

using video cameras in waterproof housings, though 

measurement 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 to 

intertidal zones. Not being used underwater, our 

method 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 is 

in 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 greater 

than 0.1 are present, they determine the majority of 

the 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 from 

ripple wave length) and taxonomic richness, number 

of individuals, and diversity (DeFelice and Parrish 

2001).

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, the 

abundance of Manila clams was small compared to 

that in other stations (Figure 4). On the other hand, 

ripple steepness was no less than 0.14 in the trough 

on the middle sandbar and the crest of the landward 

sandbar. The abundance of Manila clams there was 

much 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 against 
Saito et al. 41

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Langan 43

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 established 

an 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 used 

in nearshore ocean fisheries was equipped to tend submerged longlines. Gear and technology used in 

surface 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 and 

has 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 yield 

has 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.
44

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, and 

mussels 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 an 

estimated 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. Pacific 

and Grays Harbor counties in southwest Washington rely on the shellfish culture industry to support a 

significant 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.

Dewey
45

Relationship Between Gametogenesis and Food Quality in Sea Urchin Gonads

Tatsuya Unuma

Fisheries Research Agency

Japan Sea National Fisheries Research Institute

Suido-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 Group

Durham, 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 the 

year. 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 and 

transferred 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 urchins 

with 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, knowledge 

of unique characteristics of the sea urchin gonad 

as food products would be useful for successful aquaculture. Here we describe the features of 

gametogenesis, describe the relationship between 

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 release 

gametes to the outside world by a separate gonoduct. Each gonad consists of hundreds of acini and 

resembles a bunch of grapes. Two sacs (Figure 1b) 

of tissues (outer and inner) compose the wall of each 

Unuma and Walker
46

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 

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, after 

Strenger (1973), c) Lobe of a sea urchin gonad stained with phalloidin to show muscles on the exterior surface of the GHS of the 

inner 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 with 

permission from Walker et al. 2006.

from oogonia to ova in the ovary and from spermatogonia to fully differentiated spermatozoa in the 

testis) and somatic cells called nutritive phagocytes 

(NPs) that are present in both sexes (Walker 1982). 

NPs are versatile cells that provide a structural and 

nutritional microenvironment for GCs throughout 

gametogenesis (Figure 2) (Walker et al. 2005). NPs 

have multiple functions. NPs may contain amitotic 

oogonia or spermatogonia and growing primary 

oocytes 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 same 

NPs may also simultaneously contain additional 

GCs at several earlier stages of oogenesis within 

smaller, 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 to 

enormous numbers of spermatogenic cells at diverse 

stages (Figure 2b) (Walker et al. 2005).

Stages in Gametogenesis

During the annual reproductive cycle, gonads 

of 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 gonad 

of red sea urchins during gametogenesis as classified 

into five stages by Fuji (1960) with modifications for 

that species (Unuma et al. 1996, Unuma 2002).

Stage 1. Before gametogenesis: In both sexes, each 

acinus is filled with NPs (eosinophilic cells). In 

ovaries, a few young oocytes are present at the periphery of the acini. Hematoxylin-stained round 

spots, residue from phagocytized ova (Masuda 

and Dan 1977, Tominaga and Takashima 1987), are 

occasionally present centrally in the ovarian lumen. In testes, detection of spermatogenic cells is 

difficult at this stage in paraffin sections. Instead, 

many hematoxylin-stained speckles, residue from 

phagocytized 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
47

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 positions 

of germ cells relative to groups of NPs are shown, especially the continuous incubation chamber 

that 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 the 

lumina. At stage 4, the lumina are filled with ripe ova or spermatozoa. At stage 5, the lumina contain a few residual ova or spermatozoa. NP = nutritive phagocyte, OC = oocyte, OV = ripe ovum, SG 

= spermatogonium, SC = spermatocyte, and SZ = spermatozoon. Inset a, round spot representing 

a phagocytized residual ovum. Inset b, amorphous speckles representing phagocytized residual 

spermatozoa. Scale bar represents 100 μm. Modified with permission from Unuma 2002.

useful feature to distinguish testes from ovaries.

Stage 2. Beginning of 

gametogenesis: Many 

developing oocytes or 

clusters of spermatogonia 

are present at the periphery of the acini, and the 

gonadal lumina are still 

filled with NPs.

Stage 3. Middle of 

gametogenesis: NPs are 

replaced with ripe ova or 

spermatozoa in the center 

of 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 are 

gradually 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 already 

lost nutrients, are present 

only at the periphery of 

the acini.

Stage 5. After spawning: 

The gonadal lumina have 

numerous empty spaces 

and a few residual ova or 

spermatozoa. NPs gradually phagocytize residual 

gametes and begin to 

grow as they store nutrients. After this stage, 

gonads return to Stage 1 

and a new cycle starts.

Unuma and Walker
48

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 food 

and increase in size. About 80% of the total protein 

contained in NPs is a glycoprotein with a molecular 

mass of about 170 kDa (Unuma et al. 2003). This 

protein was originally identified as the predominant 

component of yolk granules in sea urchin eggs and 

termed the major yolk protein (MYP) (Yokota and 

Sappington 2002). Unlike other oviparous animals 

where the yolk protein is female-specific, both male 

and 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 two 

months 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 period 

of gametogenesis of red sea urchins is shorter than 

those of other species, where gametogenesis usually 

takes more than three months.

Relationship Between Gametogenesis and 

Food 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 the 

oozing of gametes (Unuma 2002). The tissues of the 

gonads become fragile as gametogenesis proceeds.

Unuma and Walker

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 of 

gametogenesis 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 of 

NPs. 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 the 

gonoduct, and the gonads cannot maintain their 

consistency. This phenomenon, common among 

all the edible sea urchins, considerably reduces the 

commercial value of the gonads. To suppress this 

gamete ooze, alum, an aluminum compound, is usually used in Japan. After removing the gonads from 

the testis, they are lightly washed in salt water and 

then 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 the 

bitterness of mature ovaries in some species. In the 

red sea urchin and the green sea urchin (Hemicentrotus pulcherrimus, another popular species in the 

southern part of Japan), mature ovaries have an extremely bitter taste, unlike the mature testis. Recently, pulcherrimine, a novel sulfur-containing amino 

acid, has been isolated as the bitter component from 

the 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 urchins 

has not been identified. The bitterness of mature 
Unuma and Walker 49

Figure 5. Deterioration of commercial quality in the gonads of 

red sea urchins. Mature testes were taken out of the testis and 

placed in a petri dish for 30 minutes. The testes appeared to be 

melting because of oozing sperm. The same process also occurs 

in the mature ovaries. Reprinted with permission from Unuma 

2002.

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.

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). Both 

methods prolong the season during which large 

gonads that contain fewer GCs can be harvested. 

However, the acceleration of NP growth is easier 

than the suppression of gametogenesis and is now 

being put to practical use. In contrast, the suppression of gametogenesis, which is more effective at 

yielding large gonads with fewer GCs, is technically 

difficult and requires much additional research before it can be put to practical use.

Use of Formulated Feed to Accelerate NP 

Growth

Most 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 shortened 

commercial season compared to that of other species 

where ovaries at the end of gametogenesis are not 

bitter. 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 with 

the advance of gametogenesis, subsequent spawning causes a large year-to-year decrease in the size 

of the gonad (Unuma 2002). Following spawning, 

it takes time for the gonads to grow to marketable 

size 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 a 

longer 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 is 

important to produce large gonads that contain few 

GCs. If the gonad can be enlarged without increase 
50 Unuma and Walker 

et al. 2001, Pearce et al. 2002, Hammer et al. 2006) 

and fish meat (Agatsuma and Nishikiori 1991). This 

implies that protein ingestion and assimilation are 

limiting 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 of 

the 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 be 

lower 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 of 

commercial quality gonads (Unuma 2002). Control 

Figure 7. Gonad indices 

of 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 the 

young red sea urchins of 

15 mm testis diameter. 

Casein was used as the 

protein source for the 

formulated feed. Based 

on data from Akiyama et 

al. 2001.

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 is 

suggested 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 brood 

stock of red sea urchins and green sea urchins is 

induced by manipulating water temperature (Ito et 

al. 1989, Masaki and Kawahara 1995, Noguchi et al. 

1995). Alternatively, for culturing adult sea urchins, 

suppression of gametogenesis is required. In order 

to 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-scale 

aquaculture because of high energy costs. Economical methods, such as use of warm effluent from an 

electric power station or cold seawater from the deep 

sea, are essential to control water temperature in 

aquaculture facilities.

Böttger et al. (2006) investigated the effects of 

invariant summer photoperiod and progressing 

ambient (fall/winter) photoperiod on the gonads of 

northern green sea urchins. Maintenance under invariant photoperiod yielded gonads containing fewer GCs than NPs (unripe), while maintenance under 

ambient 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 under 

invariant summer photoperiod may improve the 

food quality and prolong the harvest season of the 

species in a land-based aquaculture system.

Production of Infertile Sea Urchins

Production of infertile sea urchins may also be 

utilized to suppress gametogenesis. To generate 

infertile adults, triploidy is often induced in other 

organisms, 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 51

Figure 8. Plastic sections of A) a representative northern green

sea urchin ovary and B) testis maintained for 5 months under 

invariant 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 the 

spermatogenic 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) and 

new (NO) primary oocytes in NP incubation chambers. The testicular lumen is filled with new spermatozoa and the NPs (dark 

granule containing cells) are reduced in size with only a slender 

strand 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.

in generating triploid sea urchins is that sea urchin 

eggs complete both meiotic divisions before spawning (Walker et al. 2006). Therefore, retention of the 

polar body, a popular method of generating triploid 

fish and shellfish, cannot be applied to sea urchins. 

Production of a tetraploid sea urchin by suppressing 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 in 

generating 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 the 

use 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 urchins 

accumulate nutrients in NPs as normal diploid sea 

urchins do.

Conclusions

Intragonadal nutrient storage and its utilization 

for gametogenesis are linked processes in sea urchin 

reproduction. This situation, which is not found 

in 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.
52 Unuma and Walker

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Unuma and Walker
55

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 with 

which 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 in 

polyculture 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 with 

industry 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 been 

successful 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
56

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 weight 

m-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, light 

availability, 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.

Langdon and Evans
57

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 circulation 

and exchange are needed, in order to improve and 

preserve the quality of the seawater and seabed in 

such regions.

A large amount of work has been conducted to 

develop 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 seawater 

exchange, while others directly improve seawater 

and sediment conditions. Use of tidal and ocean 

currents or wave induced currents may be one of 

the possible solutions to the problem. However, the 

driving 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 basins 

through the use of wave power have been constructed for fisheries in ports and harbors. We have 

developed a seawater exchange breakwater in which 

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, Japan

ohmura 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 such 

as ports and harbors—including aquaculture grounds—seawater circulation and exchange needs to be 

enhanced. It may be possible to use a seawater exchange structure driven by wave motion. Among the 

many kinds of seawater exchange structures that have been developed, seawater exchange structures 

with 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.

the unidirectional current is excited by vortex flows 

(Ohmura et al. 2005). I have also developed another 

seawater 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 with 

blockwork mounds may be one of the most effective 

(Figure 1). The structure was originally developed 

by Yamamoto et al. (1992) as a breakwater in which 

one-way flow is excited in a wave chamber due to 

wave breaking or wave overtopping. When water 

level 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 seawater 

exchange structures with blockwork mounds.

Ohmura
58

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

Function Works (energy)

Enhancement of Seawater exchange structure (wave, tide, current) 

seawater exchange 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 oxygen Seawater exchange structure (wave)

to seawater 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 Tideland (solar, wave, tide)

by benthos and Sand beach (solar, wave, tide)

plankton 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 Sediment dredge

salts from sediment Sand capping

Supply oxygen

also help supply oxygen to the seawater. It is known 

that when the distance from seawater level to the top 

of 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 with 

blockwork 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 of 

the modified structures (Ohmura 2007).

Physical Model Tests

Experimental setup. Experiments were conducted in a wave basin at the National Research 

Institute 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 long 

conduits 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
59

outer face of each mound had a 1:2 slope in order 

to enhance more wave setup in the wave chamber. 

Each conduit was made of PVC pipe with a diameter 

of 0.1 m and a length of 4.3 m. Model B used a rear 

chamber as shown in Figure 4. The specifications 

of model B were the same as model A except for the 

rear chamber. The conduit was made of PVC pipe 

with 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 regular 

waves 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 and 

each 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 front 

of the wave paddle and at the exit of the pipe by capacitance-type wave gauges, and velocity in the center of 

the 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 should 

be 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 relationship 

between the spatially averaged velocity U and the 

center velocity ,uc is given by:

 (2)

where Q is the volume rate of discharge, A is the 

area of pipe, and r is the radius of pipe.

Figure 2. Plane view of a wave basin.

Figure 3. Plane view and cross section of model A with three 

different crests.

Ohmura
60

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, g

is the gravitational acceleration, D is the diameter of 

pipe, and n is the Manning’s roughness coefficient.

Consequently, relation between the spatially averaged velocity U and the center velocity uc is given 

by:

 

 (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

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 regular 

waves 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 regular 

waves may be given by:

 

 

 (6)

where hc is the time-averaged surface elevation 

in 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 from 

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

Figure 5. Photograph of seawater exchange structures with 

blockwork mounds in the experiment.

Ohmura
61

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 for 

incident wave Q1 and the discharge for return flow 

from 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 return 

flow, h is the time-averaged surface elevation in 

wave 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 at 

exit, 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 each 

mound can be calculated by the discharge model 

described above. The volume rate of discharge from 

the rear water chamber may also be expressed as 

given by Equation 9 and Equation 10 by substituting the time-averaged surface elevation at the rear 

chamber hwc into h. It is also necessary to take into 

Ohmura

account 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 volume 

rate 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, and 

hwc + hc,k ≥ 0.

Results and Discussion

Figure 7 shows the results on the mass of water 

transport from each mound Q for model A. From 

the figures, it is easily seen that the discharge from 

each mound appears effective at a different water 

level. When water level is comparatively low, the 

discharge from low crest mounds shows high performance. On the other hand, when water level is 

comparatively high, the discharge from high crest 

mounds shows high ability. The reason is that the 

wave setup in the wave chamber that induces the 

discharge for inlet flow from each mound is influenced by the crest elevation from the still water 

level. The volume rate of discharge becomes gradually larger for longer and larger waves.

Figure 8 shows the dimensionless volume rate 

of discharge from each mound Q* for model A as a 

function of crest elevation by deepwater wave height 

hc/H0. The volume of water transport for unit width 

Q/B is normalized by the amount HL/2π, which is 

equivalent 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 of 
62

hc/H0. From the figure, it can be seen that the value 

of the dimensionless surface elevation becomes 

maximum 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 is 

linear over the range of –1.2 < hc/H0 < 1.2, which is 

the same tendency as shown in Figure 8.

Figure 10 shows the comparison of measured 

and calculated time-averaged surface elevation in 

wave 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 

Figure 6. Time-averaged phenomena in wave chamber.

Figure 7. Volume rate of discharge for inlet flow from each 

mound for model A.

Ohmura

Figure 8. Dimensionless volume rate of discharge for inlet flow 

from each mound for model A.

Figure 9. Time-averaged surface elevation in wave chamber for 

model A.
63

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, the 

calculated values are overestimates compared with 

the value from the experiment, so there needs to be a 

correction to increase accuracy.

Figure 12 shows the comparison of measured and 

calculated volume rates of discharge from the rear 

chamber for model B. In the calculation, fe, fo, fb, and 

hout for pipes from the rear chamber were chosen as 

1.0, 1.0, 0.256, 0.0, respectively, and CI, CR, ffe, ffo, and 

ffb for pipes from each mound were 1.0, 1.0, 1.0, 1.0, 

and 0.0, respectively. From the figure, we see that 

both measured and calculated volume rates of discharge from the rear chamber appear reasonable over 

a range of water levels. However, when the water 

level is relatively high, say levels 6 and 7, the volume 

rate of discharge decreases. It should be noted that 

the calculated values show fairly good agreement 

Ohmura

Figure 10 (at right).

Comparison of 

measured and 

calculated timeaveraged surface 

elevation in wave 

chamber for model 

A.

Figure 11 (at right).

Comparison of 

measured and 

calculated volume 

rate of discharge 

for inlet flow from 

each mound for 

model A.

Figure 13. Calculated volume rate of discharge for inlet flow 

from each mound for model B.

Figure 12. Comparison of measured and calculated volume rate 

of discharge from rear chamber for model B.

compared to the values from the experiment.

Figure 13 shows the calculated volume rate of 

discharge 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 structures 

in order to prevent return flow from the rear water 

chamber. The present discharge model may be useful in predicting the performance and specifications 

of the structures.
64

Conclusions

Our results confirm that both of the models are 

effective not only for wide tidal change but also for 

long 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, although 

correction may be needed for more accuracy. In addition, the present discharge model may be helpful 

in predicting the effective specifications for modified seawater exchange structures using blockwork 

mounds. It is hoped that the technology of seawater 

exchange structures can play a significant role on 

aquaculture grounds in the near future.

References

Ohmura, Y. 2002. Development and testing of the 

seawater 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 remote 

submerged 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 of 

Fisheries Engineering, Aquaculture and Fishing Port 

Engineering 10:39–55 [in Japanese].

Yamamoto, M., M. Nakaizumi, M. Manabe, and A. 

Moriguchi. 1992. Development of the breakwater 

with submerged mound for interchanging sea water. 

Bulletin of National Research Institute of Fisheries 

Engineering 13:11–30 [in Japanese].

Ohmura
Yoshimatsu et al. 65

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 and 

supports profitable coastal fishing. Short-neck clam culture is a key component and rapidly increasing 

area 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 the 

capture 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 have 

been reared successfully for algal foods (i.e., mainly diatoms), these are expensive to produce and do not 

always 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 food 

supplement 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 of 

short-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).
66 Goldberg

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 be 

highly automated, economic to operate, and generate little or no waste discharge. Producing large seed 

bay 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 physiological 

responses of scallops to different culture parameters, overcoming unexplained winter mortalities, and 

defining 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.
Widman 67

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 part 

of that country’s aquaculture production. It has many characteristics that make it a prime candidate for 

aquaculture: 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, which 

currently restricts bay scallop aquaculture to a single-season endeavor. When scallops were grown in 

lantern 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 this 

time.

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 water 

and 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 accumulation 

of toxic nitrogenous compounds. Recently we found that scallops were most susceptible to ammonia in 

comparison to nitrite or nitrate. We have developed automated demand feeding systems to minimize the 

nitrogen 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.
68

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 two 

commercially 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 are 

often 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 five 

flagellates. 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, were 

observed 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 this 

end, an enzyme-linked immunosorbent assay, also known as an ELISA, is currently under development 

to measure cortisol concentrations in bivalve hemolymph.

Milke et al.
69

Biotechnology Branch Overview on Probiotic Bacteria

and 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, she 

isolated a number of bacterial strains from within the shells of healthy mollusks and screened these for 

biological effects on oyster larvae and on bacterial pathogens of oyster larvae. Promising strains have 

been 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, but 

that 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 HAB 

taxa, including dinoflagellates, prymnesiophytes, and raphidophytes. Our main accomplishments in this 

area 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 and 

disease problems in hatcheries, either on-site or remotely. These activities serve industry throughout the 

United States, not just in the northeast region.

Wikfors
70

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 food 

are 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 are 

often seen when growing cultures outdoors can affect the growth of a phytoplankton feed.

Meseck
71

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 some 

coastal 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 such 

interactions 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 of 

phosphate 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 of 

proteins 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 controlled 

environment 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 was 

quantified by analyzing inflows, outflows, and settling. The removal of phytoplankton by scallops was 

quantified, 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
72

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, are 

developed 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 have 

special 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 aquaculture 

industry. However, prefiltration is absolutely essential in aquaculture applications. Industrial process 

control 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.

Smith
73

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 of 

inbreeding depression which could be manifested as slower growth, decreased viability, disease susceptibility, or overall decreased production. An example of inadequate attention to genetic consequences of 

culture 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 in 

combination with varying degrees of success as measured by different responses for hatchery culture and 

in 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.