U.S. DEPARTMENT OF COMMERCE
National Oceanic and Atmospheric Administration
National Marine Fisheries Service

NOAA Technical Memorandum NMFS-F/SPO-222

This page intentionally left blankMarine Aquaculture in a Changing Environment

Proceedings of the 46th U.S.-Japan
Aquaculture Panel Symposium
Held in Mystic, CT
NOAA Northeast Fisheries Science Center
November 14-15, 2018
Kristen Jabanoski1, Clete Otoshi2, Erick Sturm3 and Mike Rust2 (editors)
1

Contractor with A.I.S., Inc.
NOAA National Marine Fisheries Service
Northeast Fisheries Science Center – Milford Laboratory
212 Rogers Avenue
Milford, CT 06460
2

NOAA National Marine Fisheries Service Office of
Aquaculture1315 East-West Highway
Silver Spring, MD 20910
3

Southwest Fisheries Science Center
Fisheries Ecology Division
110 McAllister Way
Santa Cruz, CA 95060
NOAA Technical Memorandum NMFS-F/SPO-222
October 2021

U.S. Department of Commerce
Gina M. Raimondo, Secretary of Commerce
National Oceanic and Atmospheric Administration
Dr. Richard W. Spinrad, NOAA Administrator
National Marine Fisheries Service
Janet Coit, Assistant Administrator for Fisheries

SUGGESTED CITATION:
Kristen Jabanoski, Clete Otoshi, Erick Sturm, and Mike Rust (editors).
2021. Marine Aquaculture in a Changing Environment: Proceedings of the
46th U.S.-Japan Aquaculture Panel Symposium, Mystic, CT, November
14-15, 2018. U.S. Dept. Commerce, NOAA Tech.Memo.
NMFS-F/SPO-222,140 p.
A COPY OF THIS REPORT MAY BE OBTAINED FROM:NOAA National Marine Fisheries Service
Office of Aquaculture1315 East-West Highway
Silver Spring, MD 20910
OR ONLINE AT:
https://spo.nmfs.noaa.gov/
Reference throughout this document to trade names does not imply
endorsement by the National Marine Fisheries Service, NOAA.
Cover photo of historic canned oyster collection at Copps Island Oysters,
LCC, in Norwalk, Connecticut. Photo credit: NOAA Fisheries

Table of Contents
Introduction
Michael B. Rust

1

The Influences of Environmental Changes on Japanese Nori Mariculture
Mahiko Abe, Noboru Murase, Masahiro Kobayashi, Eiji Fujiyoshi and Motoya
Tamaki2

Development of Environmentally Friendly, Cost-Effective and Nutritionally
Balanced Alternative Protein Based Diets for High Value Marine Fish Culture
in Recirculating Aquaculture Systems
Md Shah Alam, Wade O. Watanabe and Patrick C. Carroll

7

Seaweed Aquaculture in a Warming Environment—Building Resilience with
Saccharina angustissima (Laminariales, Phaeophyceae)
Simona Augyte & Charles Yarish

20

Scallop Farming in Maine, a 20-year Overview
Hugh S. Cowperthwaite, Dana L. Morse, and Melissa L. Britsch
The Influence of Climate and Environment on the Spawning, Condition, andLarval Set of Naturalized Pacific Oysters Crassostrea gigas in a U.SWest Coast Estuary
Brett R. Dumbauld, Jennifer L. Ruesink, Alan J. Trimble, and Zachary Forster
A Review of the Potential of Macroalgae Feedstuffs to Reduce Methane
Emissions from Ruminant Livestock
Luke Gardner, Scott Hamilton, Michael Graham, Stacey Gunter, and Kenneth
Kalscheur
Condition Index and Fecundity of Manila Clam Ruditapes philippinarum
Related to Habitat Environment
Natsuki Hasegawa, Toshie Matsumoto, Satoshi Watanabe, Kazuhiro Hanyu, Dai
Miyawaki, Ryogen Nambu, and Masami Hamaguchi
Ocean Acidification Experiment in Seagrass-Oyster Ecosystems Using a
Novel System to Conduct In-Situ CO2 Enrichment
Masakazu Hori, Tomohiro Kuwae, and Masahiro Nakaoka

iii

25

34

49

56

64

Effects of Ocean Acidification on the Early Developmental Stages of theCommercially Important Gastropods, Ezo Abalone and Horned Turban,
in Japan
Ryo Kimura, Hideki Takami, Toshihiro Onitsuka, Tsuneo Ono, and Yukihiro Nojiri
Chemical Changes in the Environment: What Does This Mean for Shellfish?
Shannon L. Meseck, Mark Dixon, Yaqin Li, George Sennefelder, Dylan Redman,
Renee Mercaldo-Allen, Paul Clark, Lisa Milke, Gary H. Wikfors
Current Trends in pH and Ocean Acidification in Aquaculture Sea Areas in
Gokasho Bay, Japan
Masayuki Minakawa, Satoshi Watanabe, Yuka Ishihi, Natsuki Hasegawa, andToshie Matsumoto
Ecological Interactions Among Wildlife and Shellfish Farms: A Case Study
from New Jersey, USA, on Horseshoe Crabs and Oyster Farms
Daphne Munroe
Use of Point-of-View Video Cameras to Assess Fish Interactions with Oyster
Aquaculture Cages
Gillian Phillips, Renee Mercaldo-Allen, Peter J. Auster, Paul Clark, Erick Estela,
Yuan Liu, Lisa Milke, Dylan Redman, and Julie Rose

68

72

80

89

97

Vibrio coralliilyticus Induction of Virulence Toward Larval Oysters and Corals
at Elevated Seawater Temperatures and Potential Mitigation Strategies
104
Gary P. Richards and Blake Ushijima
Cyst Distribution Patterns of the Paralytic Shellfish Poisoning Plankton
Species Alexandrium catenella and A. pacificum off the Pacific Coastof Eastern Japan
Tomoko Sakami, Tetsuroh Ishikawa, and Toru Udagawa
Trends in Aquaculture Production in Japan
Satoshi Watanabe and Tomoko Sakami

111
118

Preliminary Experiments on Using the Seaweed Extract, AMPEP, to Enhance
Thermal Tolerance and Growth of the Sugar Kelp, Saccharina latissima,
126
from New England
Schery Umanzor, Sookkyung Shin, Michael Marty-Rivera, Simona Augyte, Charles Yarish,
and Jang K. Kim
Challenges to Establishment of Tetraploid Breeding Stocks for Shellfish
Aquaculture
Huiping Yang

iv

134

Introduction
The impacts and interactions between environmental change and aquaculture arediverse and many are likely unknown. What we do know is that nutrient pollution isdriving eutrophication and dead zones, ocean acidification is changing waterchemistry and climate change is already influencing our food supply, fresh water
availability, weather and way of life. Aquaculture will be impacted by, and can also
impact, these environmental changes over various scales. Aquaculture of finfish,shellfish and seaweed have different threats, benefits and opportunities related toenvironmental change, but in the right location, and with the right management they
can be mostly beneficial to the environment. Because of the significant aquacultureindustry impacts caused by environmental change, and the potential for aquaculture
to provide some mitigation, research on this topic was a priority shared by the
Fisheries Research and Education Agency (FREA) of Japan, the U. S. National
Oceanic and Atmospheric Administration (NOAA), and the U.S. Department ofAgriculture. These Proceedings represent the second of three consecutive years ofmeetings where the theme is “Aquaculture in a Changing Environment”. This year,
the research focused on the impacts to aquaculture production due to environmental
change (e.g., ocean acidification impacts on shellfish aquaculture) and science tomitigate these impacts (counter measures). Proceedings from the previous
Symposium were published by FREA (45th UJNR), where the focus was on thepotential of aquaculture to mitigate impacts of environmental change (e.g.,
sequestering carbon, bioextraction of nutrients and CO2, antacid, oxygen
production). Looking forward, we will seek to explore science and technology that
address these issues and will enable aquaculture to prosper in a changing
environment and be a part of the solution to reducing the human impacts ofenvironmental change.

Michael Rust
NOAA Fisheries Office of Aquaculture
United States Panel Chair

1

The influences of Environmental Changes on Japanese Nori Mariculture
Mahiko Abe1*, Noboru Murase1, Masahiro Kobayashi2, Eiji Fujiyoshi3, and Motoya Tamaki3
1

National Fisheries University, FRA, Shimonoseki, Yamaguchi 759-6595, Japan
National Research Institute of Fisheries Science, FRA, Yokohama, Kanagawa 236-8648, Japan
3
Seikai National Fisheries Research Institute, FRA, Nagasaki, Nagasaki 851-2213, Japan
2

Corresponding author: abemahi@fish-u.ac.jp
Keywords: Pyropia tenuipedalis, breeding, cell selection, low salinity tolerance

ABSTRACT

nori are active at low water temperatures. Even if
some measures are taken to control herbivorous
fish, birds would alternatively eat nori. It is urgently
necessary to develop effective measures to control
these herbivores.

Nori mariculture has a long history of over one
hundred years and is one of the most importantfisheries industries in Japan. Recently, noriproduction has been decreasing due toenvironmental changes, such as seawatertemperature rise and low nutrient levels. As for theseawater temperature rise, the start of norimariculture is delayed because of high watertemperature staying above 23ºC in autumn, thereby
shortening the season of nori mariculture. Spores
start to be released when the water temperature
decreases below 23ºC. Low nutrient levels inambient waters cause bleaching of nori thalli,
reducing the quality and price of nori. Currently,
new culture species and strains of nori around thecoasts of Japan are being explored. The thallus has asimple structure and cells within the thallisometimes have mutations. Mutant cells may havethe potential for tolerance to high water temperatureand low nutrient level, as well as other favorable
characteristics. More recently, shortening of thethalli is observed frequently. One of the causes ispredation by herbivorous fishes (e.g.
Acanthopagrus schlegelii, Mugil cephalus
cephalus) and ducks (Anas penelope, Anas acuta).
In Japan, it has been reported that herbivorous
fishes cause the disappearance of marine forests,
which is referred to as “iso-yake”. The duration of
iso-yake caused by herbivorous fish has been
prolonged because of increased water temperatures.
While the activities of the iso-yake causing fish are
reduced below 17ºC, the herbivorous fishes eating

INTRODUCTION

Figure 1. Photograph showing nori mariculture in Fukuoka
Prefecture.

Nori mariculture has a long history and many
farmers and researchers have developed innovativetechnologies which made nori mariculture one of
the most important fisheries industries in Japan(Fig. 1). However, nori production has decreased
due to environmental changes, such as seawatertemperature rise and low nutrient levels. At present,
nori mariculture of Pyropia yezoensis is mainly
used and P. tenera is used in limited areas*. Culture
strains have been repeatedly selected within P.
yezoensis for a long time and, as a result, the strains2have lost genetic diversity. Dissolved inorganic
nitrogen (DIN) levels started to decrease (i.e.
oligotrophication) in the 1980s in the western partof Japan, and in the 1990s in the eastern and central
parts of the Seto Inland Sea (Abo and Yamamoto,2019). After the late 1990s, disappearance ofmacroalgal beds, generally called “iso-yake” inJapanese, has spread along the coast of westernJapan because of increasing sea water temperature(Yoshida et al., 2019). Nori production had thehighest value in 2001 and has been decreasing until
present. However, in the nori mariculture industry,
it was not until recent years, when the production
fell below the level of demand of 8 billion sheets,that the effects of oligotrophication and watertemperature rise became an issue. Therefore, many
researchers are searching earnestly for measures toaddress reduced nori mariculture production. In thepresent paper, we introduce the history, problems
and solutions of nori mariculture.

scales using machinery for harvesting, choppingand drying of the nori. Nori mariculture has become
one of the most important fisheries industries inJapan with many innovations.

Figure 2. Photographs showing well grown nori thalli (a), bleachedthalli (b) and products of well grown and bleached thalli (c). Red
arrow shows product of bleached thalli.

Background on nori mariculture in Japan
In Japan, the use of nori has a long history of over
two hundred years. In the Bunka-Bunsei period(1804 – 1830), Katsukawa Shunsen designed theukiyo-e print of nori making (Miyata and Tomizuka,
1997). People during this era harvested nori
growing in the wild. The procedure for nori making
then is similar to that in the present, such as
chopping, forming and drying, except thateverything was done by hand. It is thought thatsights of nori-making were common scenery in the
Edo period (1603 – 1867). In the early 1900’s, norimariculture began and was cultured on “sodahibi”,
which are made of tree branches and set around thecoast (Okamura, 1909). However, at that time,
nobody knew where the spores came from, and nori
was harvested, chopped and formed into sheets byhand like in the Edo period. In recent years, norimariculture has changed. Dr. Kathleen Mary Drew
Baker’s discovery of the microscopic conchocelis
phase within the life cycle of nori (Drew 1949)
made it possible for Japanese researchers toestablish methods of maintaining different strains ofnori. Nori mariculture in the present uses nets
instead of “sodahibi” and is operated on larger

Problems of nori mariculture
Nori mariculture is one of the largest fisheries
industries in Japan. However, nori production has
decreased for the past decade from 10 billion sheetsin the 1990’s to below 8 billion sheets in recent
years. Nori production further decreased to 6.3
billion sheets in 2018. The causes of the decrease innori production are mainly seawater temperaturerise and low nutrient levels.
Nori mariculture starts when the seawatertemperature decreases below 23°C. The start of norimariculture is delayed by increasing watertemperatures, staying above 23°C in autumn, due toglobal warming. Nori mariculture used to begin in
the first quarter of October in the 1980’s, but began
in the second to the last quarter of October in the2010’s. On the other hand, the timing of springseawater temperature rise is unchanged, which
results in a shortening of the culture period for nori.
In Japan, the oligotrophication of coastal waters dueto the regulation of wastewater discharge isbecoming problematic. Low nutrient levels in the3ambient water cause bleaching of nori thalli,
reducing the quality and price of nori. Generally,
nori thalli grow with a dark black color (Fig. 2-a, c),but in an oligotrophic environment, thalli grow witha light brown color (Fig. 2-b, c). Bleached thalli are
2 to 3 JPY cheaper per sheet than dark black thalli.

used for mariculture have been screened by
selective breeding for over 40 years, making genetic
diversities of the strains poor. Moreover, breeding
methods such as cell fusion and mutation mentioned
above require special machines and techniques.
Bangiaceae including the genus Pyropia contains 30
species, and many of them are used as local foodresources. However, the distribution of nori species
in many local areas is unclear. Different nori species
growing in the wild have a wide variety ofcharacteristics and they may be candidates for new
culture species suited for different environmental
conditions. In fact, P. tenuipedalis has been cultured
for a few years on an experimental scale (Fig. 3).

Potential Solutions to the Problems
Many researchers are trying to develop new
breeding strains of nori with tolerances toenvironmental changes, such as high watertemperature and low nutrient levels. In addition toCountermeasures for oligotrophication, fertilization,
optimal management of wastewater, and plowing of
the sea bottom have been attempted in several water
bodies. Since nutrients are used not only by nori but
also by phytoplankton, which could cause adverse
effects to the environment, these methods must
always be accompanied by a survey of theoccurrence of phytoplankton (Mine, 2019).
Moreover, development of polyculture techniques
combining bivalve aquaculture (e.g. oyster andclams) with nori mariculture has been attempted.
Bivalves feed on phytoplankton, which competewith nori for nutrients, and their excreta is supplied
to nori as nutrients. Although these attempts are in
the trial phase, environmental improvement
utilizing biological functions is promising.

Figure 3. Photograph showing experimental mariculture of new
culture species Pyropia tenuipedalis.

conventional selective breeding, cell selection
within thalli and cell fusion using protoplasts andmutation breeding with heavy ion beams are also
carried out at several institutes (Sakaguchi 2011;
Niwa et al., 2011). In China, pigment mutants using
60
Co-γ ray irradiation were isolated (Yan and Aruga,
2000). Field use of the strains obtained by cellfusion and mutation breeding is not yet permitted inJapan. However, strains obtained by cell selection
within thalli have begun to be used in field culture(Sakaguchi, 2011; Abe et al., 2019). It is thoughtthat cell selection within thalli is a useful techniquefor development of new strains and its use in thefield has no legal restrictions so far. In addition,
many researchers are exploring alternative culture
species in the genus Pyropia. Strains of P. yezoensis

Figure 4. Photographs showing nori culture net with well grownthalli and the specimens (a) and culture net with shortened thalli andthe specimens (b).

4An emerging problem
In recent years, many incidents of shortening of nori
thalli have been observed in Japan (Fig. 4). Until
now, it was thought that one of the major causes ofthe shortening was low salinity of ambient water,
based on the symptom of the cell damage (Kirita
and Matsui, 1993). However, occurrences of theshortening have also been observed without low
salinity conditions. Field observations at norimariculture sites using video and time-lapse
cameras have revealed another cause of theshortening: predation by herbivorous fishes (e.g.
Acanthopagrus schlegelii and Mugil cephalus
cephalus) and ducks (Anas penelope and Anasacuta). In Japan, it has been reported thatherbivorous fishes (e.g. Calotomus japonicus,
Siganus fuscescens and Kyphosus bigibbus) cause
the disappearance of marine forests, which isreferred to as “iso-yake”. The duration of“iso-yake” caused by herbivorous fish has beengetting longer because of seawater temperature risebecause of global warming. The activities of fishcausing “iso-yake” are reduced below 17ºC,
allowing macroalgae to thrive in the winter.
However, the fishes and ducks causing theshortening of nori can actively graze nori evenbelow 17ºC. The seawater temperature typicallyranges from 10ºC to 20ºC during nori culture
season, and grazing pressure can cause an enormous
loss of nori thalli. It is necessary to develop
effective measures to lessen predation damage by
these herbivorous fishes and ducks immediately.

Abo, K. and T. Yamamoto. 2019.
Oligotrophication and its measures in the Seto
Inland Sea, Japan. Bull. Jap. Fish. Res. Agen.
49: 21-26.
Drew, K.M. 1949. Conchocelis-phase in the lifehistory of Porphyra umbilicalis (L.) Kütz.
Nature 164: 748-749.
Kirita, M. and T. Matsui. 1993. Effect of exposurein air and specific gravity change of seawateron growth of the sporelings of Porphyra.
Suisanzoshoku 41: 281-286. (in Japanese withEnglish abstract)
Mine, T. 2019. Current situation and problems of
Nori mariculture in Japan. (Nihon ni okeru
Nori yoshoku no Genjyou to Kadai). Frontierof Pyropia biology (AMANORISEIBUTSUGAKU no SAIZENSEN, Mikami
K. Ed.) Globe EDIT, Germany:.7-23. (inJapanese)
Miyata, M. and T. Tomizuka. 1997. Ukiyo-e“Painting of Nori Making”, made by
KATSUKAWA SHUNSEN. Nat. Hist. Res.,Special Issue 3: 129-134.
Niwa, K., T. Yamamoto, H. Furuita, and T. Abe.
2011. Mutation breeding in the marine crop
Porphyra yezoensis (Bangiales, Rhodophyta):
Cultivation experiment of the artificial red
mutant isolated by heavy-ion beammutagenesis. Aquaculture: 314: 182-187.
Okamura, K. 1909. Asakusa-nori. HakubunKan,
Tokyo, 374p. (in Japanese)
Sakaguchi, K. 2011. New nori strain with highwater temperature tolerance in Mie prefecture“MIENO AKARI” (Mie Ken no Kousuion
Taisei KuroNori no Shin Hinshu “MIE NOAKARI”) Norin Suisan Gijyutsu Journal 34:
42-43 (in Japanese)
Yan, X.-H. and Y. Aruga. 2000. Genetic analysis ofartificial pigmentation mutants in Porphyra
yezoensis Ueda (Bangiales, Rhodophyta).
Phycol. Res. 48: 177-187.
Yoshida, G., H. Shimabukuro, S. Kiyomoto, T.
Kadota, T. Yoshimura, N. Murase, M. Noda,
S. Takenaka, Y. Kono, T. Tamura, N. Tanada,
X. Yu, N. Yoshie, and X. Guo. 2019.
Assessment and future prediction of climate* Pyropia tenuipedalis, P. yezoensis and P. tenera inthis paper were replaced to Neopyropia
tenuipedalis, N. yezoensis and N. tenera by Yang etal. (2020), respectively.

REFERENCES
Abe, M., T. Ohashira, N. Murase, and M. Kishioka.
2019. Influences of salinity on growth of two
Pyropia tenuipedalis strains selected in low
salinity condition. Bull. Nat. Fish. Univ.
(submitted)

5change impacts on the macroalgal bed
ecosystem and cultivation in the Seto Inland
Sea. Bull. Jap. Fish. Res.Agen. 49: 27-34.

in Yamaguchi Prefecture, Japan. The habitat of thisspecies is more brackish as compared with that of P.
yezoensis, which is the common species in Japanesenori mariculture. In this paper, the authors
investigated the relationship between salinity andgrowth of P. tenuipedalis and P. yezoensis. It was
revealed that P. tenuipedalis tolerates lower salinity
in comparison with P. yezoensis.

ANNOTATED BIBLIOGRAPHY OF KEY
WORKSAbe, M., M. Kobayashi, E. Fujiyoshi, M. Tamaki,
N. Kikuchi, and N. Murase. 2013. Use of
PCR- RFLP for discrimination of JapanesePorphyra and Pyropia species (Bangiales,
Rhodophyta). J. Appl. Phycol. 25: 225-232.
DOI. 10.1007/s10811-012-9856-7.

Abe, M., Y. Fujita, M. Kobayashi, E. Fujiyoshi, M.
Tamaki, Y. Fukui, M. Satomi, and N. Murase.
2015. Effects of antibiotics on survival andgrowth for Pyropia yezoensis protoplasts.
Aquacult. Sci. 63: 1-8.
The nori thallus sometimes has cell mutations. For
the breeding of Pyropia species, it was necessary todevelop an axenic cell culture method. In this paper,
we investigated a way of isolating axenic
protoplasts of P. yezoensis and the effects of twelve
antibiotics on the survival and growth of the
protoplasts. The axenic protoplasts were able to beisolated by a series of treatments with sodium
citrate seawater. The growth rates of the protoplasts
treated with FRM and ABPC were 1.1–2.7 times
higher than that of the control, while PCG and SM
suppressed growth. The authors succeeded indeveloping an axenic culture method for Pyropia
species, but the survival, growth and morphogenesisof the cells in the axenic culture varied.
Improvement of the methodological stability isnecessary.

In this paper, the authors developed a PCR-RFLP
method useful for discrimination of 16 Japaneseand two non-Japanese Porphyra and Pyropia
species. These species have simple morphologicalfeatures, making morphological species
identification difficult. The two primer sets on the
mitochondrial DNA used in this study were able toamplify single fragments with PCR (partial
mitochondrial DNA related to ATP6 gene and trnC
– rns). All 18 species were successfully
distinguished using a combination of five
restriction enzymes (TaqI, SspI, AciI, Cfr13I andAluI). It was concluded that PCR-RFLP analysis isa useful tool for discrimination of wild strains ofPorphyra and Pyropia species for potential use inmariculture.
Nakayama, T., M. Abe, N. Murase, and Y. Shikano.
2017. Influence of salinity on growth of red
alga Pyropia tenuipedalis and Pyropia
yezoensis foliose thallus. Aquacult. Sci. 65:
321-330.

Yang L.-E., Y.-Y. Deng, G.-P. Xu, S. Russel, Q.-Q.
Lu, and J. Brodie. 2020. Redefining Pyropia(Bangiales, Thodophyta): four new genera,
resurrection of Porphyrella and description ofCalidia pseudolobata sp. nov. from China. J.
Phycol., 56: 862-879.

Pyropia tenuipedalis is a new culture species of nori6

Development of Environmentally Friendly, Cost-Effective and Nutritionally
Balanced Alternative Protein Based Diets for High Value Marine Fish Culture in
Recirculating Aquaculture Systems
Md Shah Alam*, Wade O. Watanabe, and Patrick C. Carroll
Center for Marine Science, Aquaculture Program, University of North Carolina Wilmington, 601 S. College
Rd., Wilmington, NC-28403, USA, Tel. 910-962-2945
Corresponding author: alamm@uncw.edu
Keywords: Alternative protein sources, fish meal replacement, black sea bass, southern flounder, andcost-effective aquafeeds

ABSTRACT“environmentally friendly” technology for
producing farmed fish.

With very little domestically grown seafood, alongwith increasing exploitation of wild stocks, the US
annual seafood trade deficit now exceeds $15
billion. Economics, environmental issues and fish
feed are factors vital to the sustainability of theaquaculture industry in the US and Japan, and thesefactors increase the pressure on aquaculturists todevelop eco-friendly and cost-effective aquaculture
practices. One of the challenges faced by theaquaculture industry is to prevent the pollution ofwater by artificial feed. Fish meal is the main
protein source used in aquafeed and is made fromsmall pelagic species of forage fish. Two nutrientsin fish meal, nitrogen and phosphorus, have a great
impact on the environment, and their release canresult in eutrophication. Fish meal could also
contain polychlorinated biphenyls (PCBs), dioxins,
and other harmful chemicals, and become a vector
of contamination in farm-raised finfish. Alternate
protein sources such as terrestrial animal and plant
protein sources can reduce the amount of wild fishused as protein, and potentially reduce the nutrient
levels in effluent waste. One of the goals of ourresearch at the University of North Carolina
Wilmington Center for Marine Science is to develop
sustainable marine finfish feed with reduced
inclusion of fish meal in recirculating aquaculture
systems, which are widely considered to be an

A series of experiments were conducted to test theeffects of different dietary levels of soybean meal,ultra-low gossypol-based glandless cottonseed
meal, and poultry meal in the diet of southern
flounder and black sea bass reared in a recirculating
aquaculture system for 8-10 weeks. Growth
performance, feed utilization, digestibility, and thebiochemical composition of fish tissues were
evaluated.
Results suggest that about 68 and 39% fish meal
protein could be replaced by soybean meal protein
and 82 and 100% of fish meal protein could be
replaced by poultry meal protein in the diet of black
sea bass and southern flounder, respectively. Results
also suggest that fish meal protein could be replaced
up to 100 and 75% by glandless cottonseed meal
protein in black sea bass and southern flounderdiets, respectively. These findings reveal
species-specific differences in effective substitution
levels of alternative protein sources to fish meal
protein, and provide a basis for developing
environmentally- sound and cost-effective
predominantly terrestrial plant and animal
protein-based diets for high value marine fishculture in the USA and Japan.

7

INTRODUCTION

fish has been improved by reducing or eliminating
gossypol through traditional genetic selection or
through genetic modification (GMO). Regular highgossypol CSM (R-CSM) protein has been used toreplace fish meal protein at maximum levels of 35%
in grass carp (Zheng et al., 2012) and 30% inparrotfish feeds (Lim and Lee, 2009). However,research on fish meal replacement by low-gossypol
based CSM is very limited on marine fish species.

In terms of dietary composition, protein is the single
largest and most expensive component in fish feed.
In aquaculture feed, fish meal is a popular source ofhigh-quality protein and highly digestible essential
amino and fatty acids (Cho and Kim 2011). By
replacing fish meal in aquaculture diets, alternate
terrestrial plant and animal protein sources canlower feed costs, reduce the amount of wild fishused as protein, and potentially reduce the nutrient
levels in effluent waste (Trushenski et al. 2006).
However, for most species, there is a limit to howmuch fish meal can be replaced by alternative
protein sources without negatively affecting fish
growth and feed efficiency (Gatlin et al. 2007).

Poultry by-product meal
Terrestrial animal protein sources have several
advantages, including a similar amino acid profile
to fish meal, availability, and relatively low cost.
Poultry by-product meal (PBM) is a protein source
produced from waste and by-products of processedchickens, possibly including heads and feet, but
excluding feathers and intestines. Like otheranimal- based protein feedstuffs, it has a highprotein content, but can vary in compositional
quality and lacks certain essential amino acids(Tacon et al. 2006). PBM has been usedsuccessfully to replace fish meal at high levels of
dietary inclusion for a number of finfish species. In
gilthead seabream, 50% of the fish meal protein was
successfully replaced with PBM protein without areduction in growth (Nengas et al. 1999). In
juvenile red drum, 67% of the fish meal protein was
replaced by PBM protein with no reduction in
growth (Kureshy et al. 2000).

Soybean meal
Soybean meal is considered to be one of the mostsuitable ingredients for replacing fish meal incommercial fish feed. Compared with other plant
protein sources, soybean meal has high protein
content, very low carbohydrate and fiber, highdigestibility, and a good amino acid profile (Hardy
2006; Gatlin et al. 2007). Soybean meal is used infish feed not only because of its high protein
content, but also owing to its worldwide
availability.
Low-gossypol cottonseed meal
Cottonseed meal (CSM) has received limited
attention as a potential fish meal replacement inaqua feeds; however, CSM protein is anincreasingly attractive alternative to fish meal
protein due to improved plant production andprocessing methods. Due to the large global
production of cotton, as well as the large quantities
of cottonseed by-products, CSM is much cheaper
per unit of protein than fish meal and other fishmeal replacements. An important constraint to theuse of CSM in animal feeds is gossypol, aterpene-based secondary metabolite that has animportant role in the cotton plant's defense
mechanisms against pests and possibly diseases(Romano and Scheffler, 2008). The utilization of
CSM as an ingredient in the feeds of animals andBlack sea bass
Black sea bass Centropristis striata is acommercially important marine finfish species thatcommands a high market price. This species
inhabits the coastal waters of the eastern USA, fromthe Gulf of Maine to Florida. The abundance of
black sea bass along the U.S. East Coast has been
declining since the 1950s (NOAA 2012; ASMFC
2016). Potential for limited market supplies and forhigher prices of ocean caught black sea bass in the
future are important economic incentives toinvestigate the feasibility of black sea bass
production via aquaculture to help meet market
demand (Watanabe 2011; Watanabe et al. 2016).
Hatchery-raised black sea bass juveniles originating

8

Table 1. Experimental designs.

from captive wild- caught broodstock are routinely
cultured through the market stage in a recirculating
aquaculture system at the University of North
Carolina Wilmington (UNCW) Aquaculture Facility(Watanabe et al. 2003; Watanabe 2011; Watanabe etal. 2021 ).
Southern FlounderThe southern flounder Paralichthys lethostigma is aflatfish in the family Paralichthyidae. It can befound in coastal waters from Albemarle Sound,
North Carolina, through the South Atlantic stateswith the exception of South Florida. Southern
flounder landings have declined, leading to more
stringent fishery regulations and interest in culturing
native flatfishes for stock enhancement or food fish
production. The development of intensive culture
methods for southern flounder in the southeastern
U.S. is of great interest because of its euryhaline
character and its status as a highly desirable foodand recreational species and potential forcommercial culture. Hatchery methodology for
spawning and larval rearing is well investigated(Daniels and Watanabe, 2003; Watanabe et al.,2006; Wright- Moore et al., 2019).

Experimental Tested
fishalternatives

Replacement ReferenceslevelsBlack sea
bass

soybeanmeal

0-100%

Alam et al.
2012

Southern
floundersoybeanmeal

0-100%

Alam et al.
2011

Black sea
bass

lowgossypol
cottonseed
meal

0-100%

Andersonet al. 2016

Southern
flounderlowgossypol
cottonseed
meal

0-100%

Alam et al.
2018

Black sea
bass

poultry by
product
meal

0-100%

Dawson etal. 2018

Southern
flounderPoultry by
product
meal

0-100%

Dawson,2012

Experimental diets and designs
A series of feeding trials were designed to test thesubstitution limits of soybean meal, CSM and PBM
protein in replacement of menhaden fish meal
protein (FMP) in the diet of juvenile black sea bass
and southern flounder (Table 1) (Alam et al. 2011,2012, 2018, Dawson, 2012, Dawson et al. 2018,
Anderson et al. 2018).

MATERIALS AND METHODS
Experimental animals and system
Juvenile black sea bass and southern flounder were
cultured from eggs supplied by photothermallyconditioned captive broodstock held at the UNCW
Center for Marine Science, Aquaculture Facility(Wrightsville Beach, NC). Adults were induced tospawn using luteinizing hormone-releasing
hormone analogue (LHRHa) implants (Watanabe etal., 2003). Eggs were hatched and reared throughjuvenile stages in 150 L tanks using protocols
established at UNCW (Watanabe, 2011; Carrier et
al. 2011, Russo et al. 2017). All the experiments
were conducted in a controlled environment
laboratory consisting of twenty-four 75-L
rectangular (76 x 32 x 43 cm) glass tanks, supported
by a recirculating aquaculture system.

Studies with soybean meal
Three experiments were conducted to determine theextent to which FMP can be replaced by solventextracted soybean meal protein (SBP) in the diet ofjuvenile black sea bass. In all experiments, diets
were formulated to replace FMP by SBP from 0 to
100% and supplemented with squid meal, krill
meal, and attractants (Alam et al. 2012). For
southern flounder, two feeding experiments wereconducted replacing FMP with SBP at 0, 10, 20, 30,
40, 50, 60 and 70% with or without supplemental Lmethionine and L-lysine (Alam et al. 2011).

9

Studies with low-gossypol CSM
Eight diets were formulated for juvenile black sea
bass to replace FMP by three CSM proteins: a CSM
prepared from glandless cottonseed meal (GCSM,
50.4% crude protein, CP), a CSM that had beensolvent extracted with acidic ethanol to remove thegossypol (SCSM, 53.8% CP), and a CSM preparedfrom regular (glanded) cottonseed (RCSM, 45%
CP). Three diets replaced 50, 75 and 100% of FMP
with GCSM, and three diets replaced 50, 75 and100% of FMP with SCSM. One diet replaced 100%
FMP with RCSM protein (Anderson et al. 2016).
For southern flounder, CSM proteins from GCSM,
genetically-modified low-gossypol seed (GMOCSM, 56.0% CP) and R-CSM were evaluated toreplace FMP. Six diets replaced 50, 75 and 100%
FM protein with GCSM or GMO-CSM protein(Alam et al. 2018).

individual tanks were lot weighed every two weeks
to monitor weight gain throughout the experiment.
Mortalities were also recorded for each tankthroughout the experiment. Water quality wasmonitored throughout the experiment on a weekly
basis and maintained at optimum levels (Watanabe
et al. 2011). Temperature, salinity, dissolved
oxygen, and pH were monitored using a multiparameter probe (YSI, Yellow Springs, Ohio, USA).
Ammonia and nitrite were monitored using a
HACH spectrophotometer (DR 2010, Loveland,Colorado, USA).
Biochemical and statistical analysis
At the end of the feeding trial, five fish from eachtank were collected for biochemical analysis todetermine proximate composition (moisture, ash,lipid, and protein-AOAC, 2000) and fatty acidprofiles of the whole bodies of the fish. Fatty acidanalysis was conducted in triplicate as described in
Dawson et al. (2018) and Alam et al. (2012). Total
amino acid analysis of diets was conducted in
duplicate (AAA Laboratories, Damascus, Oregon,
USA). At the conclusion of the experiment, fish
were reared for an additional 14 days on theirtreatment diets to determine the apparent
digestibility coefficient (ADC) of crude protein in
the experimental diets using 0.5% chromic oxide as
inert marker. Chromic oxide content wasdetermined using a spectrophotometer through a
modified Furukawa and Tsukahara (1966) method.
Statistical analysis was performed using the JMP 7
statistical software (SAS Institute Inc., ver. 13,
Cary, North Carolina).

Studies with PBM
Two feeding trials were conducted to determine themaximum substitution limits of poultry by-productmeal (PBM; 66% CP) protein for fish meal (Fishmeal; 59% CP) protein in the diet of juvenile black
sea bass and southern flounder. Eight diets were
formulated for each experiment to replace FMP
with feed-grade PBM protein at 0 (control), 40%,
50%, 60%, 70%, 80%, 90%, and 100% in black sea
bass and southern flounder diets (Dawson et al.
2018, 2012).
All experimental test diets were prepared at the
UNCW Aquaculture Facility as described by Alamet al. (2011, 2012, 2018) and stored in a freezer (-20oC). All diets were isonitrogenous and isolipidic foreach experiment. A control diet (0% SBM, PBM
and CSM) was formulated with high FMP and otherpractical protein sources. L-methionine and L-lysine
were supplemented to the diets to equal the control
diet.

RESULTS AND DISCUSSION
Experiments with soybean meal protein(SBP)
No significant differences in body weight gain, feed
efficiency, and survival were observed among fish
fed diets replacing FMP with SBP at 0 to 70%(Alam et al. 2012). In addition, body weight gain
was not significantly different in fish fed
supplemental methionine and lysine in 70% SBP
diets compared to fish fed 0% SBP diets. No

Feeding trials
Experimental diets were fed to triplicate groups offish twice a day (0900 and 1500 h) to apparent
satiation (i.e., as much as they can consume withminimal wastage) for six to ten weeks. Fish in10

significant differences were observed inwhole-body n-3 polyunsaturated fatty acids among
the treatments. Broken-line regression of thespecific growth rate data suggested that the
maximum level of FMP replacement with SBP inblack sea bass diets was 67.6–68.4% with 7.5%

leading to higher n-3 PUFA levels in the SBP diets.
All three feeding trials showed that black sea bass
juveniles are able to utilize high levels of soybean
meal in their diet without reducing growth
performance.

SGR(%d-1)

In southern flounder, no significant differences wereobserved in body weight gain, specific growth rate,
feed intake, feed conversion and protein efficiency
ratios, and the whole-body proximate composition
among fish fed diets replacing 0–40% of FMP with
SBP (Alam et al. 2011). When compared with fish
fed 0% SBP, weight gain decreased significantly forthe fish fed more than 40% SBP. Broken-line
regression showed that the optimum levels of FMP
replacement with SBP in the diet of southern
flounder without and with supplemental methionine
and lysine were 35.1% and 38.9%, respectively,
when diets contained 9% squid meal and 5% krill
meal. Maximum replacement level of FMP with
SBP without supplementing amino acids forsouthern flounder (40%) in this study was similar towhat was found in cobia (40%; Zhou et al. 2005)
and slightly lower than reported in the gilthead sea
bream (45%; Martinez-Llorens et al. 2008). In bothexperiments (with or without amino acids),
whole-body moisture, protein, and lipid contents
were not affected by SBP replacement of FMP up to
40%, similar to what was reported in yellow croaker(Ai et al. 2006), rainbow trout (Bureau et al. 2000),and Indian major carp (Khan et al. 2003).

Figure 1. Broken-line regression analysis between specific growthrate (SGR) and % FMP replaced by % SBP with supplemental squidmeal, krill meal and attractants in the diets of juvenile black sea bass
(Alam et al. 2012).

squid meal and 5% krill meal in the diet (Fig. 1) and
57.2–58.0% without squid and krill meal
supplementation (Alam et al. 2012). In black sea
bass diets supplemented with squid meal, krill meal,
and attractants, the maximum replacement level of
FMP with SBP (67.6–68.4%) was higher thanreported for other marine finfish species such as
Japanese flounder (45%, Kikuchi 1999); giltheadseabream (45%, Martinez-llorens et al. 2008); cobia(50%, Zhou et al. 2005), and Atlantic cod (50%,
Walker et al. 2010), but lower than that found for
freshwater fishes such as common carp (100%,
Viola et al. 1982) and Nile tilapia (100%, Deyab etal. 2002). In general, the fatty acid composition of
the fish whole body reflected the composition of
their diets. Linoleic acid (18:2[n-6]) was higher in
the SBP diets as well as in the whole body of fish
fed those diets. Studies on yellow perch (Kasper et
al. 2007), pike perch (Schultz et al. 2005), andJapanese flounder (Lee et al. 2000) also foundhigher levels of linoleic acid in fish fed diets
containing SBP or soybean oil. As SBP content wasincreased in the diets, fish oil was increased tocompensate for the low lipid content of SBP,

Experiments with glandless cottonseed
meal (CSM)
Growth performance of black sea bass juveniles fed
diets replacing between 50 and 100% of FMP
protein with low-gossypol GCSM, or replacing 50
to 75% of FMP with low-gossypol SCSM protein
was not impaired compared to fish fed a FMP-based
control palatability of the RCSM diet was
attributable to the anti-nutrient compound gossypol.
No significant differences in survival, feed
conversion ratio (FCR), protein efficiency ratio(PER), or whole body protein or lipid composition
were observed among the fish fed the low-gossypol
diets. Gossypol (25.9 mg/kg) was only detectable in11the livers of fish fed the high- gossypol RCSM diet.
Fish whole body essential amino acid compositions
did not differ significantly among treatments.
Whole body n-3 PUFAs decreased, while n − 6
PUFAs increased with increasing CSM protein in
the diets. The apparent digestibility coefficient ofprotein was high (83.1– 87.1%) for all treatments(Anderson et al. 2016). For juvenile black sea bass,
75% FMP in the diet can be replaced with
low-gossypol CSM protein prepared bysolvent-extraction, and 100% of FM can be replacedwith low-gossypol CSM protein prepared fromglandless seed with no adverse effects on survival,
growth or feed utilization. These results indicatethat the CSM protein from glandless seed was
utilized as efficiently as FMP by juvenile black sea
bass. In comparison, low-gossypol CSM protein has
been used to replace FMP at levels ranging from 20
to 100% for hybrid striped bass (Sullivan andReigh, 1995), channel catfish (Li et al., 2008),
rainbow trout (Lee et al., 2006), Florida pompano(Riche and Williams, 2010; Cook et al., 2016),
white shrimp (Siccardi et al., 2012; Richardson etal., 2016) and black sea bass (Anderson et al.,2016).

the control diet (79.4%) (Alam et al. 2018).
Arginine levels in the diets increased as CSM wasincreased, consistent with the high arginine
concentrations found in CSM. Liver gossypol was
only detectable in fish fed the 100% R-CSM diet
(37 μg/g). Replacing up to 75% FM protein by
GCSM- or GMO-CSM protein did not affect wholebody omega-3 PUFAs, or liver gossypol. The
results suggest that up to 75% of fish meal proteinreFigure 2. Effects of replacing FMP with CSM protein of different
sources in southern flounder diets: Glandless, GMO = geneticallymodified (glandless), and Regular (glanded). A control fish mealbased diet (0% CSM) was also tested. Bars represent percent bodyweight gain of juvenile flounder after 8 weeks of feeding. Values aremean ± SEM of triplicate tanks.Means with different letters differ
significantly (Alam et al. 2018).

For southern flounder, growth performance (final
weight and percent weight gain) of juveniles fed
diets replacing from 50 to 100% FMP with GCSM-,
GMO-CSM, or R-CSM protein was not different
from fish fed a control FMP-based diet (Figure 2;
Alam et al. 2018). However, in the present study,
replacing FMP with 100% GCSM, GMO or R-CSM
appeared to lower growth performance (albeit notsignificantly), suggesting that longer-term studies
are needed. After eight weeks of feeding, survivalof fish ranged from 80 to 91%, with no treatment
differences. Apparent protein digestibility of diets
was significantly higher for the fish fed 75% and100% GCSM and 100% GMO-CSM protein diets(83.5, 83.5 and 86.5%, respectively) compared withmay be replaced by GCSM or GMO-CSM proteinin the diet of juvenile flounder without adverse
effects on growth performance and bodycomposition. In black sea bass juveniles, replacing
100% FM protein with GCSM protein did not alter
growth performance or survival when diets were
supplemented with lysine (Anderson et al., 2016),
results similar to what was found in southern
flounder in the present study. These findings
suggest that CSM protein, regardless of source, wasmore digestible than menhaden FM in juvenile
southern flounder.

12

FMP in the diet (Riche 2015); however, growth
performance was reduced when PBM protein
replaced 100% of the fish meal protein (Rossi andDavis 2012). The results demonstrated that FMP
can be replaced by feed-grade PBM protein injuvenile black sea bass diets at levels as high as
81.8% without adversely affecting survival, growth,
feed utilization, fish biochemical composition, or
ADC of protein or lipid. Poultry byproduct meal is ahighly effective protein source for alternative
protein-based feed formulation for black sea bass.
Juvenile southern flounder fed diets with PBM
protein in substitution for FMP at levels up to 100%
showed comparable growth (i.e., fish weight andpercent weight gain) to fish fed the control FMP
diet (Dawson, 2012). Many marine carnivorous
species such as cobia (Zhou et al., 2011) andhumpback grouper (Shapawi et al., 2007) have beenreported to show no differences in growth when fed
diets with substantial levels of PBM (60 and 75%,
respectively) in replacement of FMP, but reduced
growth when FMP was completely replaced with a
100% feed- grade PBM diet (Shapawi et al., 2007).
Feed utilization (feed intake, FCR, and PER) in
juvenile southern flounder fed diets replacing FMP
with up to 100% PBM protein was not significantly
different from fish fed a control fish meal diet(Dawson, 2012). This suggests that palatability,
digestibility, and assimilation of feed were notcompromised by substitution of PBM protein for
FMP in the diet at any replacement level (Dawson2013). These results are very similar to thosereported in African catfish (Abdel-Warith et al.,2001) and sunshine bass (Webster et al., 1999).
Whole body moisture content of juvenile southern
flounder was not affected by level of PBM protein
replacement for FMP. Dietary fatty acidcomposition was reflected in the whole body fatty
acid profile (Dawson 2012). The ADC of protein bysouthern flounder was not affected by PBM protein
substitution for FMP at levels of up to 100%,
suggesting PBM protein is as digestible as FMP in
juvenile southern flounder. In conclusion, PBM
protein completely replaced FM protein in the dietof juvenile southern flounder without adverse
effects on growth or feed utilization, indicating an

Figure 3. Broken-line regression analysis between body weight gain
and % FMP replacement by PBM protein in the diets of juvenile
black sea bass.

Experiments with poultry by-product meal
(PBM)
For black sea bass, no significant differences inbody weight gain were observed in fish fed the
40–90% PBM protein diets compared with thecontrol diet. However, body weight gain of fish fed
the 100% PBM protein diet was significantly lower
than in the control group. Regression analysis withbody weight gain indicated that PBM protein canreplace FMP in black sea bass diets at levels as highas 81.8%, with no reduction in fish growth
performance (Fig 3) (Dawson et al. 2018). For fish
fed diets with up to 90% PBM protein, feed
conversion and protein efficiency ratios were notsignificantly different from fish fed a control 100%
FM-protein-based diet (Dawson et al. 2018).
Apparent digestibility coefficients of dietary protein
remained high (81.6– 87.0%) under all levels of
FMP replacement with PBM protein. After thefeeding trial, whole body and muscle protein
content and the concentrations of whole body n-3
polyunsaturated fatty acids showed no significant
differences among the treatments at FM protein
replacement levels up to 90%. For the marine
finfish totoaba, PBM protein was a good source ofnutrients in juvenile diets at a FMP replacementlevel of 67%, whereas fish fed a 100% PBM protein
diet showed the slowest growth and highest
mortality (Zapata et al. 2016). In the marine fish,
Florida pompano, growth performance was
unaffected when PBM protein replaced 67% of the13

exceptional ability of this species to digest and toassimilate PBM protein.

low-gossypol cottonseed meal as alternative
protein sources in the diet of juvenile southern
flounder Paralichthys lethostigma reared in arecirculating aquaculture system. Aquaculture
489: 36–45.
Alam, M.S., W.O. Watanabe, K.B. Sullivan, T.C.
Rezek, and P.J. Seaton. 2012. Replacement ofmenhaden fish meal protein by solvent
extracted soybean meal protein in the diet ofjuvenile black sea bass Centropristis striata
supplemented with or without squid meal, krill
meal, methionine and lysine. North American
Journal of Aquaculture 74:2, 251-265.
Alam, M.S., W.O. Watanabe, A.R. Myers, T.C.
Rezek, P.M. Carroll, and S. Longfellow. 2011.
Effects of replacement of menhaden fish meal
protein by solvent extracted soybean meal
protein supplemented with or without Lmethionine and L-lysine on growth
performance and body composition of juvenile
southern flounder. N. Am. J. Aquacult.
73:350– 359.
Anderson, A.D., M.S. Alam, W.O. Watanabe, P.M.
Carroll, T.C. Wedegaertner, and M.K. Dowd.
2016. Full replacement of menhaden fish meal
protein by low-gossypol cottonseed flour
protein in the diet of juvenile black sea bass.
Aquaculture 464: 618-628.
AOAC (Association of Official Analytical
Chemists). 2000. Official Methods ofAnalysis, 17th edition. Association of Official
Analytical Chemists, Arlington, Virginia,
USA.
ASMFC (Atlantic States Marine FisheriesCommission). 2016. Overview of Stock Status
Black Sea Bass, Centropristis striata.
http://www.asmfc.org/files/Meetings/2016Su
mmerMtg/ASMFC_StockStatus_June2016.
pdf.
Bureau, D.P., A.M. Harris, D.J. Bevan, L.A.
Simmons, P.A. Azevedo, and C.Y. Cho. 2000.
Feather meals and meat and bone meals fromdifferent origins as protein sources in rainbow
trout (Oncorhynchus mykiss) diets.
Aquaculture 181: 281-291.
Carrier, J.K. III, W.O. Watanabe, M. Harel, T. F.

CONCLUSION
The results demonstrate that juvenile black sea bass
and southern flounder are able to utilize high levelsof soybean meal, low-gossypol CSM, and PBM
protein in their diet without reducing growth
performance. About 68 and 39% fish meal protein
could be replaced by soybean meal, 82 and 100% offish meal protein could be replaced by poultry mealin the diet and 100 and 75% fish meal protein could
be replaced by glandless cottonseed meal protein in
the diet of black sea bass and southern flounder,
respectively. These findings provide a basis fordeveloping environmentally-sound and costeffective predominantly terrestrial plant and animal
protein-based diets for high value marine fishculture.

ACKNOWLEDGEMENTS
We thank the following organizations for funding
support: NOAA Marine Aquaculture Program,North Carolina Sea Grant Fisheries Resources Grant
Program, Cotton Incorporated, United States
Department of Agriculture National Institute ofFood and Agriculture, Marine Biotechnology inNorth Carolina (MARBIONC), New Hanover Farm
Bureau, UNCW Center for Marine Science andNorth Carolina Biotechnology Center.

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Romano, G.B. and J.A. Scheffler. 2008. Lowering
seed gossypol content in glanded cotton(Gossypium hirsutum L.) lines. Plant Breed.
127 (6): 619-624.

Rossi, W. Jr. and D. Davis. 2012. Replacement offishmeal with poultry by-product meal in thediet of Florida pompano Trachinotus carolinus
L. Aquaculture 338-341:160–166.
Russo, D.J., W.O. Watanabe, S.T. Kinsey, and P.J.
Seaton. 2017. Effects of feeding frequency oflive prey on larval growth, survival, resistanceto hyposalinity stress, Na+/K+ ATPase activity,and fatty acid profiles in black sea bass
Centropristis striata. Aquaculture 470: 56-67.
Schultz, C., U. Knaus, M. Wirth, and B. Rennert.
2005. Effects of varying dietary fatty acidprofile on growth performance, fatty acid,body and tissue composition of juvenile pikeperch (Sander lucioperca). Aquacult. Nutr. 11:
403-413.
Shapawi, R., W.K. Ng, and S. Mustafa. 2007.
Replacement of fish meal with poultry byproduct meal in diets formulated for the
humpback grouper, Cromileptes altivelis.
Aquaculture 273: 118-126.
Siccardi, A.J., C.M. Richardson, M.K. Dowd, andT.C. Wedegaertner. 2012. Glandless
cottonseed meal replaces fishmeal in shrimp
diet research. Global Aquaculture Alliance.
Sullivan, J.A. and R.C. Reigh. 1995. Apparent
digestibility of selected feedstuffs in diets forhybrid striped bass (Morone saxatilis♀ x
Morone chrysops♂). Aquaculture 138: 313322.
Tacon, A.G.J., M.R. Hasan, and R.P. Subasinghe.
2006. Use of fishery resources as feed inputs
to aquaculture development: trends and policy
implications. FAO Fisheries Circular, 1018,
1-99.
Tomas, A., F. De La Gandara, A. Garcia-Gomez, L.
Perez, and M. Jover. 2005. Utilization ofsoybean meal as an alternative protein sourcein the Mediterranean yellowtail, Seriola
dumerili. Aquacult. Nutr. 11: 333-340.
Trushenski, J.T., C.S. Kasper, and C.C. Kohler.
2006. Challenges and Opportunities in Finfish
Nutrition. North American Journal of
Aquaculture 68: 122-140.
Viola, S., U. Mokady, U., Rappapor, and Y. Arieli.
1982. Partial and complete replacement of fish16

meal by soybean meal in feeds for intensive
culture of carp. Aquaculture 26: 223– 236.
Walker, A.B., I.F. Sidor, T. O’Keefe, M. Cremer,
and D.L. Berlinsky. 2010. Partial replacementof fish meal with soy protein concentrate indiets of Atlantic cod. N. Am. J. Aquacult. : 72:
343-353.
Wright-Moore, W.D., W. O. Watanabe, A.J.
Bourdelais, M.S. Alam, T.C. Rezek, P. M.
Carroll, and C.A. Woolridge. 2019. Spawningperformance and egg quality of wild-caught
and first generation southern flounder
Paralichthys lethostigma broodstock inducedwith piscine and mammalian GnRH analogs.
Aquaculture 506: 367-379.
Watanabe, W.O., L.J. Theodore, D.L. Berlinsky,
C.A. Woolridge, K.R. Stuart, K.A. Copeland,and M.R. Denson. 2003. Volitional spawningof black sea bass Centropristis striata inducedwith pelleted luteinizing hormone releasing
hormone-analogue. J. World Aquacult. Soc.
34: 319-331.
Watanabe, W.O. 2011. Species profile: black sea
bass. Southern Regional Aquaculture Center
Publication No. 7207.
Watanabe, W.O., P.M. Carroll, M.S. Alam, C.F.
Dumas, J.E. Gabel, T.M Davis, and C.D.
Bentley. 2021. The status of black sea bass,
Centropristis striata, as a commercially readyspecies for U.S. marine aquaculture. Journalof the World Aquaculture Society.
https://doi.org/10.1111/jwas.12803
Watanabe, W.O., C.A. Woolridge, and H.V. Daniels.
2006. Progress toward year round spawning ofsouthern flounder broodstock by manipulationof photoperiod and temperature. J. World
Aquacult. Soc. 37: 256-272.
Webster, C.D., L.G. Tiu, A.M. Morgan, and A.
Gannam. 1999. Effect of partial and total
replacement of fish meal on growth and bodycomposition of sunshine bass Morone
chrysops x M. saxatilis fed practical diets. J.
World Aquacult. Soc. 30: 443-453.
Zapata, D. B., J.P. Lazo, S.Z. Herzka, and M.J.
Teresa Viana. 2016. The effect of substituting
fishmeal with poultry by-product meal in diets

for Totoaba macdonaldi juveniles. Aquacult.
Res. 47: 1778–1789.
Zheng, Q., X. Wen, C. Han, H. Li, and X. Xie.
2012. Effect of replacing soybean meal withcottonseed meal on growth, hematology,
antioxidant enzymes activity and expression
for juvenile grass carp, Ctenopharyngodon
idellus. Fish Physiol. Bioch. 38: 1059-1069.
Zhou, Q. C., K.S. Mai, B.P. Tan, and Y.J. Liu. 2005.
Partial replacement of fishmeal by soybean
meal in diets for juvenile cobia (Rachycentron
canadum). Aquacult. Nutr. 11: 175-182.

ANNOTATED BIBLIOGRAPHY OF KEY
WORKS
Alam, M.S., W.O. Watanabe, P.M. Carroll, J.E.
Gabel, M.A. Corum, P. Seaton, T.C.
Wedegaertner, K.S. Rathore, and M.K. Dowd.
2018. Evaluation of genetically- improved(glandless) and genetically- modified
low-gossypol cottonseed meal as alternative
protein sources in the diet of juvenile southern
flounder Paralichthys lethostigma reared in arecirculating aquaculture system. Aquaculture
489: 36–45.
One of the biggest operational expenses in marine
recirculating aquaculture systems (RAS) is highquality feed. Traditionally, fishmeal (FM) is used as
the main source of protein in marine fish diets due
to its high protein content, amino acid profile,digestibility, and palatability. Unfortunately, as theaquaculture industry continues to grow it is creatinga higher demand for FM worldwide. Limited
supplies along with increasing demand are causing
the price of FM to continually increase. In an aim toinvestigate the replacement of FM by alternative
protein sources in southern flounder diets, glandless
cottonseed meal was tested at our University ofNorth Carolina (UNCW)-Aquaculture Facility.
Cottonseed meal (CSM) proteins from geneticallyimproved (glandless) low-gossypol seed (GI-CSM,
52.1% crude protein, CP), genetically-modified
low-gossypol seed (GMO-CSM, 56.0% CP) andfrom an untreated regular (glanded) seed (R-CSM

17

49.9% CP) were evaluated to replace fish meal(FM) protein (59.5% CP) in juvenile southern
flounder Paralichthys lethostigma diets. The results
suggest that up to 75% of fish meal protein may bereplaced by GI- or GMO-CSM protein in the diet ofjuvenile flounder without adverse effects on growth
performance and body composition. Cottonseed
meal is a potentially cost- effective alternative plant
protein source for use in aquafeeds. These ultra-low
gossypol cottonseed flour proteins could be
inexpensive protein sources for the commercial
culture of southern flounder and other finfish
species.

60, and 70%. In experiment 3, body weight gain
was not significantly different for fish fed
supplemental methionine and lysine in 70% SBP
diets compared with fish fed 0% SBP diets. No
significant differences were observed inwhole‐body n‐3 polyunsaturated fatty acids among
treatments in experiment 2. Broken‐line regressionof the specific growth rate data suggested that the
maximum level of FMP replacement with SBP inblack sea bass diets was 67.6–68.4% with 75 g/kg
squid meal and 50 g/kg krill meal in the diet and
57.2–58.0% without squid and krill meal
supplementation.

Alam, M.S., W.O. Watanabe, K.B. Sullivan, T.C.
Rezek, and P.J. Seaton. 2012. Replacement ofmenhaden fish meal protein by solvent
extracted soybean meal protein in the diet ofjuvenile black sea bass Centropristis striata
supplemented with or without squid meal, krill
meal, methionine and lysine. North American
Journal of Aquaculture 74:2, 251-265.

Dawson, M.R., M.S. Alam, W.O. Watanabe, and P.
Carroll. 2018. Evaluation of poultry byproduct meal as an alternative to fish meal in
the diet of juvenile black sea bass reared in arecirculating aquaculture system. North
American Journal of Aquaculture 80:74–87.
Feeding trial was conducted to determine themaximum substitution of limits of poultry byproduct meal (PBM) protein for fish meal (FM)protein in the diet of juvenile black sea bass
Centropristis striata (family Serranidae). Eight isonitrogenous (44% CP) and iso-lipidic (13%, crude
lipid) diets were formulated to replace FM proteinwith PBM protein at 0 (control), 40, 50, 60, 70, 80,
90, and 100% in black sea bass diets. Diets were fed
twice daily to triplicate groups of juveniles toapparent satiation for 8 weeks in a recirculating
aquaculture system. Final survival was excellent(95-100%) in all diet treatments, with no significant
differences. No significant differences on bodyweight gain were observed in fish fed the 40-90%
PBM protein diets compared to the control diet.

Three experiments were conducted at our UNCWAquaculture Facility to determine the extent towhich menhaden fish meal protein (FMP) can be
replaced by solvent‐extracted soybean meal protein(SBP) in the diet of juvenile black sea bass
Centropristis striata. Diets were formulated
replacing FMP by SBP at 0, 10, 20, 30, 40, 50, and
60% (experiment 1) and 0, 60, 70, 80, 90, and 100%(experiment 2), with supplementation with squidmeal, krill meal, and attractants in bothexperiments. Experiment 3 was designed to replace
FMP by SBP at 40, 50, 60, 70, and 80% without
supplemental squid and krill meal and at 60% and
70% with supplemental methionine and lysine.
Diets were fed twice daily to triplicate groups offish (N = 15 per group) in 75‐L tanks containing
recirculating seawater. Fish were fed for 6, 10, and8 weeks in experiments 1, 2, and 3, respectively. No
significant differences in body weight gain, feed
efficiency, and survival were observed among
treatments in experiment 1. In experiment 2, no
significant differences in percent weight gain wereobserved among fish fed diets replacing FMP at 0,

However, body weight gain of fish fed the 100%
PBM protein diet was significantly lower than in thecontrol group. Regression analysis with bodyweight gain indicated that PBM protein can replace
FM protein in black sea bass diets at levels as highas 81.8%, with no reduction in fish growth
performance. For fish fed diets with up to 90%
PBM protein, feed conversion and protein

18

efficiency ratios were not significantly different
from fish fed a control 100% FM protein-based diet.
Apparent digestibility coefficients of dietary protein
remained high (81.6-87.0%) under all levels of FM
replacement with PBM protein. After the feeding
trial, whole body and muscle protein content andthe concentrations of whole body n-3 PUFAs
showed no significant differences among thetreatments at FM protein replacement levels up to
90%. PBM is a promising alternative protein source
for sustainable diet development in black sea bass.

19

Seaweed Aquaculture in a Warming Environment—Building Resilience with
Saccharina angustissima (Laminariales, Phaeophyceae)
Simona Augyte* and Charles Yarish
Department of Ecology & Evolutionary Biology, University of Connecticut, 1 University Place, Stamford, CT
06901-2315, USA
Corresponding author: simona.augye@uconn.edu
Keywords: aquaculture, climate change, kelp, Saccharina angustissima

ABSTRACT

fluctuations. Overall, this domesticated kelp species
has great potential as an economically valuable sea
vegetable in the Gulf of Maine. Nevertheless,because of its restricted geographic distribution,
care must be taken to protect the donor population.
Although additional work needs to be done to bringthis kelp to market, growing it also offers a suite of
ecosystem services, including nutrient bioextraction
and temporary habitat formation for sea animals.

Locally sourced, high quality sea vegetables, inparticular kelp, are sparking consumer interest anddemand in the New England region, USA. The
narrow-bladed sugar kelp, Saccharina angustissima,with one narrow (1.6 ± 0.7 cm wide) and one long(average 1.8 ± 0.96 m, up to 4.4 m) blade ismorphologically and genetically distinct from
Saccharina latissima, which grows much wider (2030 cm) and is usually shorter (up to 3.5 m) in
length. In the intertidal, S. angustissima is adapted
to withstand extreme hydrodynamic forces an orderof magnitude higher than the subtidal populations ofsugar kelp that are usually found in more shelteredlocations. It is restricted in distribution in midcoastal Maine and occurs in the low intertidal,
exposed to high ocean swells. In collaboration withkelp farmers from Maine Fresh Sea Farms andMaine Coast Sea Vegetables, we successfully
domesticated this kelp, growing it to full maturity
on longlines to be used commercially for its
desirable culinary traits. Based on harvest resultsfrom two growing seasons, biomass yields of thiskelp were up to 24.1 (±6.3) kg m-1 of line with aplant density of 400 plants m-1 of line. The
phenotypic characteristics that make this kelp
exceptionally adapted to extreme sea conditions
were preserved at the open-water farm sites.
Experiments with the microscopic gametophyte
stages of S. angustissima showed that it has thecapacity to withstand some temperature

INTRODUCTION
Global aquaculture production continues to increase
with wild fisheries stagnating. Sustainablecultivation can be a significant contribution for foodsecurity. Seaweeds, including those incorporated
into integrated multi-trophic aquaculture (IMTA)
and nutrient bioextraction, contribute to food andfeed production. Currently, on a global scale,seaweed production is valued at 11 billion USD(FAO 2018) with 95% of cultivation taking place indeveloping countries where it provides income tomillions of families. The global industry isdominated by a few main seaweed species with apredominance of Saccharina japonica, Undaria
pinnafida, Pyropia/Porphyra spp., Gracilaria spp.,
Eucheuma/Kappaphycus spp. (Kim et al. 2015;
Buschmann et al., 2017). Seaweed aquaculture isalso gaining interest in the US and Europe. In New
England, USA, kelp farming is developing as anenvironmentally and economically sustainableaquaculture activity that is revitalizing the working
20

waterfronts by providing economic opportunities
that are ecologically sound (Augyte et al, 2017;
Yarish et al. 2017).

(Koehl 2008). The research conducted here using
both genetics and sea farming allowed us to test theassumption that this unique kelp had a genetic basis
for its morphology and was not just phenotypicallyplastic based on the exposed conditions of where it
was found. It was also unknown if this species
could be domesticated using traditional kelp
cultivation methods.

MATERIALS AND METHODS
Reproductive material was collected in Harpswell,
Maine, USA, following low tides in OctoberNovember when the kelp was at peak sorus
production. Following standard cultivation
protocols, kelp was grown in nursery conditions at
the UCONN Stamford Marine Biotechnology
Laboratory on seedstring before being outplanted at
two open-water sea farms in Maine. Sporophytes of
the common sugar kelp, S. latissima, with parental
meiospores obtained from subtidal populations from
Casco Bay, Maine, were grown alongside the S.
angustissima sporophytes to test how environment
affects morphology. The wave energy at both farmsites was low compared to what the kelp population
experiences in the field. The resulting lengths andwidths of the two morphologies were compared atharvest. The kelp was grown to maturity on longlines for 6 months. In the lab, experiments were run
to expose the microscopic gametophyte stages todifferent temperature and light levels to better
understand the ecophysiological tolerance of theselife stages.

Figure 1. Saccharina angustissima bed in the field showing long andnarrow morphology.

The rare kelp, Saccharina angustissima (F.S.
Collins) Augyte, Yarish & Neefus, is an endemic
narrow-bladed species found on localized andexposed islands and promontories on the contiguous
mid-coastal areas of Maine, USA (Figure 1,
Mathieson et al. 2008, Augyte et al. 2018). S.
angustissima, has one blade that is very narrow (1.6
± 0.7 cm wide) and long (average 1.8 ± 0.96 m, up
to 4.4 m) that is morphologically and geneticallydistinct from Saccharina latissima, which grows
much wider (20-30 cm) and is usually shorter (up to
3.5 m) in length (Augyte et al. 2018). Many studies
on kelps have identified phenotypic plasticity as adriver of the great range of morphological variation
seen in thallus shapes as well as macroalgal
production and physiology when exposed to various
environmental parameters, specifically strong wave
exposure (Hurd 2000, Blanchette et al. 2002,
Fowler-Walker et al. 2006). Transplant experimentsof kelp (in the order Laminariales) from exposed
environments with rapid water movement toprotected ones with slow flow, often result in blade
morphology changing from narrow, thick, flat
blades to wider, thinner, and more undulate blades

RESULTSThe results of the common garden experiments at
two sea farms revealed that the sporophyte S.
angustissima blade retained its length to width ratio
and did not become wide like the sporophytes of S.
latissima (Figure 2). However, some characters,
including thickness of the blade, were lost, while
ruffles formed on blade edges.

21

sporophytes grew best at temperatures of 8-13°C at
the lowest irradiance of 10-μmol photons m- 2 s-1(Augyte et al. Accepted). Light had a significant
effect on both male and female gametophyte grown
only at the higher temperatures. Temperatures of
8-15°C and irradiance levels of 10- 100 μmol
photons m-2 s-1 were conditions for the highest
juvenile sporophyte growth (Figure 3). Finally,
calculations estimating the nutrient bioextraction
capability of the cultivated S. angustissima kelp
harvested in June reveal N removal of 88.7 kg ha-1
and C removal of 1666.7 kg ha-1 (Augyte et al.
2017).
Figure 2. Saccharina angustissima from the sea farm in Sorrento,
Maine, USA. (Photo courtesy of Sarah Redmond).

DISCUSSION
Both farms grew significantly narrower blades of S.
angustissima than of the sugar kelp, S. latissima.
Common garden experiments with the twomorphotypes identified trait stability for length andwidth, while blade ruffles and thickness varied withthe environment. Overall, this unique kelp form has
emergent potential as a crop for the Gulf of Maine,with several sea farmers growing it for commercial
purposes. In terms of microscopic stages, high lightintensity effects might pose stress on S.
angustissima, although, as an intertidal species, it
could also be better adapted to temperature and lightextremes than its subtidal counterpart, S. latissima.

These results confirmed that environmental cuesalone were not wholly responsible for the uniquemorphology in S. angustissima and suggested agenetic basis. Peak meristematic growth rates forblade length occurred from March through April at
2.85 (± 0.34) cm day-1. The kelp was harvested
from May through June with yields of up to 17 (±4.4) kg m-1 of line and plant density of 330 plants
m-1 of line at the Bristol farm and yields of 13.3 (±6.2) kg wet weight m-1 line and a plant density of
400 plants m-1 of line at the Sorrento farm. Second
season yields at Sorrento were on average 24.1(±6.3) kg m-1 of line.

REFERENCES
Augyte, S., C. Yarish, S. Redmond, and J.K. Kim.
2017. Cultivation of a morphologicallydistinct strain of the sugar kelp, Saccharina
latissima forma angustissima, from coastal
Maine, USA, with implications for ecosystemservices. J. Appl. Phycol. 29: 1967–1976.
Augyte, S., C. Yarish, and C. D. Neefus. Thermal
and light impacts on the early growth stages of
the kelp Saccharina angustissima(Laminariales, Phaeophyceae). Accepted in
ALGAE.
Augyte, S., L. Lewis, S. Lin, C.D. Neefus, and C.
Yarish. 2018. Speciation in the exposed

Figure 3. Saccharina angustissima juvenile sporophyte grown in lab
conditions.

The microscopic gametophytes and juvenile

22

ANNOTATED BIBLIOGRAPHY OF KEY
WORKSintertidal zone: the case of Saccharina
angustissima comb. nov. & stat. nov.
(Laminariales, Phaeophyceae). Phycologia
57(1):100–112.
Blanchette, C. A., B. G. Miner, and S. D. Gaines.
2002. Geographic variability in form, size andsurvival of Egregia menziesii around Point
Conception, California. Marine Ecology
Progress Series 239:69-82.
Buschmann, A. H. et al. 2017. Seaweed production:
overview of the global state of exploitation,
farming and emerging research activity. Eur. J.
Phycol. 52: 391-406. FAO 2018. The State ofWorld Fisheries and Aquaculture 2018 Meeting the sustainable development goals.
Rome. Licence: CC BY- NC-SA 3.0 IGO.
Fowler-Walker, M.J., T. Wernberg, and S. D.
Connell. 2006. Differences in kelp
morphology between wave sheltered andexposed localities: morphologically plastic or
fixed traits? Marine Biology 148:755-767.
Hurd, C.L. 2000. Water motion, marine macroalgal
physiology, and production. Journal of
Phycology 36(3):453-472.
Kim J.K., G.P. Kraemer, and C. Yarish. 2015. Use
of kelp aquaculture in Long Island Sound andthe Bronx River Estuary for nutrient
extraction. Mar Ecol Prog Ser 531:155–166.
Koehl, M. a R., W. K. Silk, H. Liang, and L.
Mahadevan. 2008. How kelp produce blade
shapes suited to different flow regimes: a new
wrinkle. Integrative and Comparative Biology
48(6):834–51.
Mathieson, A.C., E.J. Hehre, C.J. Dawes, and C.D.
Neefus. 2008. An historical comparison ofseaweed populations from Casco Bay, Maine.
Rhodora 110(941):1–102.
Yarish, C., J.K. Kim, S. Lindell, and H.
Kite-Powell. 2017. Developing anenvironmentally and economically sustainablesugar kelp aquaculture industry in southern
New England: from seed to market.
Department of Marine Sciences.
https://opencommons.uconn.edu/marine_sci/4

Augyte, S., C. Yarish, S. Redmond, and J.K. Kim.
2017. Cultivation of a morphologicallydistinct strain of the sugar kelp, Saccharina
latissima forma angustissima, from coastal
Maine, USA, with implications for ecosystemservices. Journal of Applied Phycology 29:
4.10.1007/s10811-017-1102-x.
Consumer interest and demand for North Atlantic
sourced sea vegetables drives opportunities foraquaculture development in the northeast USA. The
unique morphology and desirable culinary traits of
the wild narrow-bladed kelp, Saccharina latissima
forma angustissima, were successfully translated
into a cultivated crop on two geographically distinct
open-water farms in Maine, USA. Environmental
conditions, growth, and tissue analysis were
quantified. Peak meristematic growth rates for blade
length occurred from March through April at 2.85(±0.34) cm day−1. The kelp was harvested from
May through June with yields of up to 17 (±4.4) kg
m−1 of line and plant density of 330 plants m−1 ofline at the Bristol farm and yields of 13.3 (±6.2) kg
wet weight m−1 line and a plant density of 400
plants m−1 of line at the Sorrento farm. Second
season yields at Sorrento were on average 24.1(±6.3) kg m−1 of line. Both farms grew significantly
narrower blades of f. angustissima than of the sugar
kelp, S. latissima. Common garden experimentswith the two morphotypes identified trait stability
for length and width, while blade ruffles andthick-ness varied with the environment.
Calculations estimating the nutrient bioextraction
capability of the cultivated f. angustissima kelp
harvested in June reveal N removal of 88.7 kg ha−1
and C removal of 1666.7 kg ha−1 (combined farmsite averages). Overall, this unique kelp form has
potential as a new aquaculture crop for the Gulf ofMaine while providing several coastal ecosystemservices.

23

latissima f. angustissima (Collins) A. Mathieson to
specific rank as S. angustissima (Collins) Augyte,
Yarish & Neefus comb. nov. & stat. nov

Augyte, S., L. Lewis, S. Lin, C.D. Neefus, and C.
Yarish. 2018. Speciation in the extremely
exposed intertidal: the case of Saccharina
angustissima (Collins) Augyte, Yarish &Neefus comb. nov. et stat. nov. (Laminariales,
Phaeophyceae). Phycologia 57: 100-112.

J. K. Kim, G.P. Kraemer, and C. Yarish. 2015. Useof sugar kelp aquaculture in Long Island
Sound and the Bronx River Estuary for
nutrient extraction. Marine Ecology Progress
Series 531:155-166.

Saccharina latissima is a perennial kelp with acircumboreal distribution from the North Pacific to
the North Atlantic coasts. Our study clarified thetaxonomy of the morphologically distinct
Saccharina latissima forma angustissima (Collins)
A. Mathieson from the low intertidal zone on
exposed islands and ledges of Casco Bay, Maine,USA. To identify genetic divergence between thetwo morphotypes, S. latissima and S. latissima f.
angustissima, we used a multilocus phylogenetic
approach. Genetic analysis suggested low
divergence between the two forms. However, there
was as much or more genetic divergence between S.
latissima and S. latissima f. angustissima as there
were between other taxonomically accepted speciesof Saccharina. To investigate sexual compatibility
between the two forms, we made reciprocal crossesof the gametophytes and observed sporophyte
formation. All crosses were successfully grown to
the juvenile sporophyte stage, suggesting that thetwo are reproductively compatible in vitro. It is
unknown if the two populations freely hybridize inthe field. Last, we compared wave action, theecological factor most likely driving the uniquemorphology, at exposed sites with S. latissima f.
angustissima and protected sites with S. latissima.
The mean wave force at the exposed site was over
30 times higher in magnitude than at the protectedsite respectively, during the summer. The significant
differences in morphology, the lack of specimens
with intermediate morphologies, and the results of acommon garden experiment suggest that themorphological differences in S. latissima f.
angustissima are heritable with a genetic basis.
Therefore, on the basis of our molecular evidence
coupled with ecological studies, we are elevating S.

The present study revealed an even higher nutrient
bioextraction capacity in the cold-water species
Saccharina latissima at three sites - the mouth of
the Bronx River Estuary (Bronx, NY; BRE),
western Long Island Sound (Fairfield, CT; WLIS),
and central Long Island Sound (Branford, CT;
CLIS), during winter and spring of the 2012−2013
growing season. These sites differ in temperature(BRE > CLIS > WIS), salinity (BRE < WLIS =CLIS) and nutrients (BRE >> WLIS = CLIS). It
was estimated that S. latissima could remove up to180, 67 and 38 kg N ha−1 at BRE, WLIS and CLIS
respectively, in a hypothetical kelp farm systemwith 1.5 m spacing between longlines. In the same
hypothetical kelp farm system, the estimated carbon
sequestration values are 1350 (BRE),1800 (WLIS)
and 1100 (CLIS) kg C ha−1. The potential monetary
values of N sequestration by the sugar kelp are up to$1600 (BRE), $760 (WLIS) and $430 (CLIS)
ha−1, if incorporated into the State of Connecticut
Nitrogen Credit Trading Program and acarbon-pricing scheme. The potential economic
values of C sequestration are $30−300 (BRE),$40−400 (WLIS), and $24−240 (CLIS) ha−1.
These results suggest that seaweed aquaculture is a
useful technique for nutrient bioextraction in
urbanized coastal waters, such as LIS and BRE.
Alternation of the warm- and cold- water species
would maximize nutrient bioextraction and augment
other ecosystem services, producing economic
benefits for the region while helping to manage
non-source eutrophication.

24

Scallop Farming in Maine, a 20-year Overview
Hugh S. Cowperthwaite1*, Dana L. Morse2, and Melissa L. Britsch3
1

Coastal Enterprises, Inc. 30 Federal Street, Brunswick, ME, 04011, USA
Maine Sea Grant College Program, and University of Maine Cooperative Extension. Darling Marine Center,
193 Clark's Cove Road, Walpole, Maine, 04573, USA
3
University of Maine, Darling Marine Center, 193 Clark's Cove Road, Walpole, ME 04573, USA
2

Corresponding author: hugh.cowperthwaite@ceimaine.org
Keywords: scallop, aquaculture, Maine, Aomori

ABSTRACT

dedicated license for scallop spat collection isunderway. Altogether, fishermen, farmers,
economic development groups, scientists andregulators are engaged in pursuit of developing
Maine's scallop aquaculture industry. These
collaborations - combined with expertise fromJapanese colleagues - are a powerful aggregation ofexpertise to solve the many problems involved.
Ex-vessel value for Maine scallops has risen forover a decade, though state and federal landings
have increased, reflecting the value that consumers
place on Maine scallops specifically; and that trend
is expected to continue (Hale Group 2016). Trials
by CEI and collaborators indicate that the Japanese
production machinery appears to work well, andgrowth rates from ongoing studies and older works
appear to fall within acceptable limits, often inexcess of 25mm/year shell height (Cowperthwaite,
unpublished; Morse, unpublished; Davidson et al.
2014; Pottle and Hastings 2001). Lastly, allindications point to a growout period between two
and three years from spat (seed) to commercially
viable harvest size. Aquaculture practitioners in
Maine desire to incorporate Japanese expertise so
the industry can ramp up effectively and provide
new sources of protein. We seek ongoing advice
with lease site set-up and husbandry, growout
techniques, biofouling and predators, equipment
and machinery, handling, processing, food safety
and packaging. This innovative and unique project
offers a one-of-a-kind exchange experience that is

Climate change has profoundly impacted capture
fisheries in the Gulf of Maine. Notably, the largest
fishery in the United States is in flux: landings oflobster (Homarus americanus) have shifted
northeast in recent years. These challenges – which
are not unique to Maine and are also experienced by
Japanese fisheries - have caused great uncertainty
among fishermen in Maine. Consequently, some areturning to aquaculture to compliment fishingincomes. Maine’s fishing and aquaculture industries
have benefited from exchanges with Japan’s wellestablished scallop industry, and we continue tolearn directly from the Japanese. Coastal
Enterprises Inc. (CEI), Maine Sea Grant (MESG)
and others are working to introduce
commercially-viable scallop aquaculture into thestate of Maine, building on a 20- year-old
foundation of expertise. Maine's sister-state
relationship with Aomori Prefecture is central tothis process, with an origin dating to the wreck of
the Bath-built Cheseborough, which foundered off
the coast of Aomori in 1889. Currently, several
projects are being carried out in Maine, developing
the culture sector for scallops in coastal waters. An
aquaculture cooperative in Maine focused onscallops is now in place. Optimization of spatcollection has been ongoing work over several
years. Biotoxin monitoring in scallop tissues
enables some producers to access the live market. A
25

helping to further strengthen ties between the
United States and Japan. The introduction ofJapanese technical knowledge and equipment to ourMaine scallop industry is the first of its kind in the
United States and is groundbreaking for theAmerican aquaculture community.

Maine & Aomori to remember and recognize thetragedy as an event that brought our cultures
together, and eventually a sister state agreement was
signed. Over the years there have been several
cultural, education and trade exchange programs.
This laid the foundation for the opportunity to learn
about their scallop industry (Fig.1).

Maine/Aomori relationshipGroups from Maine visited Aomori, Japan in 1999,
2010 and 2016 with the primary focus on scallop
aquaculture. Specific topics included: spatcollection, growout techniques, productdevelopment, and environmental interactions.
Aomori is the northernmost prefecture on theJapanese mainland of Honshu (just south of
Hokkaido) and includes some of the mostproductive scallop grounds in all of Japan.

Following is a timeline of some important events inthe development of scallop culture in Maine. In
many cases, the scientific questions being asked are
direct outputs from technology transfer from Japan.
1999
The delegation from Maine that visited Aomori in
May of 1999 was an important event in theadvancement of scallop farming and stockenhancement the US. Thirteen individuals fromindustry, science, management and education
travelled for a week to Aomori Prefecture, wherestops at processors, fishermen's cooperatives, andscientific offices provided the US delegates withcritical first-hand information about spat collection
and growout of scallops. Equipped with thisexperience, there were a number of subsequent
outreach efforts all along the coast, to introduce thematerials and methods of seed collection to Maine's
fishermen. Importantly, such events also involved
scientists and regulators, which allowed thelicensing process for such work to proceedsmoothly, and to assist other scientists in joining theoverall pursuit of diversifying the scallop industry
in Maine. Another principal outcome was the
publication of a report that covered the major
findings of the trip, entitled: The culture of Japanesescallops (Patinopecten yessoensis) (Beal et al.,1999).

Aomori has a population of ~1.5M people and whenoverlaid on the US (from a geographic standpoint)
it’s at the same latitude as the state of New York.
There are several similarities Aomori shares withMaine, including rural communities with natural
resource and tourism-based economies
encompassing fishing, farming and forestry.
Maine has had a longstanding connection with
Aomori; one that began with a shipwreck. On
October 31, 1889, the Maine-built Cheseborough,was caught by a typhoon off the coast of the
Aomori Prefecture. The ship was destroyed andonly four of the (23 member) crew survived. In
1994, a Sister City agreement was signed between

2000-2003
In the years immediately after the 1999 delegation,
there were upward of 100 Maine fishermen who
participated in scallop spat collection, along
virtually the entire Maine coast, and several
reseeding efforts - mostly of the informal kind were undertaken. Of the quantifiable efforts that
occurred, re-seeding success was undemonstrated

Figure 1. The Cheseborough, built in Bath, Maine, and lost off thecoast of Aomori Prefecture, Japan, October 31, 1889.

26

(Schick and Feindel, 2005). Given this lack of clear
proof that reseeding worked, most fishermen
eventually stopped participating in spat collection.
However, this period introduced spat collection to awide segment of fishermen in Maine, and theprocesses and terminology involved became much
more familiar territory for fishermen. In addition,spat collection provided a mechanism by which theearly life stages of scallops became more clear, as
well as the interplay between spawning populations,oceanographic conditions, substrate and larval
behavior. Regular communications and interactions
with Canadian scientists, fishermen and gear
suppliers also grew during this time; all of whichbecame resources for Maine participants. As aresult, those in Maine involved in scallop spatcollection and aquaculture became much moreconnected to efforts elsewhere, and in essence
greatly broadened their professional networks.

Maine's coastal waters, and it opened the door forsmall trials. Consequently, familiarity withaquaculture increased for many fishermen, and thepassing of time allowed fishermen to better
understand the opportunities and mechanics ofaquaculture, and not to see the new industry as
merely a threat to their existing businesses.
2011-2019
Between 2011 and 2019, there were several
significant events in both the relationship between
Maine and Aomori, and the development of scallop
farming in Maine.

In 2001, the consulting company Gardiner Pinfold
completed a document entitled: Development
Potential of the Maine Scallop Industry. The report
included the potential for the development of an
aquaculture industry, and identified farm production
as an area of growth, though with caveats on sale ofwhole products and the size of the market.

Figure 2. 2016 Maine Delegation (front row) at the Aomori
Prefecture Welcome Reception.

2016
At the invitation of Shingo Mimura (Governor of
Aomori Prefecture), Coastal Enterprises, Inc. (CEI),
the Friends of Aomori (FOA), and Maine Sea Grant
assembled a ten-member group of shellfish
aquaculture industry professionals from Maine tovisit Aomori, Japan, in the fall of 2016. The purposeof the visit was to expose Maine aquaculturists to
Aomori’s well-established scallop aquacultureindustry experts for an intense one-week techtransfer exchange trip to help advance thedevelopment of Maine’s scallop aquacultureindustry. The Japanese are recognized world leadersin aquaculture and through the existing relationships
Maine has with Aomori (Friends of Aomori, MaineAomori Sister-State Advisory Council) we
strengthened our relationship with Aomori
prefecture staff and private businesses throughaquaculture. (Fig. 2)

2001 also saw a report from Tom Pottle and his
collaborators on a demonstration project in
Cobscook Bay (Pottle and Hastings, 2001). The
project examined growth rates and density in caged
scallops and was notable in that it provided aroadmap for the testing needed to sell whole
scallops. This roadmap and regulatory process wasinvaluable for efforts that came later.
2004-2010
The mid- to late-2000's saw relatively low activity
in scallop reseeding and aquaculture, althoughinvolvement in spat collection continued each yearwith a small group of fishermen. Scallops wereadded to the species allowed within the Limited
Purpose Aquaculture License (LPA) in 2008. Thiswas important because the LPA was an easy way for
new entrants to experiment with aquaculture in27

Aomori Agenda
Our agenda included visits to learn about scallop
processing, the Fisheries Research Institute for ascientific and regulatory overview, a few different
fishing cooperatives, observing aquaculture gear onland, a few trips out on scallop farms to witness
ear-hung scallops being washed and stocking
densities being reduced in pearl nets. Lastly, we
spent two days on land viewing several
demonstrations of specialized scallop aquaculturemachinery including: scallop graders, an age pin
automatic pin setter, scallop drilling machine, andscallop cleaning machines. We learned about theuse of various equipment installed on scallop
vessels to accommodate long line culture, as well as
specific gear used in the water including: spatcollection gear, pearl nets, lantern nets and ear
hanging gear.

Figure 3. Location and map Aomori Prefecture, Japan.

Our visit focused on Mutsu Bay, which issurrounded by Aomori Prefecture. The bay
experiences three foot (1 m) tides and encompasses~650 square miles (1683 ~km2). Tsugaru Strait is tothe north, the Sea of Japan to the west, and thePacific Ocean to the east. (Fig 3)

Mutsu Bay
A typical long line (in Mutsu Bay) is 200m (~650

Figure 4. Schematic of a typical longline.

28

Figure 5. The principal steps of Japanese scallop culture, and thetimes of year at which they occur.

feet). Farms are sited in water that is typically atleast ~70 feet (23 m) deep or greater. All stages ofcollection and growout can occur on the same line
including: spat collection bags, pearl nets, lantern
nets and the ear-hanging technique. Each long line
is held taut with tensioning buoys and suspended~10-20 feet below the surface of the water. Dropperlines with ear hung scallops or nets are kept ~10feet (3m) off the bottom to avoid predators.

Scallop Culture in Mutsu Bay
The basic process of scallop culture in Mutsu Bay isillustrated along a 12-month timeline. Spatcollection bags are set in April-May. Aomori
Prefecture and fishing co-ops collectively monitor
environmental conditions in the bay to best predict
approximate settlement and when to set bags.

Anchors weigh between 130-220 lbs. (59-100 kg)
and each long line has three anchors on each end(Fig 4). A long line is lowered and raised at a single
point along the line to tend to the gear. The line isthen secured on the bow and stern of the vessel with
automated star wheels, which allows the vessel toslowly work its way along the line. Biofouling ispartially mitigated by raising and lowering the linein the water column at different times of the year.
High water temperatures are also a reason to lower
the line to cooler waters.

In July and August juveniles are transferred out ofspat collection bags and put into pearl nets ~80-100
animals per net chamber. In October, the juveniles
are then transferred into clean pearl nets and thestocking density is reduced to ~15-20 animals per
net chamber. In February and March, the scallops
are then transferred to either lantern nets or the ear
hanging technique for further growout. At the
beginning of the second year, some half-maturescallops are harvested and sent to market, others aregrown out for the remainder of year two and somefor a third year for the largest meats. (Fig 5)29

approximately 21 ft2 of substrate. Collectors were
hauled in June and July of 2017, and comparisonswere made via ANOVA for the number of scallops
retained, their size frequency, and the numbers ofother bivalve settlers in the bags, by mesh type anddepth. Depth significantly affected the number ofscallops and other bivalves per collector (ANOVA,
p < 0.001 & p = 0.027), but not scallop shell height(ANOVA, p = 0.91).

Commercial Efforts in Maine
The effort to test Japanese scallop equipment in
Maine with Atlantic Sea Scallops was led by CEI.
CEI has been working with two commercial farms:
Bangs Island Mussel and Pine Point Oyster Co.
This involved drilling and pinning on ear hanging
lines to determine: how the shells hold up to
drilling, the optimum time to drill, and to track
mortality. Several months later we returned to theear hung scallops to run them through the washmachine to clean off the biofouling organisms.
Observations were made about the organisms
encountered, the fragility of shells going throughthe machine, pins breaking off, mortality, cleaning
frequency and growth rates. We’ve also been
tracking labor costs and market price paid at final
sale of the scallops.

Additionally, the number of mussels was
significantly higher near the surface of the water(ANOVA, p = 0.001, Table 1). The mean number ofscallops per collector (reported as the average, ± thestandard error) was highest at mid depths with ¼”
mesh (12293 ± 2671). The mean number of all otherbivalves per collector was highest at mid depths(1084 ± 150), and the mean number of mussels percollector was highest near the surface of the watercolumn (393 ± 94). Mussels were analyzed
separately because they can cause substantial
damage to scallop spat. The mean scallop shellheight across all depths was 9.41 ± 0.29 mm (Fig
1).

Spat bag materials: 2016-2018
Two seasons of spat collection experiments were
undertaken to examine the efficacy of collection
substrate on numbers of scallops retained, size
frequency, and presence of other settlers. The
rationale behind the experiments was that thestandard substrate - Netron - has grown very
expensive in recent years. We undertook two
seasons of experiments to compare Netron to lessexpensive options.

Retrieval date had no effect on the mean number ofscallops per collector (t-test, p = 0.24) nor the meanshell height of scallops (t-test, p = 0.49). Retrieval
date affected the mean number of other bivalves percollector (t-test, p = 0.004), but because scallops
grow slowly, the two-week difference in collection
date did not have a large effect and we treated alldata equally, with four replicates of each factor.

In September and October of 2016, four lines ofspat collectors were deployed in the coastal watersof Maine, approximately three miles southeast ofCape Elizabeth, in approximately 50m of water.
Each line supported 12 spat collectors; four at each
depth of approximately 40m off bottom, 30 m offbottom, and 24 m off bottom. Each level contained
one collector filled with the following substrates:
Netron (serving as the control), and one each ofIndustrial Nettings 1/6” (OV-7100), ¼” (OV-7822),½” (OV-1581). Each collector contained

Two spat bags were lost during the experiment: oneat the surface with ¼” mesh and one at mid depthwith ½” mesh. Notably, blue mussels (Mytilus
edulis) were observed to settle preferentially on thebags nearest the surface (Fig 6).

30

Table 1. Summary of effects of mesh type and depth on scallop
catches in spat collectors, 2016-2017.

greater set depth and that the OV-7822 appears to
perform as well as the more expensive Netron(Table 2). In coming months, we will analyze thedata using ANOVA, to test for statisticaldifferences.

Depth above seafloor (total depth 150')

Mesh
Type

High (132')

Mid (108')

Low (84')1/2"7171 ± 2068

6947 ± 735

3773 ± 1537

1/4"

10980 ± 2803

12293 ± 2671

4620 ± 931

Depth above sea floor (total
depth = 150’)

1/6"7798 ± 1366

8626 ± 1009

3923 ± 1001

High(132’)

Mid(108’)

Low(84’)

Mean byMaterial

Netron6771 ± 1155

7525 ± 871

4294 ± 1409

Netron7594.643668.573726.00

4966.40¼”(OV7822)7038.575255.83

1831.674709.69

Plurima

2879.032326.252495.50

2566.93R94499.365251.25

2862.504237.70

Mean bydepth

5502.90

4150.482728.92

Table 2. Mean number of scallops retained in collectors by depth andby substrate type, 2017-2018.

Mesh
Type

Exploring the feasibility of whole, live
scallop markets
Maine fishermen and scallop farmers have shown
interest in pursuing markets for live and whole
scallops, such as indicated by the Pottle andHastings project (2001) and the Gardiner-Pinfold
report (2001), but biotoxins have presented apersistent threat to exploring this opportunity.
Specifically, saxitoxin and domoic acid are
differentially present in scallop tissues, and resideprincipally in the digestive gland and the mantle,
where they can persist for weeks and months.
Moreover, as the scallop metabolizes thesecompounds, they may transform from low-toxicity
forms to high-toxicity forms over time, even in theabsence of a harmful algal bloom (Bricelj andShumway, 1998). Therefore, any meaningful effort
will need to include a rigorous testing program toensure the safety of any product other than adductor
muscles.

Figure 6. Mussel settlement on spat collectors by depth, 2016-2017.

Given the results of the 2016-2017 experiment, werepeated the experiment, with slightly different
variables. Collectors were set in the same locations,and set in Sept and October of 2017, and retrievedin June and July of 2018. We repeated the same
depths as previously, but our substrate types
changed, to include: Netron (control), OV-7822 (asin 2016/2017), and two similar agricultural nettings- Tenax Plurima and Tenax R-9. All bags contained
approximately 21 ft2 of netting.
Although the data are not completely analyzed todate, we examined the same variables of depth andmesh type on the catches of scallop spat, size
frequency and the presence of other settling
bivalves. Data thus far indicates that for this site,
the number of scallops collected goes down with

Such work has been ongoing since May of 2017, on
three sites along the coast of Maine. In brief, thetesting and communications mechanisms created31during the Pottle and Hastings project have beenrevisited and updated and have allowed whole
scallops to go to market in each winter between
2017 and 2019. We have documented that toxicity
will vary strongly by site, and that regular testing
can provide the necessary confidence in productsafety for regulators, producers and consumers.
While testing using the mandated
High-Performance Liquid Chromatography (HPLC)
method is expensive, it has been helpful insupporting the market exploration of whole and live
scallops, and has provided producers withadditional income, while adding valuable
information about the requirements of scale to beable to afford testing costs.

This paper is a critical review of the biotoxin
dynamics of several species of bivalve molluscs,
including the sea scallop, Placopecten
magellanicus. Specifically, the paper reviews theglobal distribution, sources of variation in toxicity,
anatomical partitioning, metabolism anddetoxification kinetics of toxins important in
Paralytic Shellfish Poisoning (PSP). As it relates tothe scallop aquaculture effort in Maine, the paper
provides details showing that scallops can rapidly
toxify, that toxins are spread differentially between
tissues within the scallop, and that toxins can takean extended time (weeks or months) to be fully
metabolized. Moreover, the paper notes that toxinschange form during metabolism, and can go fromless-toxic forms to more-toxic. The practical
implication is that even in the absence of a toxic
algal bloom, a scallop can test above regulatory
limits, through this biotransformation process.

ANNOTATED BIBLIOGRAPHY OF KEY
WORKS
Beal, B.F., M. Hastings, W. Hopkins, S. Inches, D.
Morse, K. Porter, T. Pottle, S. Rappaport, L.
Taylor, and J. Trenholm. 1999. The culture ofJapanese scallops (Patinopecten yessoensis).
Report of the Maine delegation to Aomori
Prefecture, Japan, May 14-21, 1999. The
Maine Aquaculture Innovation Center, 49p.

Davidson, L.A., R. Nowlan, and M. Niles. 2014.
Open ocean sea scallop (Placopecten
magellanicus) trials in Chaleur Bay:
Comparing culture gears and husbandry
practices. Canadian Technical Report ofFisheries and Aquatic Sciences 3121. 26p.

This report summarizes the findings of thedelegation from Maine to Aomori Prefecture in
1999. It reviews the history of the scallop
aquaculture industry and fishery in Japan, and thetechnologies, management, science and to somedegree the products and processing that havesupported the development of this sector. In
particular, there is attention paid to the spatcollection process for scallops, underlaid byreviewing the biology of Patinopecten yessoensis,
and the subsequent steps and equipment used inscallop farming in Aomori.

This report examined the use of several different
gear types in raising sea scallops; some weresuspended and some on the seabed. The main
evaluation was a comparison of alternate gears
versus the traditional lantern net. Lantern nets have
low unit cost and good performance, but high labor
cost for maintenance. The study identified cost andgrowth tradeoffs for the different methods used, andrecommended cost analyses at given sites for the
methods in question. Additionally, the study
identified spat collection in the region as being highenough to support ongoing activities.

Bricelj. M. and S.E. Shumway. 1998. Paralytic
shellfish toxins in bivalve molluscs:
occurrence, transfer kinetics andbiotransformation. IN: Reviews in Fisheries
Science 6(4): 315-383.

Gardiner-Pinfold Consulting Economists, Ltd.
2001. Development Potential of the Maine
Scallop Industry. Report to the Maine Dept. of
Marine Resources. 60p.
This report examines the status of the scallop
32

resource in Maine, and mechanisms by which theresource can be supported to gain maximum value.
They address details of the product relative to themarket, an examination of the wild fishery and its
structure, and the possibility of cultured scallops.

purpose-built vessel was constructed for the project,and growth was found to be acceptable at alldensities tested. A protocol and Memorandum ofUnderstanding was developed with the Maine Dept.
of Marine Resources as a prerequisite for
permission to sell whole scallop products.
Bi-weekly testing of scallop tissues revealed that
regulatory limits for saxitoxin and domoic acid
were not exceeded over the duration of the study.

Hale Group. 2016. Farmed Shellfish Market
Analysis. A report to the Gulf of Maine
Research Institute, Portland, ME. 65p.

Schick, D. and S. Feindel. 2005. Maine scallop
fishery: monitoring and enhancement. Finalreport to the Northeast Consortium. Award#013-138-2051-30. 73p.

The analyses for farmed product in this study
addressed eastern oysters, blue mussels and sea
scallops. Authors summarized the large domestic
market for scallop products in the US, and thelandings for US vessels as well as imports. They
project that supply over the next 15 years - the timeof the planning horizon - will stay even or perhaps
decline somewhat, and that Maine-produced
scallops command a price premium. They also notethat the industry is still in early phases ofdevelopment, and much needs to be done to capture
the market opportunity.

This project created and refined a methodology forevaluating scallop enhancement efforts in coastal
Maine, paired with information about the fishingfleet, and the spatial patterns of the scallop resourcein terms of size, meat yield, relative abundance,
recruitment and associated fauna. A GIS database ofsuitable spat collection areas was created, and spatcollection field trials were undertaken. Field
deployment of collected spat followed. Collections
were generally positive, with some spat collectors
containing over 10,000 individuals, althoughcollections varied widely by location. Results of reseeding efforts were mixed, with an admission that
more complete observation of re-seeded areas was
warranted.

Pottle, T.J. and M. Hastings. 2001. Sea Scallop
Demonstration Project, Final Programmatic
Report. Submitted to National Fish andWildlife Foundation. 10p, with appendices.
This project brought forth important processes forthe sale of whole scallops in Maine, and tested
culture gear and density in a scientific setting. A

33

The Influence of Climate and Environment on the Spawning, Condition, andLarval Set of Naturalized Pacific Oysters Crassostrea gigas in a U.S. West Coast
Estuary
Brett R. Dumbauld1*, Jennifer L. Ruesink2, Alan J. Trimble2, and Zachary Forster3
1

Agricultural Research Service, U.S. Dept. of Agriculture, Hatfield Marine Science Center, Newport, OR
97365, USA
2
3

Department of Biology, University of Washington, Seattle, WA 98195, USA
Washington Dept. of Fish and Wildlife, Ocean Park, WA. 98640, USA

Corresponding author: brett.dumbauld@usda.gov
Keywords: Pacific oyster, condition index, Willapa Bay, spawning, climate

ABSTRACT

acidification, where aragonite saturation state is
reduced and larval oysters are unable to deposit
shell, but evidence for direct effects of carbonate
chemistry in estuaries like Willapa Bay is moreequivocal due to complex interactions with otherfactors. Variables like temperature, salinity, andphytoplankton as food, as well as other sources oflarval mortality, are still related to the proximity ofthe ocean and cold upwelled water, but have beenless studied in this estuary. Some of these factors
also influence gametogenesis and spawning in adultoysters. We summarize historical records forspawning and setting in this naturalized Pacificoyster population, including a long term record ofoyster condition. We propose a new effort toevaluate some of these additional factors andcompare these records with data from locations inJapan, where these oysters were originally sourced,
and other locations with similar climate records
where Pacific oysters have been introduced. Thiseffort will help understand and potentially mitigate
for the effects of anthropogenic changes to thesesystems and an uncertain future climate.

The Pacific oyster Crassostrea gigas wasintroduced to the US west coast in the early 1900’s
where it replaced the native oyster Ostrea lurida
and has become the mainstay for the shellfish
aquaculture industry. Pacific oysters only regularly
spawned and became “naturalized” in several
discrete estuarine locations where conditions
allowed for both adult oyster conditioning andspawning and larval survival, retention andsettlement. The shellfish industry relied on“natural” set at these locations and/or continued toimport seed from Japan until the advent of hatchery
technology in the late 1970’s. Since that time, theindustry has gradually shifted to hatcheryproduction of oyster larvae, with large operators
establishing their own in-house hatcheries andsmaller companies incorporating remote setting
facilities and purchasing product from hatcheries
that specialize in producing late stage eyed larvae.
Most operators continued to take advantage of“wild” set until a prolonged set of failures in larval
production termed the seed crisis occurred in both
hatcheries and in these estuaries like Willapa Bay,
Washington, where wild set was and continues to bea feature of management on state established oyster
reserves. The failures at hatcheries have beenclearly linked to water chemistry and ocean

INTRODUCTION
The Pacific oyster Crassostrea gigas wasintroduced to the US west coast in the early 1900’s
where it replaced the native oyster Ostrea lurida
34and has become the mainstay for the shellfish
aquaculture industry (Lindsay and Simons 1997;
Robinson 1997; Shaw 1997; Steele 1964). Pacificoysters only regularly spawned and became“naturalized” in several discrete estuarine locations
where conditions allowed for both adult oyster
conditioning and spawning and larval survival,
retention and settlement (Chapman and Esveldt
1943; Quayle 1988). The shellfish industry relied on“wild” set at these locations with the mostconsistent supply of US seed oysters produced in
Dabob Bay in Washington state and/or continued toimport seed from Japan until the development ofconsistent hatchery technology in the late 1970’s(Chew 1984; Hi Im et al. 1976; Langmo and Clark
1979). Since that time, the industry has graduallyshifted to hatchery production of oyster larvae, withlarge operators establishing their own in-househatcheries and smaller companies incorporating
remote setting facilities and purchasing productfrom hatcheries that specialize in producing latestage eyed larvae (Nosho and Chew 1991).Most
operators continued to take advantage of “wild” set
until a prolonged set of failures in larval production
termed the seed crisis occurred in both hatcheries
and in these estuaries like Willapa Bay, Washington(Hales et al. 2017). Wild set, however, continues tobe an important feature of management of Pacificoysters on state established oyster reserves in
Willapa Bay (Dumbauld et al. 2011).

1964), with sampling in Willapa Bay beginning in
1955 and continuing with some gaps throughpresent day.
Recent failures of Pacific oyster larval production at
US West Coast shellfish hatcheries have beenclearly linked to water chemistry and ocean
acidification, where aragonite saturation state is
reduced and larval oysters are unable to deposit
shell (Barton et al. 2015). Evidence for direct
effects of carbonate chemistry on larvae and wild
set Pacific oysters in estuaries like Willapa Bay,
however, is more equivocal due to complex
interactions with other factors (Hales et al. 2017;
Ruesink et al. 2018). Upwelled oceanic water,which already has a low aragonite saturation state,enters this coastal estuary during summer months(Banas et al. 2007; Roegner et al. 2002), and while
this low aragonite saturation state has beenenhanced by recent atmospheric CO2 contributions(Hales et al 2017), other riverine drivers influence
this chemistry up estuary (Ruesink et al. 2018).
Variables like temperature, salinity, andphytoplankton as food have also been shown to varyalong the estuarine gradient (Ruesink et al. 2003;
Ruesink et al. 2015) and potentially influence
gametogenesis and spawning in adult oysters. Thisestuarine gradient is well known to the shellfish
growers, in part as the fattening line in Willapa Bay(Banas et al. 2007; Dumbauld et al. 2011). Oyster
growers typically plant or harvest juvenile seedoysters that are grown south of this imaginary line(from just North of Nahcotta to just north of the
Nemah River on the East side, Fig. 1) and transplantthese oysters to better “fattening” beds located northof this line for eventual harvest. This line also
serves as a rough boundary for the locations ofsuccessful spawning and setting of Pacific oysters
in this estuary, with the majority of naturally set
oysters occurring south of the line (Dumbauld et al.
2011; Ruesink et al. 2018). Thus older oysters northof the line have a high condition index or meat tovolume ratio while those south of the line a much
lower condition, in part because they spawn during
the summer season and release gametes.

Due at least in part to the longstanding importanceof the oyster industry in Washington state, arelatively substantial effort was devoted tounderstanding or at least monitoring and forecastingoyster spawning, larval abundance, and larval set,with water chemistry laboratories established atboth the Washington Department of Fish andWildlife (then Fisheries) field stations located on
Willapa Bay (Nahcotta) and Dabob Bay (Pt.
Whitney) (Lindsay et al. 1959; Packer and Mathews
1980). In addition to monitoring water chemistry
and oyster larvae during the summer spawningseason, a long term monitoring program wasundertaken to examine the condition or “fatness” ofadult oysters (Westley 1959; Westley 1961; Westley35

Condition index has been relatively extensively
evaluated as a tool for examining the effects ofenvironment on oysters and other bivalveselsewhere (Dutertre et al. 2009; Filgueira et al.
2013; Rheault and Rice 1996; Soniat et al. 1989),
and the Willapa Bay record was previously
examined as well (Schoener and Tufts 1987;
Schumacker 1999; Strickland and Ebbesmeyer
1995). We are unaware, however, of any temporal
records like this that now exceed six decades. We
characterize spatial trends that are related to theslightly less complete record for spawning andsetting of this naturalized Pacific oyster populationin this estuary, and propose a new examination of
the relationship between condition and similar
records of ocean climate in the Northeast Pacific.

Pre 1999 values are converted to gravimetricequivalents using the relationship developed by
Schumacker (1999): Gravimetric = Westley/1.180 0.905 (n= 504, r2 =0.9126). Twenty oysters were
sampled monthly at each of four sites in most years
from 1954 to present (Stony Pt., Nahcotta,
Oysterville, and Stackpole; Fig. 1). Expanded
surveys at six additional sites were conducted by
WDFW biologists from 1969-1978, with four of
those notably located south of the “fattening line”
described above, near the south end of the estuary(Refuge, Bear River, Smokey Hollow and Naselle,Fig. 1). We used these data to examine spatial andannual temporal patterns in condition index values
and group data (selecting portions of thegametogenic cycle = months) to average and obtain
annual means and better examine the longer 60 year
record. Some simple univariate analysis (ANCOVA
with month as a co-variate) were conducted to testfor differences by site. We calculated and examined
the long term anomaly = deviation from the meanfor each of these records. Finally, we examined two
similar long term records of environmental datafrom the Eastern North Pacific: the El-Nino
Southern Oscillation (ENSO; data accessed athttps://www.ncdc.noaa.gov/teleconnections/enso/in
dicators/soi/) and the Pacific Decadal Oscillation(PDO; data accessed at
http://research.jisao.washington.edu/pdo/PDO.latest) and calculated similar anomalies to visuallycompare with the oyster condition index record andevaluated these relationships with simple multiple
regression. We also used the condition index datafrom one site (Nahcotta) where oysters appear tospawn on a regular basis (e.g. condition drops
dramatically between consecutive months) todetermine whether these climate anomalies werecorrelated with the month(s) during which thisapparent spawning occurred. Finally, we calculated
a similar anomaly for the less continuous long term
record of oyster larval set in Willapa Bay, collectedby WDFW biologists and ourselves over time forcomparison. We expect the mostly visual
examinations presented here to instruct new
quantitative time series analyses like thoseundertaken by previous authors where time lags and

MATERIALS AND METHODS
While all measures of bivalve condition relate someestimate of meat biomass to the volume of the shellcavity, multiple methods have been used to makethese assessments (Abbe and Albright 2003; Crosby
and Gale 1990; Lawrence and Scott 1982).
Condition of oysters in Willapa Bay from 1954 –1998 was measured using the Westley method(Westley 1961) which uses Archimedes principle toobtain the shell cavity volume and is calculated asfollows:

Oysters were pooled to make these assessments sono estimate of error was obtained for these monthly
values. After a detailed comparison with othermethods, a consistent relationship was establishedbetween conditions collected using this techniqueand the gravimetric method, which is much easier to
conduct on individual oysters and less prone to error(Schumacker 1999). Thus, the gravimetrictechnique was adopted and all values from 1999 –present were calculated as follows:36

processes can be incorporated and further assessed(Schoener and Tufts 1987).

locations for both C. virginica (Abbe and Albright2003; Austin et al. 1993; Galtsoff 1964; Medcof

RESULTS AND DISCUSSION

Figure 1. Average oyster condition at 10 sites in Willapa Bay,
Washington, monitored from 1968-1978 (bars = SE’s). Note estuarine
gradient with higher values near the estuary mouth and lower values
and seasonal drop during summer at sites south of an estuarine
fattening line during late spring and summer (May – Sept).

and Needler 1941; Rainer and Mann 1992; Rheault
and Rice 1996; Soniat et al. 1989) in the US as well
as C. gigas in Asia and multiple locations
worldwide where it has been introduced(Chavez-Villalba et al. 2003; Enriquez-Diaz et al.
2009; Kang et al. 2000; Mason and Nell 1995;
Ubertini et al. 2017). While condition was
significantly different amongst years (ANCOVA,
F1,9 = 29.7, p <0.001), it was highest at sites near theestuary mouth and north of the “fattening” line
(Stackpole, Oysterville, Stony Point, and Bay
Center) where it also fluctuated less, especiallyduring the summer season. Oyster condition was
significantly lower at the southern end of theestuary (Nahcotta, Smokey Hollow, Bear River,
Naselle, and Refuge; ANCOVA, Site F1, 8 = 406.1,
p< 0.001) where there was often a more dramatic

The seasonal pattern of oyster condition was
similar, but average condition varied by site overthe 10 year interval with data from 10 sites in
Willapa Bay (1968-1979, Fig. 1B). As might be
expected, oyster condition increased from seasonallows in January and February to highest values insummer (June – August), and then declined again
over fall months. This seasonal cycle has been well
documented, and is related to changes in the
reproductive cycle and quantity of associatednutrients including carbohydrates, proteins andminerals in the tissues and loss of meat weight
when/if the gametes are spawned at numerous
37drop during summer, presumably due to spawning
events at these sites. This type of regional variation
has also been well studied and related toenvironmental parameters like temperature, salinity
and food (phytoplankton or chlorophyll) that
influence oyster growth and reproduction (Brown
and Hartwick 1988; Dutertre et al. 2009; Gasmi etal. 2017). We suspect that both temperature andfood are the key factors in Willapa Bay, but thatwater residence time is also important. A north tosouth gradient in chlorophyll a, phytoplankton
production, and phytoplankton species composition
has been observed in Willapa Bay that is driven by
intrusion of oceanic water into the estuary during
spring and summer months (Newton and Horner
2003; Roegner et al. 2002; Ruesink et al. 2003).
When a non-conservative tracer for Chl-a wasadded to a detailed oceanographic model created forthis estuary (Banas et al. 2007), results suggested
that there was a large difference in water residence
time between areas north of a mid- estuary line,
such that benthic grazers, like oysters south of thisline, would not experience these intrusions ofoceanic phytoplankton as often and potentially besubject to drawdowns of this production by grazers
north of the line. Growers have recognized thisfeature for some time and traditionally plant small
seed oysters to grow to harvest size and/or placeshell on their beds to encourage natural settlementof these oysters south of this line and thentransplant them to areas north of the line before
marketing them.

0.13) and lowest at Nahcotta (6.64 ± 0.09), andfluctuated over time – declining on average from1954 – 1997 and increasing since then (Table 1, Fig.
2).
Table 1. Average oyster condition index (±SE) for winter and summer
months at each site. These values represent the entire time series(1954-2017) for the first four stations, but only the shorter period(1968- 1978) for the latter six sites.

Based on the observed seasonal cycle, we divided
the data for the extended 1954-2017 time series into
two separate time segments that reflect oyster
physiology and ocean estuary conditions instead ofcalendar year: 1) winter and early spring months(October through April, termed “winter”) during
which oysters resorb gametes and accumulate
glycogen and other reserves and 2) late spring andsummer (May through September, termed“summer”) during which oysters produce gametes
and potentially spawn. The average condition
during winter at the four sites measured over thislonger time frame was highest at Stackpole (11.02 ±
38

Site

Winter CI (Oct- Summer CI
April)(May-Sept)

Stackpole

11.02 (0.13),
n = 355

12.70 (0.18),
n = 237

Oysterville

10.02 (0.14),
n = 314

11.57 (0.21),
n = 213

Stony Point

8.56 (0.11),
n = 392

10.67 (0.17),
n = 266

Nahcotta

6.64 (0.09),
n = 391

8.06 (0.14),
n = 269

Bay Center

8.91 (0.28),
n= 67

10.74 (0.31),
n = 48

North Long
Island9.08 (0.20),
n = 6210.42 (0.35),
n = 47

Naselle6.76 (0.21),
n = 699.55 (0.34),
n = 47

SmokeyHollow

6.66 (0.20),
n = 678.01 (0.32),
n = 47

Refuge7.18 (0.21),
n = 67

8.63 (0.35),
n = 48Bear River

5.83 (0.17),
n = 697.32 (0.33),
n = 48Figure 2. Average winter (Oct through April) oyster condition index
at 3 sites in Willapa Bay from 1954 - 2017. Note distinct differences
between sites, but similar fluctuations and long term trends with adecline from 1954 – 1997 and subsequent increase.

39

Figure 3. a) Oyster condition index anomaly at Stony Pt. site forwinter and spring months (October through April) compared with b)
Pacific Decadal Oscillation anomaly and c) Southern Oscillation
Index anomaly for the same period. Note the axis has been inverted
for the PDO and shifts are noted with vertical dashed lines andwarmest El Niño events with red arrows.

Summer values were higher but more variable,
especially at sites closer to the southern end of theestuary, where rapid declines during these months
indicated that the oysters likely spawned. Since themost complete data records were for the Stony Pt.
40

Figure 4. Oyster condition index anomaly at Nahcotta during
summer months (May through September, top) compared with Pacific
Decadal Oscillation anomaly for this same period (bottom). The axis
for the PDO has been inverted and shifts are noted with verticaldashed lines.

nearshore ocean climate. A general relationship wasapparent between oyster condition and the Pacific
Decadal Oscillation (PDO), with negative
anomalies corresponding with warm phases andpositive condition anomalies with cool phases (Fig.
3), but a breakdown in the strength of this patternoccurred after about 1997. A general visual
relationship was also evident between the winterand Nahcotta sites, we used these records tocalculate and evaluate the long term anomalies in
condition and compare them with several indices of41

Table 2. Results of simple multiple regression relating the long term
record of oyster condition index anomaly at two sites to similar
records for the Pacific Decadal Oscillation (PDO) and Southern
Oscillation Index (SOI). Results from two separate models forsummer and winter averages are shown.

oyster condition anomaly and the southern
oscillation index (SOI) anomaly, with positive SOI
values (and El Niño events) corresponding to lower
condition and negative SOI (La Niña events)
corresponding with positive condition (Fig. 3).
Though individual events, including those after
1997, seem to be correlated, no statistical
relationship emerged. Schoener and Tufts (1987)
found little relationship with sea surface
temperature when examining the same data set, but
a significant relationship with sea level height thatbest fit a phase lag with changes in sea level heightpreceding those in condition by several months.
They documented the lowest condition index onrecord to that point in time corresponding with the
1982-1983 El Niño. That low value has now beensurpassed by two even more negative condition
anomalies, one of which corresponds with the evenstronger 1997 El Niño. The previous declining trend
in oyster condition in Willapa Bay has also beencorrelated to the Pacific Northwest Index (acombined climate index derived from three moreterrestrial parameters including temperature andprecipitation) and the decline in Columbia River
discharge (Strickland and Ebbesmeyer 1995), but
we suspect the recent rebound in condition would
not track at least the latter.

Estimate SE
Model

F

P

3

10.75 <
0.001

SummerPDO-0.97

0.19 125.49 <
0.001

Summer
SOI

0.01

0.28 10.002 0.97

Site (Stony -0.05
Pt.)

0.24 10.05

0.81

Error/Total
Winter
PDO

122/
125
-0.12

0.22 32.48

0.12

Winter SOI 0.35

0.18 10.45

0.50

Site (Stony -0.06
Pt.)

0.20 10.09

0.76

Error/Total

42

df

123/
126

The relationship between PDO and the oyster
condition anomaly was significantly negative insummer (Fig. 4, Table 2). Years when a significant
drop in condition occurred during these summer
months, signaling a spawning event, were clearly
evident at the Nahcotta site, and we further
separated this data into those years occurring during
warm (PDO > 0) and cool phases (PDO< 0).
Spawning occurred during both phases, yet during
warm phases of the PDO, condition was
significantly higher in years that these eventsoccurred than when they did not (Fig. 5, ANCOVA ,F1,165 = 11.12, p = 0.001), while there was nodifference during the cool phase (ANCOVA, F1,180 =
1.78, p = 0.18). In those years when these spawning
events occurred, they often occurred earlier (Juneand July) during warm phase years and later(August) during cool phase years. This result
suggests that oceanic conditions may affect theseasonal gametogenic cycle prior to spawning.
More broadly, multiple triggers including
temperature, but also food and even disturbance
events, can be associated with spawning (Dutertre etal. 2009; Ubertini et al. 2017).

Figure 5. Average monthly oyster condition index at Nahcotta site foryears where the average PDO anomaly in the summer (May throughSeptember) was negative (top) and positive (bottom). Years with avisually distinct drop in condition (presumably due to spawning) arecontrasted with years when this was not apparent. Note the apparent
seasonal difference in timing of spawn events with more andpotentially earlier spawning events occurring during warm phase of
the PDO.

43

Figure 6. Anomaly of oyster sets (counts of oyster spat per shell) on
seasonal shell strings deployed at three sites in Willapa Bay (top)compared with the Pacific Decadal Oscillation anomaly for summer
months (May – October; bottom) with shifts noted by dashed lines.

simply more likely to result in successful spatfall inthis estuary, due to the typical three week period oflarval development during which other factors (e.g.
water retention, vertical migration, wind andweather) are involved, this relationship requires
further investigation. The PDO has been related tonumerous other biological endpoints, fromzooplankton to coho salmon (Malick et al. 2015;
Menge et al. 2008; Shanks 2013). This included

There appears to be a positive relationship between
successful spatfall resulting from these spawning
events and the PDO with larger spatfalls occurring
during warm phases of the PDO (Fig. 6). While it
may be that earlier or multiple spawning events are
44

oyster condition in one of the original
characterizations (Hare and Mantua 2000), but
defining the mechanism for this interaction withnearshore ocean conditions remains a useful
research avenue. The PDO has recently also been
related to phytoplankton community structure (Duet al. 2015) with cool phases being connected to adiatom rich community and warm phasecommunities dominated by dinoflagellates. Theseauthors have also shown that a similar community
was present during the recent presence of ananomalous warm water mass (referred to as theblob, Bond et al. 2015) in the nearshore coastal
ocean off of the US Pacific Coast and that the chl asignature and overall biomass remained high. This
could explain the breakdown we observed in therelationship between the PDO anomaly and oyster
condition at about the same point in 2013, but moreevidence should be gathered to test this hypothesis,
and we propose that a new effort to evaluate someof these additional factors be initiated. This could
include a comparison with data from locations inJapan where these oysters were originally sourced
and similar long-term records for other locations
where Pacific oysters have been introduced andlong term records are in place, in order tounderstand and potentially mitigate the effects ofanthropogenic changes to these systems and an
uncertain future climate.

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US West Coast. Multiple authors contributed to thisreview, which addresses a broad audience, butcovers the leading research on direct effects tobivalve larvae, as well as monitoring seawaterconditions and adapting to these changes.

confirm effects, they substantiate the complexity ofmeasuring these effects and attributing them to asingle cause in an estuary where conditions arevariable.
Ruesink, J.L., A. Sarich, and A.C. Trimble. 2018.
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Dumbauld, B.R., J.L. Ruesink, A.C. Trimble, andB.E. Kauffman. 2011. The Willapa Bay oyster
reserves in Washington State: Fisherycollapse, creating a sustainable replacement,
and the potential for habitat conservation andrestoration. Journal of Shellfish Research 30:
71-83.

These authors measured seawater chemistry inWillapa Bay, Washington, but unlike Hales et al(2017), they also present simultaneously collected
data on four cohorts of Pacific oyster larvae thatwere collected over three summers. The southernend of Willapa Bay has two arms which create
distinctly different characteristics because one ismuch more affected by riverine conditions thatcause reduced aragonite saturation relative to theother. Oyster settlement differed greatly between
cohorts, but they did not find differences they could
attribute to this different water chemistry, andinstead found thermal conditions were perhaps more
important.

The authors review the history of the state oyster
reserves in Willapa Bay, Washington, where Pacificoysters were introduced in 1928 and one of a few
locations on the US West Coast where a naturalized
population became established. They review therecord of larval set in this estuary and attempt tolink this to broad scale oceanographic conditions(but see Hales et al 2017 and Ruesink et al. 2018
below for a more detailed discussion of waterchemistry within this estuary).

Weisberg, S.B., N. Bednarsek, R.A. Feely, F. Chan,
A.B. Boehm, M. Sutula, J.L. Ruesink, B.
Hales, J.L. Largier, and J.A. Newton. 2016.
Water quality criteria for an acidifying ocean:
Challenges and opportunities for
improvement. Ocean & Coastal Management
126: 31-41.

Hales, B., A. Suhrbier, G.G. Waldbusser, R.A.
Feely, and J.A. Newton. 2017. The carbonate
chemistry of the "Fattening Line," Willapa
Bay, 2011-2014. Estuaries and Coasts 40:
173-186.
The authors present detailed data on seawaterchemistry (especially PCO2 and aragonite saturation
state) for Willapa Bay, Washington, where Pacificoysters have been the mainstay of the oyster
aquaculture industry for almost 100 years and there
is a long term record of spawning and setting. They
reconstruct this record for a longer historical period
and their data suggests that recent conditions
provide a smaller window of optimal conditions(low aragonite saturation state and warm enoughtemperatures for oyster spawning) than occurred
historically. While they did not sample larvae (seeRuesink et al 2018 below) and therefore can’t

This review represents a call for developing
ecologically relevant water quality criteria foracidification and augmenting coastal water quality
monitoring efforts to characterize this at appropriate
temporal and spatial scales. Historical data foroyster larval recruitment in Willapa Bay is one oftwo case examples presented that demonstrate thechallenges involved in relating biological endpoints
to appropriate water chemistry criteria in estuaries
where conditions are variable.

48

A Review of the Potential of Macroalgae Feedstuffs to Reduce Methane
Emissions from Ruminant Livestock
Luke Gardner1,2*, Scott Hamilton 2, Michael Graham2, Stacey Gunter3, and Kenneth Kalscheur4
1

California Sea Grant, Scripps Institution of Oceanography, 9500 Gilman Drive, Dept. 0232 La Jolla,
CA 92093
2
Moss Landing Marine Laboratories, 8272 Moss Landing Rd., Moss Landing, CA 95039
3
United States Department of Agriculture, Agricultural Research Service, Southern Plains Range Research
Station, 2000 18th Street, Woodward, OK 73801
4
United States Department of Agriculture, Agricultural Research Service, U. S. Dairy Forage Research Center,
Dairy Forage Research Unit. 1925 Linden Drive, Madison, WI 53706
Corresponding author: lgardner@ucsd.edu
Keywords: seaweed, climate change, methane, aquaculture, livestock

ABSTRACT

low environmental impact, coupled with thepotential to mitigate climate change impacts andbio-remediate coastal eutrophication zones makethis area of research a promising mitigation solution
for climate change. Research is required to assess
different species of local seaweed for total gas
production and digestibility when added to cattle
diets. Methane reducing candidate species also need
to be assessed for optimal culture conditions andscalability to be used as a feed additive for ruminant
livestock.

To address climate change in California beyond
carbon-based gases, the state has recently passed
legislation to reduce short-lived climate pollutants.
In particular, the state aims for a 40% methane
reduction by 2030. Several reductions are expected
to come through existing technologies associatedwith landfills and energy producers. However, thestate’s largest contributor to methane production iscattle, specifically 1.4 million resident dairy cows
and one million beef cattle. Methods to reducegases produced from cattle have largely been
unexplored in California. However, recent researchin Australia has shown that some species of
sub-tropical seaweed can reduce methane
production from cattle by up to 99% when used as afeed additive at 2% inclusion rates of organic
matter. Temperate species have not yet beenassessed for methane reducing properties, but manyof the species present in the California Current
Ecosystem are closely related to these sub- tropical
species. The nutrient-rich waters off the Californiancoast are ideal for the development of seaweed
aquaculture and this project offers an excellent
opportunity to address the underdevelopment ofaquaculture in California. Seaweed aquaculture's

INTRODUCTION
Seaweed has been harvested by humans for morethan 1700 years and has been used for a variety of
purposes, including food, animal feed, andnumerous industrial uses over this history.
However, it is only in the last few decades that
seaweed aquaculture has begun to develop. At lastcensus, approximately 27 million tons of seaweed is
cultivated per year, representing a quarter of thetotal aquaculture production worldwide (FAO,
2016). The vast majority of seaweed is produced in
China and Indonesia, generating a combined total of
23 million tons. Cultivation of seaweed in the US is
nascent and does not yet generate enough products49

to be quantifiable on a global scale. This imbalanceis largely due to the traditional and common use of
seaweed in Asian countries. However, a US market
has begun to emerge based on powerful consumer
preferences towards organic and sustainableproducts. The sustainable benefits of seaweed
aquaculture are undeniable; it requires zero
additional nutrient inputs for growth and does not
displace terrestrial crops for arable land or require
fresh water, representing an alternative biomass fornutrition, compounds and energy in the future.

emissions. Consequently, California recently
enacted legislation in 2016 mandating a 40%
reduction from present day methane emissions by
2030 (2016).
Several technologies and strategies currently exist
to mitigate methane production from sources likelandfills, yet solutions for the dairy industry are lessdeveloped. Dairy digesters, specialized structures
used to capture methane generated fromdecomposing manure, are slowly being
implemented on farms in California. However, this
only accounts for about a quarter of the methane
emitted from dairy cattle with the vast majority
being released via burps as a result of entericfermentation (EPA, 2018; Powers et al., 2014).
Enteric methane is difficult to mitigate because it
involves the natural process of a cow’s digestion.
However, research on dietary strategies have shownpromise (Hristov et al., 2015; Lee et al., 2017; Maia
et al., 2016). Some dietary practices that have beenshown to reduce methane include the addition ofionophores, fats, the use of high quality forages, andthe increased use of grains (Caro et al., 2016).
These nutritional strategies reduce methane throughthe manipulation of the ruminal environment,
inhibition of methanogens and protozoa, or by
redirection of hydrogen ions away frommethanogens. Relatively new mitigation options
have been investigated and include the addition ofsuch additives as probiotics, acetogens,bacteriocins, organic acids, and plant extracts (i.e.
condensed tannins) (Martin et al., 2010). Morerecently, researchers abroad have begun toinvestigate the effect of sub-tropical seaweed as
feed additives for ruminants, discovering that a red
macroalga called Asparagopsis taxiformis can
reduce methane production by 99% at less than 2%
inclusion in vitro and produced up to a 81%
reduction in vivo sheep fed at 3% inclusion rates (Liet al., 2018a; Machado et al., 2016). The mode bywhich A. taxiformis reduces methane production isstill unclear, but most recent research attributes the
reductions to the bioaccumulation of halogenatedmethane analogues produced as algal secondary
metabolites (Machado et al., 2018). The advantages

Furthermore, seaweed aquaculture can provide a
myriad of ecosystem benefits including:
oxygenation and uptake of nutrients by enhancing
primary production (Vásquez et al., 2014);
bioremediation of eutrophic zones, reducing
harmful algal blooms; creating food, habitat andrefuge for a diverse array of fish and invertebrates,
thereby increasing biodiversity (Almanza andBuschmann, 2013); and mitigation ofgreenhouse-gas emissions by acting as a CO2 sink
(Chung et al., 2011) and most recently by reductionof methane production from livestock (Li et al.,2018a; Maia et al., 2016).
Release of greenhouse gases into the atmosphere isa major environmental, social, and economic threat
worldwide. Methane is a particularly potent
greenhouse gas that is 25 times more efficient at
trapping heat in the atmosphere than carbon dioxide(Solomon et al., 2007). It is well documented thatruminant livestock contribute significantly to theglobal release of methane via digestion. Methane isemitted from livestock because of methanogenesis(a byproduct) in the forestomach of ruminants; asimilar process occurs in the decomposition ofmanure. The United States is the fourth largest
cattle producer in the world and cattle contribute
26% of the total U.S. methane emission (EPA,
2018). Dairy cattle are the largest methane
producers among livestock and produce between 73
and 182 kg of methane per cow annually (Dong etal., 2006). California is the dairy industry’s mostproductive state and has 1.4 million dairy cows that
account for the majority of the enteric methane50

of seaweed over other dietary manipulations is that
seaweed has shown the most significant methane
reductions with minimal declines in digestibility (Liet al., 2018b; Maia et al., 2016), and seaweed
production does not compete with terrestrial foodproduction systems.

considering the likely secondary ecosystem benefits
resulting from seaweed cultivation. California has
the potential to utilize this resource to build andgrow the nascent seaweed aquaculture sector.
Growing seaweed for livestock feed offers a unique
opportunity to circumvent many challenges
associated with terrestrial production systems, while
also mitigating methane emissions. Such a large
potential local market could help develop vital
infrastructure, skills, and distribution systems forseaweed aquaculture, if realized.

While an assessment of enteric methane production
has been done for a number of tropical species(Machado et al., 2014), nothing is known about themethane reduction properties of temperatemacroalgae. This presents an excellent opportunity
for the development of the seaweed aquacultureindustry in California, in temperate coastal watersthat are highly productive compared to tropicallatitudes, and thus provide superior conditions forseaweed culture.

Potential benefits: The potential economic benefitof this research to California’s aquaculture industry
could be vast considering the immense value of thestate’s dairy and beef industry at $21 billion dollars
annually. Beyond this, the nation’s combined cattle
industry is worth $78.2 billion, of which most cattle
are fed formulated diets for a significant period of
their life. Development and production of feed
additives in California that could be exported andsupplemented to cattle diets across the United States
would be extremely beneficial to California’s entire
economy. Additionally, the culture of seaweeds forsuch purposes in California could help develop
nascent aquaculture industries providing alternative
economic growth for coastal communities that are
significantly dependent mostly on commercial
fisheries. Outside the potential economic value of
this research, the state would also stand to benefit
environmentally by reduction of methane andabsorbing agricultural nutrient nonpoint source
pollutants as they are incorporated into seaweedbiomass. California is home to a significant numberof coastally drained waterbodies that are impaired
due to high levels of nutrients coming from irrigatedagriculture (Anderson et al., 2003). Sixteen of theseeutrophic water bodies drain into the Monterey Bay
where Moss Landing Marine Laboratories islocated. Seaweed aquaculture has been shown to beeffective in bioremediation of coastal eutrophic
areas (He et al., 2008). Development of seaweed
aquaculture could aid in reducing the impact ofeutrophication by absorbing nutrients from thewater and incorporating them into seaweedbiomass. This biomass could then be harvested for a

Scalability: In concert with identification of
methane reducing seaweed species, it is prudent totheoretically consider the scalability of seaweed
production. The United States has the world’s
largest marine exclusive economic zone, an area
equivalent to the total land area of all 50 states. The
U.S. Department of Energy estimates that the U.S.
has suitable conditions and geography for producing
approximately 500 million tons (DW) of seaweed(ARPA-E, 2017). California has the third largest
coastline in the U.S., with state waters
encompassing approximately 13,688 km2. Based ona series of assumptions, including an average
seaweed production value of 1,604 t km2 year-1(DW) (Xiao et al., 2017) and a dairy cow
consuming 9,855 kg-1year-1 (DW), a total of
140,000 t (DW) seaweed year-1 would be required tofeed the state’s 1.4 million dairy cows at a 1%
inclusion rate. Using these assumptions, a total of
86 km2, or 0.6% of state waters, is all that would be
required to culture the necessary seaweed volume toachieve approximately a 50% or more reduction of
methane emissions from California’s 1.4 million
dairy cattle, the biggest methane contributor in thestate. While this estimation is based on many
assumptions, a total of 0.6% of state waters
potentially being used for seaweed aquaculture
seems relatively tolerable, especially when51

variety of uses, including the recycling of thesenutrients back into agricultural production as a feed
ingredient.

Wattiaux. 2015. Performance, digestion,
nitrogen balance, and emission of manureammonia, enteric methane, and carbon dioxide
in lactating cows fed diets with varying alfalfa
silage-to- corn silage ratios. Journal of Dairy
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Energy Resources. United States Departmentof Energy, 2017.
Brown, R. H., Mueller-Harvey, I., Zeller, W. E.,
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Hardcastle, E. E., 2017. Facile Purification ofMilligram to Gram Quantities of Condensed
Tannins According to Mean Degree ofPolymerization and Flavan-3-ol Subunit
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Caro, D., Kebreab, E., Mitloehner, F. M., 2016.
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1967-1973.

ANNOTATED BIBLIOGRAPHY OF KEY
WORKSMachado, L., M. Magnusson, N.A. Paul, R. de Nys,
and N. Tomkins. 2014. Effects of Marine andFreshwater Macroalgae on In Vitro Total Gas
and Methane Production. PLOS ONE. 9,e85289.
The authors for the first time assess an array ofdifferent sub-tropical seaweeds for methane
reducing properties in cattle using in vitro
techniques. They find that a red seaweed species
called Asparagopsis taxiformis is the most effective
seaweed tested, reducing methane production by up
to 99%.
Maia, M. R., A.J. Fonseca, H.M. Oliveira, C.

54

Mendonca, and A.R. Cabrita. 2016. The
Potential Role of Seaweeds in the Natural
Manipulation of Rumen Fermentation andMethane Production. Sci Rep. 6, 32321.

Li, X., H.C. Norman, R.D. Kinley, M. Laurence, M.
Wilmot, H. Bender, R. de Nys, and N.
Tomkins. 2018. Asparagopsis taxiformis
decreases enteric methane production fromsheep. Animal Production Science. 58,
681-688.

This research is important in that it assesses theeffect of the basal feed source (substrate) on
methane reduction for a number of different
seaweed species. The authors find that there is asignificant effect on methane reduction based onwhether the basal feed is either grass or corn,highlighting the need to further examine theseaweed interaction with different cattle feeds.

The authors for the first time assess the effect ofseaweed on methane reduction using a wholeanimal experimental design. The article reports thatsheep fed a 3% diet of seaweed Asparagopsis
taxiformis reduce their methane emissions by 81%
without any decrease in weight gain compared tothe control treatment.

55

Condition Index and Fecundity of Manila Clam Ruditapes philippinarum Related
to Habitat Environment
Natsuki Hasegawa1*, Toshie Matsumoto1, Satoshi Watanabe1, Kazuhiro Hanyu2, Dai Miyawaki3, Ryogen
Nambu4, and Masami Hamaguchi5
1

National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 422-1
Nakatsuhamaura, Minamiise, Mie, 516-0193, Japan, 2 Suzuka Branch, Mie Prefecture Fisheries ResearchInstitute, Suzuka, Mie, 510-0243, Japan
3
Aichi Fisheries Research Institute, Gamagoori,
Aichi, 443-0021, Japan
4
National Research Institute of Fisheries Engineering, Japan Fisheries Research and Education Agency, 7620-7,
Hasaki, Kamisu, Ibaraki, 314-0408, Japan
5
National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5,
Maruishi, Hatsukaichi, Hiroshima, 739-0452, Japan* Present address: Kushiro Field Station, Fisheries Resources Institute, Japan Fisheries Research and Education
Agency, 116 Katsurakoi, Kushiro, Hokkaido, 085-0802, Japan
Corresponding author: hasena@fra.affrc.go.jp
Keywords: Ruditapes philippinarum, Manila clam, condition index, fecundity, habitat

ABSTRACT

correspondence analysis revealed that the conditionindex during both spawning seasons was positively
correlated (r = +0.48 to +0.80) with the 1st and 2nd
correspondence analysis scores on various habitat
parameters. On the other hand, the fecundity, which
is summed for each spawning season (threesampling days) and normalized to 30 mm shelllength, was negatively correlated with the 2nd
habitat score (spring: r = -0.64, autumn: r = -0.80).
These results show that the nutritional condition as
determined by the condition index is not the solefactor affecting fecundity of the clam. Actually, thefecundity was high in the intertidal zones andenclosed river mouth areas, whereas the conditionindex was higher in the subtidal zones and open
non-river mouth areas. The information obtained
about asari clam reproduction may be useful forstock enhancement by improving reproduction in
the populations. This study was conducted as part ofa research project for recovery of asari populations
by the Fisheries Agency of Japan.

Understanding reproductive traits is important for
maintenance or recovery of Manila clam Ruditapes
philippinarum fisheries production, which has
dropped considerably in Japan over the past half
century. Relationships between condition index andnumber of eggs (fecundity) of the Manilla clam
were assessed at several clam habitats in Ise Bay,
Japan, during spring and autumn spawning seasons
in 2015. Condition index was calculated from theshell size and soft body wet weight. Fecundity wasquantified using an indirect enzyme-linked
immunosorbent assay (ELISA) with a monoclonal
antibody specific to a Manilla clam yolk protein.
Land elevation (i.e. subtidal or intertidal),association with river input (river mouth or not),closeness of the shore, and chlorophyll aconcentration in the water column were the habitat
parameters analyzed in this study. A

56

INTRODUCTION

for executing stock enhancement programs for theclam.

Manila clam, Ruditapes philippinarum, alias nameshort neck clam, or asari in Japanese, is an
important bivalve species for commercial andrecreational fisheries in Japan. According to thestatistics released by the Ministry of Agriculture,
Forestry and Fisheries of Japan, fisheries productionof suspended and bottom culture of bivalve species,
such as scallop and oyster, are slightly decreasing or
staying level in Japan (Fig. 1). However, capture
fisheries production of Manila clam has dropped
considerably in Japan over the past half century(Matsukawa et al., 2008). The production peaked at
170,000 tons in 1983 and went down to 9,000 tons
in 2016, which is a 95% decrease. The Fisheries
Agency of Japan and local governments haveundertaken some projects for clam stockenhancement, but the decreasing trend iscontinuing.

One of the most important reproductive traits to bestudied is variation in fecundity (the number ofeggs) of the clam associated with environmental
factors. Some previous studies reported that
individual fecundity is simply proportional to theclam’s body weight; in other words, larger clams
spawn more eggs than smaller clams (Toba &Miyama 1994) and robust clams have more
fecundity than thin clams (Park et al. 2006,
Hasegawa et al. 2014). Therefore, individual
fecundity depends on the body weight and thenutritional condition, and fecundity per unit bodyweight is relatively constant among clams ofdifferent sizes. These statements sound intuitively
true, however, these results were obtained for clams
collected within the same habitat or cultured undersimilar rearing conditions except food level(abundance). Variation in the clam’s growthdepending on the habitat was evaluated in various
studies such as Kakino (1996). The effects ofvarious environmental factors on clam fecundity
should be further studied.

Figure 1. Fisheries production of bivalve species: Manila clam,
Japanese scallop and oysters, from the statistics released by the
Ministry of Agriculture, Forestry and Fisheries, Japan. The
production of Manila clam includes a small amount of purplish
Washington clam, Saxidomus purpurata, production.

We need more basic knowledge on the population
dynamics to improve or create newcountermeasures to mitigate the population declineof the clam. Reproductive traits may be important

Figure 2. Study sites in Ise Bay, Japan. The ID 1 on the map
represents the Manila clam, Ruditapes philippinarum, habitat in
Kusu, Yokkaichi (2: Shiroko, Suzuka, 3: Miwatari, Matsusaka, 4:
Kushida, Matsusaka, 5: Higashikurobe, Matsusaka, 6:
Offshore-Muramatsu, Ise, 7: Intertidal-Muramatsu, Ise, 8:
Kawaguchi flat, Ise, 9: Isshikimae flat, Ise, 10: Kosugaya,
Tokoname).

57

Table 1. Clam habitat environmental parameters in this study.

Habitat
ID

1

2

3

4

5

6

7

8

9

10

Land
elevation

inter

sub

sub

sub

sub

sub

inter

sub

inter

inter

River
input

none

River
mouth

River
mouth

River
mouth

none

none

none

River
mouth

River
mouth

none

6.9

7.6

4.9

6.1

6.8

6.8

4.0

3.2

8.7

0°

50°

0°

150°

210°

210°

0°

10°

180°

Chl. a µg 12.8
L-1
Openness
150°of theshore

In Japanese coastal areas, Manila clams can befound in various habitats. In Ise Bay, the clams are
distributed in habitats ranging from intertidal tosubtidal, exceeding 10 m depth, and from rivermouth to non-river offshore areas (Hanyu et al.
2017). River mouth areas have large salinity
fluctuations, while non-river areas typically havestable and high salinity water, ranging from closed
and calm areas to more open areas under the
influence of strong wave action, and from poor togood food availability. Therefore, we need toevaluate the reproductive capacity of the clam undera wide variety of environmental conditions.

shown in Table 1. Here we focused on the land
elevation, river water input, chlorophyll a level as
an index of food abundance, and openness of theshore to offshore water. Chlorophyll fluorescence ataround 1-m depth from the sea surface wasmeasured by a CTD (Rinko profiler, JFE Advantech
Co. Ltd., or DS5, Hydrolab Co. Ltd.) and
chlorophyll a concentration was estimated fromthese at each clam collection site. The meanchlorophyll a concentration in 2015 (n = 4 to 20)
was used as the chlorophyll a level at each habitat.
The openness was expressed as the angle between
two lines connecting the habitat and two points on
the coastline 1 km away from the habitat (i.e. a
modified method of that described by Ruuskanen etal. 1999).

MATERIALS AND METHODS
Study area and environmental
characteristics of habitat
Condition index and the fecundity was assessed in
Manila clams collected from 10 locations in Ise
Bay, central Japan, in 2015. The environmental
characteristics of the studied clam habitats are

Measurement of condition index andfecundity of the clams
Manila clams were collected approximately
monthly for monitoring of nutritional condition andfecundity from 10 habitats in Ise Bay between April
and November 2015. Approximately 10–30 clams
58

with a shell length (SL) of 16–51 mm (mean 31.2mm) were haphazardly collected. After measuring
the shell length, shell height (SH), shell width(SW), and the total wet weight, the clams weredissected, and soft body tissue wet weight wasmeasured. The soft body tissue of female clams(identified by the presence of oocyte in cut out
tissue) was stored at -80°C for fecundity
assessment. Condition index (CI) of the clams wascalculated as follows;

Glostrup, Denmark) was added as a color agent.
Finally, 50 µl of 2-N sulfuric acid was added as thestop solution. The optical density of the end productwas measured at 450 nm using a microplate reader.
The tissue homogenate supernatant of the clams andthe yolk protein content per egg obtained by
Hasegawa et al. (2014) was used for calculation of
the number of eggs. Clam fecundity was
represented as the number of eggs per unit of softbody weight (1 g WW).
In south-western parts of Japan including Ise Bay,the clam has two spawning seasons: spring andautumn, and the clam spawns repeatedly during
each spawning season (Matsumoto et al. 2014).
Therefore, as the representative fecundity in each
spawning season, we summed the egg number thatwas normalized to the size of the clam, 30 mm SL(for removing the effect of size), for three months ineach spawning season. However, the histologicalobservation of the gonadal development showedthat small numbers of Manila clam in Ise Bay were
sexually undifferentiated even during the spawningseasons (Matsumoto et al. 2014). Therefore, thesum of the egg number might be overestimated inour study.

𝑆𝑜𝑓𝑡 𝑡𝑖𝑠𝑠𝑢𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔 𝑊𝑊)

𝐶𝐼 = 𝑆𝐿 (𝑚𝑚) 𝑥 𝑆𝐻 (𝑚𝑚)𝑥 𝑆𝑊 (𝑚𝑚) 𝑥 105
The number of eggs in the whole soft body tissue ofindividual clams was determined using an indirect
enzyme-linked immunosorbent assay (ELISA) withmonoclonal antibodies specific to Manila clam yolk
protein following Park and Choi (2004). Frozen
whole tissue was freeze dried and homogenizedusing a sonicator in 10–15 ml buffer (20 mM TrisHCl, 150 mM NaCl, pH7.5) containing a proteaseinhibitor cocktail (#04080-11 for General use;
Nacalai Tesque Inc., Kyoto, Japan). The
homogenate supernatant was separated bycentrifugation (15,000×g for 20 min at 4°C) andstored at -80°C until analysis. The supernatants
were diluted 10-100 times with 0.5% BSA-PBS
buffer before ELISA. A 100 µl sample was added toa 96-well polystyrene microplate and incubated at
4°C overnight. After incubation, the plate waswashed twice with PBS containing 0.05% detergent(Tween20; Promega Co. Ltd., Madison, WI, USA);250 µl of 1% BSA and 5% skim milk in SBB wasadded as a blocking agent. After incubation at room
temperature for 1 hour and washing 5 times, themonoclonal antibody (Hamaguchi and Usuki, 2006)
was added to 100-µl aliquots in each well andincubated at room temperature for 2 hours. Afterwashing 5 times, a 100-µl aliquot of polyclonal goatanti-mouse immunoglobulins peroxidase conjugate(diluted 1: 4000) was added to each well. Following
incubation at room temperature for 2 hours, 100 µlof substrate- chromogen containing 3,3′,5,5′tetramethyl-benzidine (TMB+; Dako Co. Ltd.,

Analysis of the relationships between
Manila clam and habitat
To relate the condition index to habitat parameters,
and fecundity to habitat parameters, correspondence
analysis was performed using the computing
environment R (R Development Core Team, 2017).
Habitat parameters were summarized in twodimensional graphical form (1st and 2nd axis).
Correlation analysis was conducted between
condition index or fecundity and habitat scores ofthese two axes in each spawning season. As therepresentative fecundity in each of the two
spawning seasons, the egg number was summed andnormalized to 30 mm SL for three months. The
mean value of condition index for three months ineach spawning season was used as therepresentative nutritional condition of the clam.

59

RESULTS AND DISCUSSION

habitats. Moreover, the river mouth and intertidal
habitats had a negative score of the 2nd axis (belowpart) and the variation of environmental conditions,such as salinity and temperature, are considered tobe larger than those in subtidal and non-river
habitats. Therefore, it is considered that the 1st axis
is related to food availability and the 2nd axis isrelated to the magnitude of environmental stress.

Fecundity and condition index of the clam hadseasonal and spatial variations (Fig. 3). Conditionindex and fecundity were higher in spring andautumn, coinciding with the spawning season asreported by Matsumoto et al. (2014). However,there were large variations in each month. Thesevariations imply that there are spatial variations in
condition index and fecundity among the habitats,although the variation of fecundity amongindividuals was also large. Correspondence analysis
revealed that canonical correlations of the 1st and2nd axis were 0.48 and 0.31, respectively, and thecumulative contribution ratio up to the 2nd axis wasover 78%. The habitat could be summarized bythese two axes. By summarizing the set of habitat
data in a two- dimensional graphical plot, the 10
habitats were separated into 4 groups (Fig 4).
Habitats in

Figure 4. Two-dimensional graphical plot t of Manila clam,
Ruditapes philippinarum, habitats and habitat parameters in Ise Bay,
Japan by correspondence analysis. The ID 1 on plot represents the
Manila clam habitat in Kusu, Yokkaichi (2: Shiroko, Suzuka, 3:
Miwatari, Matsusaka, 4: Kushida, Matsusaka, 5: Higashikurobe,
Matsusaka, 6: Offshore-Muramatsu, Ise, 7: Intertidal-Muramatsu,
Ise, 8: Kawaguchi flat, Ise, 9: Isshikimae flat, Ise, 10: Kosugaya,
Tokoname).

Relationships between habitat scores of the 1st and2nd axis and condition index or fecundity in two
spawning seasons were different between thecondition index and fecundity and between
spawning seasons (Fig. 5). In both spring andautumn spawning seasons, the average conditionindex of the 10 habitats was positively correlatedwith the habitat scores of the 1st axis (r = +0.48 inspring and +0.79 in autumn) and the 2nd axis ( r =+0.80 in spring and +0.24 in autumn). On the other
hand, fecundity during the two spawning seasons
negatively correlated with the habitat score of the
2nd axis (r =-0.64 in spring and -0.80 in autumn).
There was only a limited relationship between
fecundity and habitat scores of the 2nd axis (r =Figure 3. Seasonal variations in condition index (a) and fecundity(b) of Manila clam, Ruditapes philippinarum, during 2015 in Ise
Bay, Japan.

non-river areas and open shore with strong wave
action had positive mean scores of the 1st axis
(right side) and food availability is considered to behigher than that in the river mouth and closed
60

+0.07 in spring and +0.11 in autumn). Different
relationships between condition index or fecundity
to habitat scores show that the nutritional condition
as determined by the condition index is not the solefactor affecting the fecundity of the clam, contrary
to the results in previous studies (Hasegawa et al.
2018).

Figure 6. Habitat types of Manila clam, Ruditapes philippinarum, on
two-dimensional graphical plot by correspondence analysis.

In conclusion, the nutritional condition as
determined by the condition index is not the solefactor affecting the fecundity of the clam. The
condition index was higher in the subtidal zones andopen non-river mouth areas, where food availability
was good. On the other hand, the fecundity washigh in the intertidal and enclosed river mouthareas, which are stressful and life-threatening to theclams. These findings on clam reproductive traits
may be useful in improving the efficiency of stockenhancement programs by maximizing the
reproductive potential of the clam populations. For
example, a habitat categorized as type A in Fig. 6
can be considered as a useful fishing ground inwhich the clam nutritional condition is good
therefore, survival rate and growth are also good but
the fecundity per body weight is not high. In habitat
type A, enforcement of catch size regulation may
improve the reproductive contribution throughincreasing the number of large clams. Habitat type
B has ideal conditions for the clam to thrive because
both nutritional condition and fecundity are high.
Creation of this type of habitat may be helpful forsustainable fishing of the clam. The clam in habitat
type C has a short life with high contribution to
reproduction because fecundity per unit body

Figure 5. Relationships between condition index or fecundity of
Manila clam, Ruditapes philippinarum, and the score of 1st or 2nd
axis of correspondence analysis.

Moreover, if the 1st and the 2nd axes are related tofood availability and magnitude of environmental
stress, respectively, condition index of the clam,which is positively correlated with the 1st axis, isconsidered to reflect the food availability. These
relationships may be intuitively acceptable.
Otherwise, fecundity, which is negatively correlatedwith the 2nd axis, is considered to reflect themagnitude of environmental stress. The fecundity
was higher in the intertidal zones and river mouthareas than in subtidal and non-river (offshore) areas.
This trend may be explained by clams exposed tostressful and hazardous habitats exerting moreenergy to reproduction than those under mild andreliable environmental conditions.

61weight is high. Improvement of food availability inthis type of habitat may increase the reproductive
contribution of the clam.

Bay, Japan. Bulletin of the Japanese Society ofFisheries Oceanography 81:110-123 (inJapanese with English abstracts).
Hasegawa, N., S. Sawaguchi , T. Unuma, T.
Onitsuka, and M. Hamaguchi. 2014. Variationin Manila clam (Ruditapes philippinarum)
fecundity in eastern Hokkaido, Japan. J.
Shellfish Res. 33: 739-746.
Kakino, J. 1996. Growth and survival of Japaneselittleneck clam Ruditapes philippinarum incage test related to the sharpness index.
Nippon Suisan Gakkaishi 62:376-383 (inJapanese with English abstracts).
Park, K.I. and K.S. Choi. 2004. Application of
enzyme-linked immunosorbent assay forstudying of reproduction in the Manila clam
Ruditapes philippinarum (Mollusca: Bivalvia)
I. Quantifying eggs. Aquaculture
241:667-687.
Park, K.I., A. Figueras, and K.S.Choi. 2006.
Application of enzyme-linked immunosorbent
assay (ELISA) for the study of reproduction in
the Manila clam Ruditapes philippinarum(Mollusca: Bivalvia): II. Impacts of Perkinsus
olseni on clam reproduction. Aquaculture
251:182-191. R Core Team. 2017. R: A
language and environment for statisticalcomputing. R Foundation for StatisticalComputing, Vienna, Austria. URL
https://www.R-project.org/.
Ruuskanen, A., S. Bäck, and T. Reitalu. 1999. A
comparison of two cartographic exposure
methods using Fucus vesiculosus as anindicator. Marine Biology 134: 139-145.
Toba, M. and Y. Miyama . 1994. Relationship ofsize to gonadal maturation and spawning in
artificially conditioned Manila clams. Nippon
Suisan Gakkaishi 60:173-178 (in Japanesewith English abstracts).

ACKNOWLEDGEMENTS
We are grateful to C. Hiro and K. Seko forassistance with analysis. Also, the members of
Kaiyo Engineering Co., Ltd, Diving Techno Co.,Ltd and Fisheries Cooperative Association around
Ise Bay for assistance with field research. This
study was supported by the Ministry of Agriculture,
Forest, and Fisheries and the Ministry of Education,
Culture, Sports, Science, and Technology of Japan.

REFERENCES
Matsukawa, Y., S. Chow, T. Katayama, and I.
Kamio. 2008. Factors responsible for the
drastic catch decline of the Manila clam
Ruditapes philippinarum in Japan. Nippon
Suisan Gakkaishi 74:137-143 (in Japanesewith English abstracts).
Matsumoto, T., M. Awaji, J. Higano, N. Hasegawa,T. Yamamoto, R. Shibata, Y. Hada, I. Sakurai,
D. Miyawaki, D. Hirai, K. Hokokawa, K.
Hanyu, N. Ikushima, J. Uchikawa, and S.
Chow. 2014. Reproductive cycle of theManila clam Ruditapes philippinarum inJapan. Nippon Suisan Gakkaishi. 80:548-560(in Japanese with English abstracts).
Hamaguchi, M. and U. Usuki. 2006. Effect ofenvironmental hormones to sex change ofRuditapes philippinarum, In: Editing
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Effects and mechanisms to fisheries species(Eds.) Environmental hormones – mechanisms
to fisheries species-. Kouseisyakouseikaku,
Tokyo, pp. 103-111 (in Japanese).
Hanyu, K., H. Kokubu, N. Hata, T. Mizuno, N.
Hasegawa, Y. Ishihi, S. Watanabe, Y. Fujioka,
J. Higano, T. Inoue, Y. Tanaka, T. Kudo, M.
Yamada, R. Nanbu, and H. Kuwahara. 2017.
Estimation of standing stock and factors
affecting the stock fluctuation of asari clam
Ruditapes philippinarum in four regions of Ise

ANNOTATED BIBLIOGRAPHY OF KEY
WORKS
Toba, M. and Y. Miyama. 1994. Relationship of size
to gonadal maturation and spawning in62

artificially conditioned Manila clams. Nippon
Suisan Gakkaishi, 60: 173-178.

Kakino, J. 1996. Growth and survival of Japaneselittleneck clam Ruditapes philippinarum incage test related to the sharpness index. Nippon
Suisan Gakkaishi, 62: 376-383.

In this study, the authors observed sexual
maturation and measured the number of eggs
released per spawned female subjected to thermal
spawning stimuli in breeding experiments forevaluating the size dependent batch fecundity ofasari clam. Smaller clams at 0 yr old (groups A andB) produced as many eggs per soft tissue weight aslarger clams at ≥ 1 yr (groups D and E). No
significant differences in egg diameter, percent
fertilization, and larval survival from straight-hinge
to pediveliger stage were observed among the clam
size groups.

The author evaluated the variation in asari clam
growth in a tidal flat in Tokyo Bay, Japan, by using
the “sharpness index” defined as width / length ofshell. The author concluded that juvenile clams
offshore grew faster than those that were shoreside,
but the juvenile survival rate in the intermediatezone of the flat was higher than those in theoffshore and shoreside areas. Juveniles having ahigh sharpness index tended to show a high survival
rate. At the same age, the juveniles with a highsharpness index grew faster than or equal to thosewith a low sharpness index

63

Ocean Acidification Experiment in Seagrass-Oyster Ecosystems Using a Novel
System to Conduct In-Situ CO2 Enrichment
Masakazu Hori1*, Tomohiro Kuwae2, and Masahiro Nakaoka3
1

National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and
Education Agency, Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan
2
Port and Airport Research Institute, National Institute of Maritime, Port and Aviation Technology, Nagase,
Yokosuka 239-0826, Japan3
Akkeshi marine station, Field Science center for Northern Biosphere, Hokkaido University, Aikkapu, Akkeshi,
Hokkaido 088-1113, Japan
Corresponding author: mhori@affrc.go.jp
Keywords: Oyster aquaculture, Zostera marina, Ocean acidification, Free ocean CO2 enrichment, Nonenvironmental risk deterrents

ABSTRACT

manipulated by FORTES. For instance, thedeterrent against the arthropod species prevents
settlement and recruitment of sessile crustaceanssuch as barnacles at low concentrations and also
removes shrimps and amphipods from the target
area at higher concentrations. Since the algal
species used as a deterrent is also taken as an oral
herbal medicine in Asian countries, the deterrent issafe for humans.

Coastal ecosystems are under the influence of
global environmental changes that threaten theirecological status and the sustainability of seafood
production. Seagrass beds, which are widelydistributed along coastlines around the world, arehighly important ecosystems, offering various typesof ecosystem services to human beings. Seagrassbeds are important for environmental change
mitigation and adaptation measures, via carbon
storage, acidification mitigation, water quality
improvement, as well as providing a nursery forcoastal marine organisms. Therefore, seagrass beds
are essential for harmonization of coastal fisheries
and aquaculture with environmental conservation,
resulting in the sustainable use of ecosystemservices. Here we developed a novel system of insitu manipulation of CO2 and nutrient enrichment todemonstrate the function of seagrass in mitigating
environmental changes. The system is called theFree-Ocean Real-Time Experimental System(FORTES). In addition, we developed nonenvironmental risk deterrents for invertebrates madefrom natural algae, specializing in arthropod andmolluscan species (PCT/JP2018/038204). Twotypes of deterrents were made: liquid and drypowdered chemicals. The liquid deterrent can be

Recently, there has been a focus on the interaction
between oyster aquaculture and seagrass vegetation
as a common practice to achieve better coastalmanagement with the framework of the sustainable
development goals (SDGs) in a changing
environment. Oysters have been grown using theoriginal oyster–seagrass interactions, including
facilitation of spat survival rate, and especiallytrophic support from eelgrass beds to oysters overan extended period of time. This is typicallyregarded as the indigenous and local knowledge(ILK) of Japanese oyster aquaculture. Using the
FORTES, we can demonstrate not only the direct
effects of environmental changes such as pCO2
increase/pH decrease, eutrophication andagrochemical inputs on oysters, but also themitigative effects of seagrass beds, which benefit
oyster aquaculture.
64

ANNOTATED BIBLIOGRAPHY OF KEY
WORKS

periods of time (weeks to months). They overcome
some of the drawbacks of laboratory experiments
and field observations by enabling (1) precise
control of CO2 enrichment by monitoring pH as anoffset of ambient pH, (2) consideration of indirect
effects such as those mediated through interspecificrelationships and food webs, and (3) relatively longexperiments with intact communities. Bringing
perturbation experiments from the laboratory to thefield is, however, extremely challenging. The main
goal of this paper is to provide guidelines on thegeneral design, engineering, and sensor options
required to conduct FOCE experiments. Anothergoal is to introduce xFOCE, a community-led
initiative to promote awareness, provide resources
for in situ perturbation experiments, and build auser community. Present and existing FOCE
systems are briefly described and examples of datacollected presented. Future developments are also
addressed as it is anticipated that the next
generation of FOCE systems will include, in
addition to pH, options for oxygen and/or
temperature control. FOCE systems should become
an important experimental approach for projecting
the future response of marine ecosystems toenvironmental change.

Campbell, J.E. and J.W. Fourqurean. 2013. Effects
of in situ CO2 enrichment on the structural andchemical characteristics of the seagrass
Thalassia testudinum. Mar. Biol. 160:
1465-1475.
Seagrasses commonly display carbon-limited
photosynthetic rates. Thus, increases in atmospheric
pCO2, and consequentially oceanic CO2(aq)concentrations, may prove beneficial. Whileaddressed in mesocosms, these hypotheses have notbeen tested in the field with manipulative
experimentation. This study examines the effects ofin situ CO2(aq) enrichment on the structural andchemical characteristics of the tropical seagrass,
Thalassia testudinum. CO2(aq) availability was
manipulated for six months in clear, open-top
chambers within a shallow seagrass meadow in the
Florida Keys (USA), reproducing the forecasted
CO2 environment for the year 2100. Structuralcharacteristics (leaf area, leaf growth, shoot mass,and shoot density) were unresponsive to CO2(aq)enrichment. However, leaf nitrogen and phosphorus
content declined on average by 11 and 21%,respectively. Belowground, non-structuralcarbohydrates increased by 29%. These results
indicate that increased CO2(aq) availability may
primarily alter the chemical composition ofseagrasses, influencing both the nutrient status andresilience of these systems.

Kline, D.I., L. Teneva, K. Schneider, T. Miard, A.
Chai, M. Marker, K. Headley, B. Opdyke, M.
Nash, M. Valetich, J.K. Caves, B.D. Russell,
S.D. Connell, B.J. Kirkwood, P. Brewer, E.
Peltzer, J. Silverman, K. Caldeira, R.B.
Dunbar, J.R Koseff, S.G. Monismith, B.G.
Mitchell, S. Dove, and O. Hoegh-Guldberg.
2012. A short-term in situ CO2 enrichment
experiment on Heron Island (GBR). Scientific
Reports 2, Article number: 413.

Gattuso, J.P., W. Kirkwood, J.P. Barry, E. Cox, F.
Gazeau, L. Hansson, I. Hendriks, D.I. Kline,
P. Mahacek, S. Martin, P. McElhany, E.T.
Peltzer, J. Reeve, D. Roberts, V. Saderne, K.
Tait, S. Widdicombe, and P.G. Brewer. 2014.
Free-ocean CO2 enrichment (FOCE) systems:present status and future developments.
Biogeosciences 11: 4057-4075.

Ocean acidification poses multiple challenges forcoral reefs on molecular to ecological scales, yet
previous experimental studies of the impact ofprojected CO2 concentrations have mostly been
done in aquarium systems with corals removed
from their natural ecosystem and placed underartificial light and seawater conditions. The Coral–
Proto Free Ocean Carbon Enrichment System (CP-Free-ocean CO2 enrichment (FOCE) systems aredesigned to assess the impact of ocean acidification
on biological communities in situ for extended

65

FOCE) uses a network of sensors to monitor
conditions within each flume and maintainexperimental pH as an offset from environmental
pH using feedback control on the injection of low
pH seawater. Carbonate chemistry conditions
maintained in the −0.06 and −0.22 pH offset
treatments were significantly different thanenvironmental conditions. The results from thisshort-term experiment suggest that the CP-FOCE is
an important new experimental system to study insitu impacts of ocean acidification on coral reef
ecosystems.

synthetic analogs as antifouling compounds:
2009–2014. Biofouling 31: 101-122.
Since early 2008, an increasing number of countries
have ratified an international treaty to ban theapplication of antifouling (AF) coatings based onorganotin compounds (e.g. tributyltin (TBT) andtriphenyltin). As a result, the demand forenvironmentally-friendly, non-toxic or low-toxic
AF compounds and technologies (green AF agents)
has become an urgent reality. Marine coatings basedon Cu2O and various other biocides have a negative
impact on the environment, and they must
eventually be replaced by new, effective, andenvironmentally-friendly AF compounds. Thismini-review describes important AF compounds
discovered from a variety of organisms from 2004
until mid-2009, and discusses recent and generaltrends in the discovery of AF compounds. Finally, aperspective on the future of AF compounddevelopment is presented. The discussion is aimed
at updating scientists and engineers on the current
challenges facing AF research.

Da Gama, B.A.P., R.C. Pereira, A.G.V. Carvalho, R.
Coutinho, and Y. Yoneshigue-Valentin. 2002.
The effects of seaweed secondary metabolites
on biofouling. Biofouling 18: 13- 20.
Antifouling activity is one poorly investigatedproperty of seaweed natural products. To determine
whether seaweeds contain chemicals able toinfluence the settlement of fouling organisms in thefield, crude organic extracts from Stypopodium
zonale, Dictyota menstrualis (Phaeophyceae) and
Laurencia obtusa (Rhodophyceae) wereincorporated at natural volumetric concentrations,
into hard stable gels that served as substrata forfouling in the experiments. Fouling organisms
settled at a significantly higher rate on plates treatedwith S. zonale extracts than on control gels, while
settlement was strongly inhibited on gels containing
K. obtusa extracts. Fouling on gels treated with theD. menstrualis extract was not significantly
different from the fouling found on control gels.
The findings suggest that the broad antifoulingproperties of the crude extract of L. obtusa inhibit
the settlement of fouling as well as hinder thedevelopment of settled fouling species, thereby
reducing the richness of species. The results imply
that L. obtusa possibly harbors powerful agents thatcan be explored for the development of antifoulingtechnology.

Stark, J.S., E.T. Peltzer, D.I. Kline, A.M. Queiros,
T.E. Cox, K. Headley, J. Barry, F. Gazeau,
J.W. Runcie, S. Widdicombe, M. Milnes, N.P.
Roden, J. Nlack, S. Whiteside, G. Johnstone,
J. Ingels, E. Shaw, L. Bodrossy, J.D.
Gaitan-Espitia, W. Kirkwood, and J. Gattuso.
2019. Free Ocean CO2 Enrichment (FOCE)
experiments: scientific and technicalrecommendations for future in situ ocean
acidification projects. Progress in
Oceanography 176: 89-107.
Free Ocean CO2 Enrichment (FOCE) experiments
are a relatively recent development in ocean
acidification research, designed to address the needfor in situ, long-term, community-level
experiments. FOCE studies have been conductedacross different marine benthic habitats and regions,from Antarctica to the tropics. Based on thisprevious research we have formed some core
operating principles that will aid those embarking
on future FOCE experiments. FOCE studies have

Qian, P., Y. Xu, Y. Li, and N. Fusetani. 2015. Minireview: marine natural products and their66

potential to provide important insight into theeffects of ocean acidification that can add to or
refine conclusions drawn from laboratory or single
species studies, because they are conducted in situ
on intact assemblages. Scaling up from suborganismal and individual effects to also include
indirect impacts on the ecosystem and ecosystemservices, make FOCE experiments essential tofilling in current knowledge gaps in theunderstanding of ocean acidification. While FOCE
systems are complex, relatively costly, andsomewhat difficult to operate, the challenges theypose are tractable and they have proven to be auseful approach in ocean acidification research. The
aim of this paper is to draw from the experiences ofpast FOCE experiments and provide practicaladvice for designing, building and operating a
FOCE experiment. Some of the most important
recommendations include: field testing the systemdesign; having a backup power supply; using
replicate treatment enclosures; monitoring andmaintaining the chemistry appropriately; allowing
sufficient time to achieve near CO2 equilibrium
conditions; and having a scientific focus with a core
set of hypotheses. Future FOCE experiments could
focus on longer durations, multiple factors, andtesting more intact benthic marine communities andecosystems. The authors hope this paper will enablefurther FOCE deployments and experiments, aswell as provide some guidelines to improve future
FOCE studies and advance ocean acidification
research.

67

Effects of Ocean Acidification on the Early Developmental Stages of theCommercially Important Gastropods, Ezo Abalone and Horned Turban, in JapanRyo Kimura1*, Hideki Takami2, Toshihiro Onitsuka3, Tsuneo Ono4, and Yukihiro Nojiri5
1Japan Fisheries Research and Education Agency (FRA), 15F Queen’s Tower B, 2-3-3 Minatomirai, Nishi-ku,
Yokohama, Kanagawa, 220-6115, JAPAN 2Tohoku National Fisheries Research Institute, FRA, Shiogama,985-0001 JAPAN3

Hokkaido National Fisheries Research Institute, FRA, Kushiro, 085-0802 JAPAN
National Research Institute of Far Seas Fisheries, FRA, Yokohama, 236-8648 JAPAN
5
Hirosaki University, Hirosaki, 036-8560 JAPAN
4

Corresponding author: rkim@affrc.go.jp
Keywords: Atlantic salmon, eastern oyster, breeding program, disease resistance

ABSTRACT

reduced developmental performance of thesegastropods in early life stages.

Commercially valuable shellfish species will beaffected more seriously by ocean acidification (OA)
in conjunction with overfishing. We investigated theeffects of OA on fertilization, larval development,
metamorphosis, and post-larval growth of valuable
gastropods; Ezo abalone Haliotis discus hannai andturban shell Turbo cornutus, using a CO2
manipulation system (AICAL pCO2 control system,
Kimoto Electric Co., Ltd, Japan). There was noeffect of exposure to <1000 µatm pCO2 seawaterobserved in fertilization, malformation, or mortality
rates in either gastropod. However, fertilization andhatching rates decreased with exposure to >1500µatm pCO2 seawater and the malformation rates at>1000 µatm increased significantly in Ezo abalone.
The diameter of larval shells in >800 µatm seawater
was significantly smaller compared to those incontrol seawater (400-450 µatm pCO2) for both
species. Scanning electron microscope images oflarval shells of Ezo abalone from >1000 µatm
showed abnormal development, suggesting
problems with shell deposition and/or increased
shell dissolution. The composition of developmental
stages of embryos in horned turban differed
between the experimental levels of pCO2 seawater.
These results indicate that pCO2 >1000 µatm

The natural habitat of these gastropods is kelp beds
where photosynthesis and respiration by macroalgae
lead to marked pCO2 diel fluctuations. Thesefluctuations are larger than the pCO2 change
projected to be caused by OA for open ocean waters
by 2100. We also conducted laboratory
investigations on the effects of ocean acidification,
in relation to diel cycles of seawater pCO2 levels
projected for the near and distant future, on theearly development of Haliotis discus hannai. Shell
length, abnormality and mortality of larval abalone
were examined under different pCO2 levels withmimicked diel cycle using the AICAL pCO2 control
system. Adult abalone, with fully matured gonads,
were induced to spawn and gametes wereartificially fertilized in concentrations of ca. 400(atmosphere-balanced, control treatment), 800 or
1200 pCO2 µatm. Sixteen hours post-fertilization,
hatched larvae were exposed to running seawater
adjusted to a constant level [ca. 400, 800 or 1200µatm pCO2] or fluctuating on a diel cycle [800±400(400-1200) or 1200±400 (800-1600) µatm pCO2]
pCO2 in 4-day experimental periods. There were no
significant differences in the mortality rate,abnormality rate and shell length of larvae amongthe 400, 800, and 400-1200 µatm pCO2 treatments.
68

METHODS

In contrast, the abnormality rate was significantly
higher in the 1200 and 800-1600 µatm pCO2
treatments than in the control treatment. Larval shelllength in the 800-1600 µatm pCO2 treatment wassignificantly smaller than in the other pCO2
treatments. The exposed duration time exceeding
the aragonite saturation state was a critical factor
determining the negative impacts on the larval
development of abalone.

INTRODUCTION
Anthropogenic elevation of the partial pressure of
CO2 (pCO2) in seawater has resulted in a loweringof seawater pH (= ocean acidification) and most
calcifying organisms investigated showed reduced
calcification in response to increased pCO2 (Fabry
et al., 2008). Kurihara (2008) reviewed ocean
acidification (OA) effects on the early
developmental stages of marine invertebrates
including bivalves (oysters and mussels); however,no reports on larval gastropods were included. Inorder to clarify impacts of OA on fishery-importantmarine gastropods, we investigated the OA effects
on the early development of Ezo abalone and thehorned turban by constant elevated pCO2 levels. We
also investigated the effects of diel fluctuating pCO2
levels on the survival and larval shell formation of
Ezo abalone.

Figure 1. Changes in partial pressure of CO2 in experimental
seawater during the diel cycle treatment experiment conducted 21- 24
December 2010 in the Shiogama laboratory. Lines represent thevalues monitored by the AICAL system every hour. Dotted lines
represent values in the constant treatment (1200, 800, 450(atmosphere balanced) μatm) and line represents those in the dielcycle treatment (400-1200, 800-1600 μatm), respectively.

We used the AICAL pCO2 control system, describedin Kimura et al. (2011), for making controlled
seawater of specific pCO2 levels. The systemshowed stable and good separation of pCO2 between
tested groups through the gas monitoring and water
sample (Kimura et al., 2011). We investigatedfertilization rate, hatching rate, malformed rate,
developmental stage retardation, and maximum
larval shell length. These methods for rearing andtesting were described in Kimura et al. (2011) for
Ezo abalone and in Onitsuka et al. (2014) for
horned turban. In the experiment testing
diel-fluctuated pCO2 level on Ezo abalone, we set
the pCO2 levels as follows: 450 (atmospherebalanced), 800, 800±400, 1200, 1200±400 µatm
(Fig. 1).

69

RESULTS
There was no effect of exposure to <1000 µatm
pCO2 seawater observed in fertilization,
malformation, or mortality rates in either species.
The fertilization rate and hatching rates decreased
with exposure to >1500 µatm pCO2 seawater in Ezo
abalone. The malformation rates at >1000 µatm
increased significantly compared to those in control
seawater in Ezo abalone. The maximum larval shelllength of larvae from >800 µatm seawater
treatments were significantly smaller compared tothose in control seawater in both species. ScanningElectron Microscope (SEM) images of larval shellsof Ezo abalone in >1000 µatm seawater showedabnormal development with shell deposition and/or
increased shell dissolution. The proportions ofembryos in horned turban that attained the 4-cellstage and 8-cell stage were significantly lower at thehigher pCO2 seawater treatments than those at thecontrol levels 2 h after fertilization. Similar trends
were observed in the ratio of veligers at 24-26 h
after fertilization. These results are described in
Kimura et al. (2011) for abalone, and Onitsuka et al.
(2014) for horned turban, respectively.

Figure 2. Relationship between larval malformation rates (upper),and shell length (lower) of Haliotis discus hannai 72 h after initiating
the cycle treatment experiment and the integral pCO2 over 1100
μatm. White- and black-colored symbols represent the results of thetreatments with a constant and cyclically fluctuating partial pressureof CO2, respectively. Each bar represents standard error.

The abnormality rate increased with the incrementof “Integral pCO2 over 1100 μatm”. Furthermore,
the larval shell length decreased as the index value
increased (Fig. 2). (The results of the dielfluctuation of pCO2 experiments were described inOnitsuka et al., 2018 and were included withanother experiment’s results).

In the diel fluctuation of pCO2 level experiment,
significantly higher malformation rates and smaller
maximum shell lengths were detected in the 1200
and 1200+-400 µatm pCO2 treatments than in the450 µatm pCO2 treatments. These negative impacts
were greater in the 1200+-400 μatm than in the1200 µatm. To test cumulative effects of elevated
pCO2, the index “Integral pCO2 over 1100 μatm”
was used because the aragonite saturation state ofseawater around the omega value of 1.0 was
equivalent to 1100 µatm pCO2. The index was
defined by the following formula:
Integral pCO2 over 1100 µatm =Σ(P-1100)i P: pCO2
over 1100 µatmi: exposed hours to pCO2 over 1100
µatm

DISCUSSION
In these experiments, our results show that pCO2
>1000 µatm reduced developmental performance of
these gastropods in early life stages (Kimura et al.
2011; Onitsuka et al. 2014). The mortality /abnormality rate, and larval shell length weresignificantly different between the 1200 and
1200±400 µatm pCO2 treatments, indicating pCO2
diel fluctuation can produce greater negative
impacts on the larval life of abalone. The exposed
duration time exceeding the aragonite saturation
state was a critical factor determining the negative
impacts on the larval development of abalone(Onitsuka et al. 2018: In the paper, the authors
introduced a new index “ECPC”, the effective70

cumulative value of seawater pCO2 over a specificcriterion value, which was calculated using thethreshold pCO2 level defined as the pCO2 value
corresponding to 1.1 of the aragonite saturation
state in seawater).

Nojiri. 2011. Effects of elevated pCO2 on theearly development of the commercially
important gastropod, Ezo abalone, Haliotis
discus hannai. Fish. Oceanogr. Vol 20: 357366.

Since mitigation measures against ocean
acidification are limited to reducing CO2 emissions,
small scale pCO2 monitoring in coastal areas isthought to be important to properly assess OA
effects on marine invertebrates.

This is one of the first papers that investigated theeffect of ocean acidification on commercially
important gastropod species. This is also a very
early paper using an AICAL system that precisely
controls the pCO2 concentration during theexperiment.

REFERENCES
Kurihara H. 2008. Effects of CO2-driven ocean
acidification on the early developmental
stages of invertebrates. Mar. Ecol. Prog. Ser.
Vol 373: 275-284.

Fabry V.J., B.A. Seibel, R.A. Feely, and J.C. Orr.
2008. Impacts of ocean acidification on
marine fauna and ecosystem process. ICES J.
Mar. Sci. Vol 65: 414-432.
Kimura R., H. Takami, T. Ono, T. Onitsuka, and Y.
Nojiri. 2011. Effects of elevated pCO2 on theearly development of the commercially
important gastropod, Ezo abalone, Haliotis
discus hannai. Fish. Oceanogr. Vol 20: 357366.
Kurihara H. 2008. Effects of CO2-driven ocean
acidification on the early developmental
stages of invertebrates. Mar. Ecol. Prog. Ser.
Vol 373: 275-284.
Onitsuka, T., R. Kimura, T. Ono, H. Takami, and Y.
Nojiri. 2014. Effects of ocean acidification on
the early developmental stages of the horned
turban, Turbo cornutus. Mar. Biol. Vol 161:
1127-1138.
Onitsuka, T., H. Takami, D. Muraoka, Y.
Matsumoto, A. Nakatsubo, R. Kimura, T.
Ono, and Y. Nojiri. 2018. Effects of ocean
acidification with pCO2 diurnal fluctuations
on survival and larval shell formation of Ezo
abalone, Haliotis discus hannai. Mar. Env.
Res. Vol 134: 28-36.

This review paper describes the effects of ocean
acidification on the early development of many
marine invertebrates including bivalves (oyster andmussel). Levels of carbon dioxide that affect thedevelopment of marine invertebrates werereviewed.
Onitsuka, T., H. Takami, D. Muraoka, Y.
Matsumoto, A. Nakatsubo, R. Kimura, T.
Ono, and Y. Nojiri. 2018. Effects of ocean
acidification with pCO2 diurnal fluctuations on
survival and larval shell formation of Ezo
abalone, Haliotis discus hannai. Mar. Env.
Res. Vol 134: 28-36.
This paper assessed the effects of constant anddiurnally fluctuating pCO2 (mimicking the rhythmof the ocean) on development and shell formation oflarval abalone using the AICAL system, which can
precisely control pCO2. It is suggested that theeffects of ocean acidification on development andshell formation of larval abalone can be determinedby intensity and time of exposure to pCO2 over thethreshold associated with aragonite saturation state(Ω-aragonite).

ANNOTATED BIBLIOGRAPHY OF KEY
WORKS
Kimura R., H. Takami, T. Ono, T. Onitsuka, and Y.

71

Chemical Changes in the Environment: What Does This Mean for Shellfish?
Shannon L. Meseck*, Mark Dixon, Yaqin Li, George Sennefelder, Dylan Redman, Renee Mercaldo-Allen, Paul
Clark, Lisa Milke, and Gary H. Wikfors
National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries
Science Center, Milford Laboratory, 212 Rogers Ave., Milford, CT 06460
Corresponding author: Shannon.Meseck@noaa.gov
Keywords: Bivalves, ocean acidification, coastal zones, aquaculture

ABSTRACT

heart, gills, gonads, and general metabolism andregulation may occur. Physiological changes could
result in slower growth, higher mortality, or
inability to grow. The influence of ocean
acidification on bivalve physiological functions isimportant to understand so that aquaculture may be
modified to respond to a changing environment.

Coastal areas, estuaries, and river deltas areexperiencing an increase in carbon dioxide resulting
from human activities. Approximately 30% of
atmospheric carbon dioxide (CO2) dissolves in theoceans. As this CO2 dissolves, it reacts with waterand produces carbonic acid (H2CO3), which
dissociates into bicarbonate (HCO3−), carbonate(CO32−), and hydrogen ions (H+). With increased
CO2 in the water, there are multiple ways thatshellfish may be affected: (1) food availability; (2)physiological responses, and (3) sedimentacidification.

Finally, the same reaction that occurs in water
during ocean acidification occurs at the sedimentwater interface, where bivalves settle, following
bacterial decomposition of settling organic matter.
As particles settle, aerobic and anaerobic reactions,combined with redox processes, result in the
production of CO2 in sediment porewater. Corrosive
sediment can impede successful settlement,
recruitment, and abundance in bivalve populations.
Nutrient-enriched coastal areas in the United States
are already experiencing sediment acidification, andthis might be limiting bivalve recruitment at the
sediment-water interface. The synthesis presented
here will focus on the myriad of ways that ocean
acidification may affect marine bivalves.

Food availability may be influenced by changes inphytoplankton community structure and nutritional
value. For example, increased carbon dioxide may
cause a shift in phytoplankton biomass andcommunity composition depending upon species’
efficiency of inorganic carbon acquisition. Shifts in
phytoplankton communities may favor
phytoplankton with more- or less- nutritional
content for support of human food production.

INTRODUCTION

In addition to potential changes in food supply,
coastal acidification may be causing physiologicalchanges in marine bivalves that affect how they
feed. Bivalve gills are constantly exposed toambient water, with the gill cilia moving water
through the shell for respiration and feeding. Under
environmental stress, physiological changes to theIncreased atmospheric carbon dioxide (CO2) isbeing absorbed by the world’s oceans and is thought
to be a concern for some groups of marineorganisms, especially those that contain structures
composed of calcite or aragonite (Andersson et al.,2008; Dickinson et al., 2013; Gazeau et al., 2013).

72

In the ocean, CO2 reacts with water to form
carbonic acid (H2CO3), which dissociates into
bicarbonate (HCO3-), carbonate (CO32−), andhydrogen ions (H+), resulting in acidification.
Currently average ocean pH is 8.1 and by 2100
ocean pH is predicted to drop to 7.7. (Caldeira andWickett, 2003; Feely et al., 2004; Raven et al.,2005; Turley and Findlay, 2016).

nitrogen (N), and phosphorus (P), are optimized(Elser et al., 2003; Elser et al., 2000).
Ocean acidification effects on phytoplankton may
include changes in cellular carbon quotas(Burkhardt et al., 2001; Burkhardt et al., 1999),
changes in phytoplankton species composition(Riebesell et al., 2013), and nutritional quality toother trophic levels (Rossoll et al., 2012). To better
understand what happens to marine phytoplankton
following increased carbon dioxide, we conducted aseries of seven experiments with temperate
phytoplankton species, grown under pre-glacial
pCO2 conditions to predicted 2100 levels of ρCO2.
Table 1 summarizes the growth rates, C:P, N:P, andPUFA, MUFA, and SFA analysis from the sevenspecies studied (King et al., 2015). This study foundno significant difference in nutritional composition
for any of these species, however some species
experienced a change in growth rate. This researchhighlighted that although there were CO2 drivenchanges in growth rate, it did not reflect changes inelemental composition or fatty acid composition.
This research highlights that the complexing ofocean acidification on marine phytoplankton, willclearly depend on how the phytoplankton
community changes. Understanding what drives
community succession will help determine howchanges in carbon dioxide might influence
nutritional quality in the future.

Environmental variations (e.g., tidal, daily, andseason changes) can influence metabolism, growth,and survival of marine shellfish (Norkko et al.,2006). Most bivalves can be classified as euryhalineorganisms (Flügel, 2013) and live in areas which
experience changes in environmental parametersthat are natural (e.g., temperature) and/or a result ofanthropogenic pollution (e.g., excess nutrients).
Marine bivalves tolerate a range of environmental
conditions; however anthropogenic pollution may
affect bivalve energy balance such that a change inphysiological processes (growth, reproduction)
could result. Increasing our understanding of howorganisms physiologically respond to specificchanges in the environment will help in assessing
how a community functions (Menge et al., 2002;
Niemi et al., 2004).
In this study, we synthesize data from our ocean
acidification experiments with phytoplankton
growth and nutrition to examine physiologicaleffects on bivalves exposed to ocean acidification,and discuss how sediment acidification may
influence settlement.

Bivalve Physiology
Future ocean acidification conditions could result in
decreased fertilization success, changes in growthrate, reduced calcification rates, and increased
mortality (Melzner et al., 2009). Our current
understanding of how shellfish physiology isaffected by ocean acidification is incomplete, butthere is a growing amount of research suggestingthat bivalves may be highly sensitive to ocean
acidification (Dickinson et al., 2013; McCorkle etal., 2015; Sugano et al., 2015; Widman Jr et al.,2012). Understanding what physiological traits are
responsible for sensitivity to ocean acidification
may provide useful information for optimizing
aquaculture practices with regard to bivalves.

Phytoplankton
The correct nutritional components ofphytoplankton are a key component regulating
physiological performance of marine bivalves. The
assimilation of key nutrients from phytoplankton,
especially polyunsaturated fatty acids (PUFA), is
required for growth and can only be acquired
through trophic transfer (Ezgeta- Balić et al., 2012;
Milke et al., 2004). Fatty acids are critical for
growth, survival, neural development, stressresistance, membrane fluidity, and enzyme activity
with enhanced growth and reproductive rates whenfatty acids, especially PUFA and carbon (C),Preliminary research suggests that under ocean
73acidification, growth rates and survival of surfclams
and sea scallops may decrease (McCorkle et al.,2015; Sugano et al., 2015; Widman Jr et al., 2012).
Furthermore, shell deformities increased as carbon
dioxide levels increased. Change in filtration rates is
one possible mechanism for reduced growth.
Preliminary short term experiments (two weeks) in
the laboratory tested how ocean acidification affects
the filtration rate of blue mussels, Mytilus edulis,
and the eastern oyster, Crassostrea virginica, undercurrent conditions and increased carbon dioxide.
Feeding rates changed with increased carbon
dioxide in both blue mussels and eastern oysters.
Measurements of cilia beat function for blue
mussels found that as carbon dioxide levels
increased cilia beats decreased, suggesting thatchanges in feeding rates might be related to changes
in cilia beat function (Meseck at al. 2020).

Sediment acidification
Acceptance or rejection of habitat by bivalves as
they settle from the water column to the benthos is
not well understood, but research suggests thatenvironmental, chemical, and physical variables atthe sediment-water interface promote settlement
and abundance of bivalves.
Recently, sediment porewater carbonate chemistry
has been linked to larval settlement and survival inlife stages of many marine bivalves (Clements andHunt, 2014, 2017; Clements et al., 2016; Green etal., 2004; Green et al., 2013). Sediment acidification
can occur at the sediment surface, as particulatematerial undergoes aerobic and anaerobic reactions,combined with redox processes, which produces
CO2. Once CO2 is produced, it reacts with water toform carbonic acid (H2CO3), and dissociates into
bicarbonate ions (HCO3-), carbonate ions (CO32-),
and hydrogen ions (H+) resulting in sedimentacidification (Green and Aller, 2001; Tang andKristensen, 2007; Wenzhöfer and Glud, 2004).
These corrosive sediments (aragonite, Ω < 1) resultin low pH conditions in the upper millimeters,
where bivalves settle, and may reduce settlement,
recruitment, and abundance in bivalve populations(Aller, 1982; Green and Aller, 2001; Zwarts andWanink, 1989).

Dynamic energy budget (DEB) models may be auseful tool to understand how changes inphysiological rates would impact marine bivalves
under ocean acidification. By understanding themetabolic costs of ocean acidification, we canmodel how biovolume, development, andreproduction might change. Further research on
how bivalve physiology is affected under ocean
acidification is necessary to understand how
aquaculture practices may be adapted to thechanging environment.

Over the last three decades, bottom water in Long
Island Sound (LIS) has been documented to have
low pH (acidified sediments) and to beundersaturated with respect to carbonate minerals

74

Table 1. Relative difference in specific growth rate, carbon:
phosphorus ratio (C:P), nitrogen: phosphate ratio (N:P), PUFA,MUFA, and SFA for each of the seven phytoplankton species using a
one way ANOVA P<0.05. The = sign means no change between
present day levels and Year 2100 pCO2 levels. Adapted from data
presented in King et al. (2015).

Species

C:P

N:P PUFA MUFA SFA

Thalassiosira =pseudonana

=

=

=

=

=

Thalassiosira Increase
rotulaDecrease =

=

=

=

Thalassiosira =weissflogii(CCMP2599)

=

=

=

=

=

Thalassiosira =weissflogii(CCMP1010)

=

=

=

=

=

Thalassiosira Decrease =oceanica

=

=

=

=

Chlorella
autotrophica

Increase

=

=

=

=

=

Dunaliellasalina

=

=

=

=

=

=

Growthratechemistry still was. The contribution of carbonate
chemistry parameters in predicting bivalve
settlement explained 44% of the total bivalve
benthic community, but the contributions ofcarbonate chemistry to settlement of individual
species varied. For the two dominant species,
carbonate chemistry explained 33% of M. arenaria
and 71% of Nucula spp. abundance (Table 3). Thesefindings were consistent with previous research in
LIS which reported increased mortality of bivalves(N. annulata and Tellina agilis) in sediments thatwere undersaturated (Ω <1) with respect tocarbonate (Green et al., 1993, 1998). Our study did
not focus on determining whether mortality or
dissolution was occurring because of undersaturated
sediments, however mean Ω values in LIS were
undersaturated during most of the sampling season.
Results from the Meseck et al (2018) study suggestthat increased pH and alkalinity may enhancebivalve community recruitment. These findings
were similar to those reported by Clements andHunt (2016) suggesting that carbonate chemistry
may influence bivalve settlement.

DISCUSSION(Aller, 1982; Green and Aller, 2001; Green et al.,1993). In Connecticut, the seafloor is leased toshellfish growers and they rely on natural reseedingof northern quahog Mercenaria mercenaria to
occur. Understanding the influence that sedimentacidification may have on natural settlement,
recruitment, and abundance of commercial bivalvesis essential for sustainable harvesting of shellfish.
During 2013, we studied the relationships between
abundance of bivalve molluscs and physical (grainsize) and chemical variables (carbonate parameters)
at the sediment-water interface on a leased shellfish
bed during the bivalve settlement period when Ω in
LIS tends to be low (Meseck et al., 2018).

Environmental conditions of ocean acidification
may influence bivalve survival, reproduction,
growth, and overall health in both direct andindirect ways. Ocean acidification may influencemultiple modes for marine bivalves. This summary
illustrates how ocean acidification may influence
phytoplankton production, shellfish physiology, andseafloor chemistry, and the importance ofunderstanding these interactions for sustainablemarine aquaculture.

REFERENCES
Aller, R.C. 1982. Carbonate dissolution innearshore terrigenous muds: the Role ofphysical and biological reworking. The
Journal of Geology 90, 79-95.
Andersson, A.J., F.T. Mackenzie, and N.R. Bates.
2008. Life on the margin: implications ofocean acidification on Mg-calcite, highBriefly, Meseck et al. (2018) found that throughout
the season, grain size in combination with carbonate
chemistry was influencing bivalve settlement. Whenthe settlement season was narrowed down to two
weeks to remove temporal effects, grain size was nolonger a significant contributor, but carbonate
75

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concentrations. Limnology and Oceanography
46, 1378- 1391.
Burkhardt, S., U. Riebesell, and I. Zondervan. 1999.
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fractionation in marine phytoplankton.
Geochimica et Cosmochimica Acta 63,
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Caldeira, K. and M.E. Wickett. 2003.
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Clements, J.C. and H.L. Hunt. 2014. Influence of
sediment acidification and water flow on
sediment acceptance and dispersal of juvenile
soft-shell clams (Mya arenaria L.). Journal of
Experimental Marine Biology and Ecology
453, 62-69.
Clements, J.C., and H.L. Hunt. 2017. Effects of CO2
driven sediment acidification on infaunalmarine bivalves: a Synthesis. Marine Pollution
Bulletin 117, 6-16.
Clements, J.C., K.D. Woodard, and H.L. Hunt.
2016. Porewater acidification alters theburrowing behavior and post-settlement
dispersal of juvenile soft-shell clams (Myaarenaria). Journal of Experimental Marine
Biology and Ecology 477, 103-111.
Dickinson, G.H., O.B. Matoo, R.T. Tourek, I.M.
Sokolova, and E. Beniash. 2013.
Environmental salinity modulates the effects
of elevated CO2 levels on juvenile hard-shellclams, Mercenaria mercenaria. The Journal of
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geochemistry and grain size.

ANNOTATED BIBLIOGRAPHY OF KEY
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Bermúdez, R., M. Winder, A. Stuhr, A. Almén, J.
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The authors conducted a field study assessing therelationship between juvenile soft-shell clamabundance and spatial, temporal, and environmental
variables in the Bay of Fundy. Sediment pH, watertemperature, location, and date was monitored in2012 during the settlement season for Mya arenaria
at four different study sites along the shore. Using
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sediment pH, and minimum air temperature best
predicted bivalve recruitment. By removing
temporal effects, there was a significant positive
relationship between sediment pH and grain size.
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might influence bivalve settlement.

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dioxide with respect to community composition andfatty acid composition (nutritional statues). Theirresults found little change in plankton community
composition from the natural condition (~347 µatm)
to predicted levels for the year 2100 (~1333 µatm).
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78

The results showed little sensitivity of common
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79

Current Trends in pH and Ocean Acidification in Aquaculture Sea Areas in
Gokasho Bay, Japan
Masayuki Minakawa1*, Satoshi Watanabe2, Yuka Ishihi2, Natsuki Hasegawa2, and Toshie Matsumoto2
1

National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 224-1 Hiruda,
Tamaki, Mie, 519-0423, Japan
2
National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 422-1
Nakatsuhamaura, Minamiise, Mie, 516-0193, Japan* Present address: Fisheries Stock Assessment Center, Fisheries Resources Institute, Japan Fisheries Researchand Education Agency, 2-12-4 Fukuura, Kanazawa, Yokohama, Kanagawa 236-8648, Japan.
Corresponding author: minmas@affrc.go.jp
Keywords: pH, AOU, ocean acidification, carbonate saturation state

ABSTRACTcalcareous organisms may be brought about by thischange.

Monthly variation in pH and carbonate saturation
state (Ω) of aragonite and calcite were investigatedin Gokasho Bay, Mie, Japan, from November 2016
to October 2019. During the winter season, pH wasalmost constant (8.02 - 8.13) in the whole bay area,
which was approximately the same level as thosereported in surface waters off the south coast ofJapan. However, pH varied greatly during summer
seasons (7.75 - 8.60). The lower value was thought
to be caused by the remineralization of organic
matter in the seabed, and the higher value was likely
derived from high phytoplankton photosynthetic
productivity. Excluding variation of pH caused byapparent oxygen utilization (AOU), acidification in
the bay water was calculated to be 8.13 pH atpresent. The pH values currently measured are
generally lower than those recorded in the 1980s,
suggesting the advance of ocean acidification (OA)
in the bay water with a reduction rate of 0.08
pH/decade. The calculated average values of Ω (>3) were relatively high in the whole bay area,
indicating calcium carbonate is supersaturated in thecurrent environment of the bay. If OA advances insurface water around Japan in the near future, thedegree of Ω in the bay water would drop with the
reduction in pH, and some negative impacts on

INTRODUCTION
Coastal environments are influenced by various
human-induced stressors. Recently, the global
reduction of pH in surface seawater, which isreferred to as ocean acidification (OA), has arisen
as a consequence of oceanic uptake ofanthropogenic CO2. It is thought that OA affects thebiogeochemical dynamics of calcium carbonate in
the oceans (Doney et al., 2009; Kawano, 2010;
Garrard et al., 2013). OA is suggested to damage
not only marine phytoplankton production but
fisheries and seafood industries (Fujii, 2018).
Shellfish aquaculture occurs in many parts ofcoastal and bay areas in Japan and there is apossibility that shellfish aquaculture will also bedamaged by OA in the near future since the shells
are composed of calcium carbonate whose
deposition is controlled by carbonate species andthe pH of seawater (Poach et al., 2019). While there
are many research studies on pH in open ocean andcoastal areas (Fujii et al., 2001; Ishii et al., 2011:
Zhai et al., 2014; Lipuma, 2016; Kubota et al.,2017), the effects of OA are not well understood in
sea aquaculture areas due to large variations in pH
80associated with many biogeochemical processes.
To obtain basic information for water quality of thecoastal sea in Japan, monthly hydrographicalobservations were conducted in a sea aquaculturearea in Gokasho Bay, Mie, Japan from November
2016 to October 2019. This paper provides recent,high-quality pH values and carbonate saturation
states (Ω) of aragonite and calcite in the bay water.
Spatial and temporal variations in pH values will be
discussed in relation to ocean acidification in
Japanese aquaculture sea areas.

tuna (Thunnus orientalis) in the main bay, andoysters (Crassostrea gigas and C. nippona) andgreen alga (Monostroma nitidum) in areas close to
the shore are operating in the bay.

METHODS
Study area
Gokasho Bay is located on the Pacific coast ofJapan, and consists of 3 coves (Gokasho, Funakoshi
and Hazamaura) and the main bay which opens to
the western North Pacific (Fig. 1). The Bay has arelatively small area of 22.2 km2 and a water
volume of 0.11 km3. Mean depth of the Bay is 12.7m, and maximum depth is 30 m at the central part ofthe bay (Toda et al., 1990). Hydrographical featuresof the bay are reported in detail by Toda et al.
(1990) and Abo (2000).

Figure 2. Sampling stations in Gokasho Bay. Black dots show thesampling stations from November 2016 to October 2018 (this study),
and Red dots from June 1986 to June 1989 (Sugiyama et al., 1991).
Major aquaculture area in the bay is also shown.

Sampling and analysis
Monthly hydrographic surveys in Gokasho Bay
were conducted from November 2016 to October
2018 from the research vessel Hamabou. Seawater
samples were collected in the bay at three depth
layers using a 1.3-L Kitahara’s water sampler at 19
stations from November 2016 to June 2017 and 20
stations from July 2017 to October 2018 (Fig. 2).
Samples were collected in 25-ml co-stoppered glass
test tubes for measurement of pH to avoid thecontamination of CO2 from the atmosphere and in
250 ml polyethylene bottles for measurements of
total alkalinity. A CTD system (RINKO profiler,
JFE Advantech) was also used to obtainhydrographic data (Watanabe et al., 2019).
Dissolved oxygen (DO) concentration obtained
from the CTD system was calibrated in thelaboratory with seawater analysis data from a DO
meter (MM-60R, TOA-DKK) with dissolved
oxygen electrodes (OE-470AA, TOA- DKK).

Figure 1. Map of Gokasho Bay.

Aquaculture is flourishing in Gokasho bay. Pearlaquaculture used to be the main industry in this area
but has decreased significantly (Yokoyama et al.,1996). Now, the aquaculture of red seabream(Pagrus major) in Hazamaura-Cove, Pacific bluefin

The pH of seawater was measured more precisely
by potentiometric method using a pH meter81

(MM-60R, DKK-TOA Co.) with a glass electrode(ELP-0320, DKK-TOA Co.). The glass electrodewas standardized against the JIS buffer scale (4.008,
6.865 and 9.180 pH at 25℃) which adapted the
NBS buffer scale. Any drift in pH was correctedagainst the seawater buffer scale (Ono et al., 1998;of pH between NBS and total scales, the systematic
error between these scales was ignored.
To calculate carbonate systems, total alkalinity wasmeasured from May 2016 to October 2018 using a

Figure 3. Vertical profile of pH at each station on August 2017 andFebruary 2018 in Gokasho Bay, Mie, Japan.

one-point titration method (Culberson et al., 1970;
Tsunogai and Noriki, 1983; Ono et al., 1998)
modified by using a small sample method (Suga etal., 2013). An aliquot (15 ml) of seawater sample
was used to measure total alkalinity. In thisresearch, systematic error of total alkalinity values
was not corrected. The precision of total alkalinity
was ±0.006 meq/L.

Dickson et al, 2007) and the precision of pH was ±0.003. For measurement of pH, different pH scales
were used. It is reported that the pH of JIS (NBS)
buffer scale are about 0.15 pH higher than that of
the total scale (Gattuso and Hansson, 2011). In thisstudy, because the large variation of pH value
observed was much higher than the systematic error82

Calcium carbonate saturation state (Ω)
Calcium carbonate saturation state of seawater (Ω)
is one of the indexes for health of the ocean
calcareous organisms. Ω was calculated using theequation as follows;・ ・ ・ (1)・where [Ca2+] is the calcium concentration calculated
from salinity (Millero, 2013), [CO32-] is the
carbonate concentration determined from observed
pH and TA (Dickson at al., 2007), and Kspcal,arago is
the solubility product for calcite and aragonite,
calculated from salinity, temperature and waterpressure (Mucci, 1983; Millero, 1983), respectively.

Figure 4. Monthly trend of pH at all data from November 2016 to
October 2018 in Gokasho Bay, Mie, Japan. The average of pH value
is also shown.

Monthly trends of pH are shown in Fig. 4 from
November 2016 to October 2018. pH values with alarge variation ranging from 7.7 to 8.6 were foundfrom spring to summer seasons, whereas thevariation was relatively small, centered around 8.1,
from autumn to winter. During phytoplankton
blooms in surface water, particularly high pH ofaround 8.6 was observed in August 2017. Low pH
values were seen in the suboxic bottom water
during these periods. Seasonal average pH was
calculated to be 8.11 ± 0.08 (1σ, n = 1384). Thislevel was similar to those reported for Japan’s
coastal waters and the western North Pacific surface
water (Kawano, 2010; Ishii et al., 2011; Japan
Meteorological Agency).

RESULTS AND DISCUSSION
An example of vertical profile of pH was shown ateach station in August 2017 and February 2018
(Fig. 3). High pH of more than 8.3 was observed forsurface water in August 2017 while pH decreased
toward the sea bottom to less than 8.0.
Vertical variation of pH tended to be greater atstations closer to the shore. However, pH in winter(February 2018) stayed constant around 8.1 because
the bay water was homogenized due to wintervertical and horizontal mixing.

Figure 5. Relationship between AOU and pH from November 2016 to
October 2018, except the data on January 2018, in Gokasho Bay,Mie, Japan. The regression line is also shown.

83

Large seasonal variations of pH and dissolved
oxygen levels come from biogeochemical andphysical processes in the bay water. The
relationship between pH and apparent oxygen
consumption (AOU) at all stations are shown in Fig.
5. In general, pH is affected by photosynthesis anddecomposition of organic matter. Whenphotosynthesis is active, CO2 is taken up from theseawater to synthesize organic matter and to releaseoxygen. As a result, AOU decreases, and pH
increases in the water column. On the contrary,decomposition of organic matter consumes oxygen
and releases CO2 to seawater. As a result, AOU
rises, and pH decreases. The observed correlation
between AOU and pH suggests that the large
seasonal variation of pH was at least partiallycaused by photosynthesis in the surface layer anddecomposition of organic matter near the bottom.

Figure 6 Comparison with pH values between the trend of pH from
June 1986 to November 1989 (Sugiyama et al., 1991) and November
2016 to October 2018 (this work) in Gokasho Bay, Mie, Japan. Since
the pH data from 1986 to 1989 was measured to be up to 2 digits
after decimal point, the overall uncertainty of the pH could be
regarded to be 0.01 pH units. Each average value of pH is also
shown.

Compared with the seasonal variation of pH values
at present, the seasonal variation reported from1986 to 1989 was larger, ranging from 7.0 to 8.8(Fig. 6). If the ratio of pH/AOU was constant in thebay water, the seasonal variation of pH could havealso been larger than that of AOU at present. Thissuggests that the production and decomposition oforganic matter were more active in the 1980s thannow. The average value of pH from 1986 to 1989
was calculated to be 8.35±0.14, which was
0.24±0.16 higher than the average value of 8.11 ±0.08 from 2016 to 2018. Assuming the pH
decreased by 0.24 in the 30 years from 1988 to
2018, the pH reduction rate is calculated to be-0.08±0.005 pH/decade. This reduction value isgreater than -0.02 ± 0.007 pH/decade of the south
west coast Japan (Ishii et al., 2011) or 0.0082 ±0.0057 pH/decade of the shelf break of the East
China Sea (Lui et al., 2015). This suggests that OA
may be progressing more rapidly in Gokasho Bay,
though a longer-term data set is required to be more
conclusive.

Marine phytoplankton have a stoichiometriccomposition of carbon to oxygen (Anderson 1995).
The slope of the regression line between pH andAOU was calculated to be -0.05 ± 0.001 pH/(mg/L)(or -0.0016 ± 0.00003 pH/(µmol/L)) (1σ, n = 1288).
The pH of surface water is dominated byphotosynthesis, decomposition of organic matterand uptake of atmospheric CO2. When AOU is incondition of 0 mg/L in sea water, the pH isapparently affected by only uptake of atmosphericCO2. Therefore, the vertical intercept of theregression line can be defined as the acidification
degree in the bay water under the conditions ofabsence of biochemical processes and AOU being 0
mg/L. In Fig. 5, the pH of acidification degree wascalculated to be 8.13 ± 0.00. These results showedthat the pH in the bay water changed with the AOU
at the ratio of - 0.05 ± 0.001 pH/(mg/L) around pHof 8.13.
A similar study was conducted in the bay from June
1986 to May 1989 by Sugiyama et al. (1991).

The monthly trends of omega calcite (Ωcal) andaragonite (Ωarago) were calculated for all data fromMay 2017 to October 2018 (Fig. 7). In summer,
high Ω value was observed in surface water and Ω
decreased with depth. The vertical variation of Ω
tended to be greater at the stations closer to theshore.

84

acidification may be advancing slowly in the bay. If
the reduction rate of pH remains at 0.08±0.005
pH/decade, Ω will be less than 1 in 50 years. In thenear future, the degree of Ω in the bay water would
drop with reduction of pH and could bring negative
impacts to the calcareous organisms of the bay.

ACKNOWLEDGEMENTS
We thank Captain Shigeya Yamamoto of Research
vessel Hamabou, and Dr. Masahiko Awaji for theirsupport with water sampling. We also thank Dr.
Tsuneo Ono for valuable discussion on the early
preparations of the manuscript.

Figure 7. Monthly trends of calcium carbonate saturation states ofseawater for calcite (Ωcal) and aragonite (Ωarago) from May 2017
to October 2018, except the data on October 2017, in Gokasaho Bay,Mie, Japan. Each average value of calcium carbonate saturations is
also shown.

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ANNOTATED BIBLIOGRAPHY OF KEY
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The authors determined that a significant trend inocean acidification is superposed on the large
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Japan, based on their observation of the carbonate
system (partial pressure of CO2, total inorganiccarbon and pH). Multiple regression analysis ofthese parameters showed that pH and the aragonite
saturation state (Ωarago) decrease at a rate of -0.020 ±0.007 pH/decade, and -0.12 ± 0.05 Ωarago/decade,respectively. If future atmospheric CO2 levels keepincreasing as predicted by IPCC scenario A1FI,
which postulates tentative fossil fuel use associatedwith very rapid economic growth, a further
reduction of -0.8 pH and -0.1 Ωarago is likely in thenext 50 years. The authors suggest that such a rapid
reduction of Ωarago could have negative impacts on avariety of calcareous organisms.

Measurement of pH and total alkalinity is the mostconvenient way to describe the carbon system in aseawater sample. In this paper, the authors
introduced a modified method for determination of
total alkalinity focused on reduced volume seawater
samples (1 mL) based on the one-point titration
method. The precision of this method is 0.1 - 0.2%
(relative standard deviation). On the other hand,with no correction, the precision was 0.1 - 1.0%.
Even though values showed a systematic error,
precision was maintained through the correction
based on concurrent measurement of commerciallyavailable standards. This method allows us todescribe the carbonate system in a small amount ofwater with sufficient precision and accuracy toinvestigate the mechanism of calcification using
only a pH meter.

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the western North Pacific Subtropical Gyre at two
separate locations, Chichijima Island (Ogasawara
Archipelago) and Kikaijima Island. A comparison

In coastal regions, the biological production is far
greater than that of the open ocean and theshort-term change of hydrographical conditions is
significant. The authors conducted continuous
measurements of salinity, pH, and dissolved oxygen

87

(DO) at three stations in Osaka Bay. Also, theycalculated the values of carbonate species in the bay
water by using the values of pH and total alkalinity.
The results showed that dissolved inorganic carbon
and DO fluctuated synchronously with highcorrelation (R2=0.97), and the records of DO andpartial pressure of CO2 (pCO2) indicated theprominent diurnal variations which correspond to
diurnal irradiation variations due to highproductivity in the bay.

88

Ecological Interactions Among Wildlife and Shellfish Farms: A Case Study fromNew Jersey, USA, on Horseshoe Crabs and Oyster Farms
Daphne Munroe
Haskin Shellfish Research Laboratory, Rutgers University, 6959 Miller Ave., Port Norris, NJ, USA 08349
Corresponding author: dmunroe@hsrl.rutgers.edu
Keywords: Oyster aquaculture, Crassostrea virginica, horseshoe crab, Limulus polyphemus, wildlife
interactions

ABSTRACTinteractions among horseshoe crabs and intertidal
oyster farms, and recently concern has been raised
about the horseshoe crab’s ability to traverse oyster
farms to reach spawning habitat. A case study
examining potential farm interactions withhorseshoe crabs was done in Delaware Bay, New
Jersey, during the 2018 crab spawning season. The
investigations included a range of controlled
experiments and surveys during high and low tideusing sonar and walking respectively, to observe
crab behavior at farm and non-farm sites. In allcases, results indicate that crabs can successfully
traverse farms and reach spawning beaches, andcrabs do not differentially use farm versus non-farmareas of the Delaware Bay mudflats. These results
provide important context for developing
frameworks for managing ecological interactions
among farms and wildlife species of concern.

Globally, shellfish aquaculture is growing rapidly.
For the first time in history, global seafood supply
from aquaculture exceeded that of wild fisheries.
Although shellfish culture is viewed as an
ecologically sound industry, as farm production
expands it faces key challenges in assuringecological and social sustainability. Understanding
the ways shellfish farms interact with coastal
wildlife, particularly species of special concern, isamong those challenges.
Farms for Eastern oyster (Crassostrea virginica) arecommonly located in shallow coastal areas along
the U.S. east coast, use a range of farm equipment,
and involve regular access to care for and harvest
livestock. In some cases, these farms are located inareas used by American horseshoe crabs (Limulus
polyphemus) as they come ashore during spring tospawn along Atlantic Coast beaches. The sandy
shores of the Delaware Bay, bordered by the statesof New Jersey to the north and Delaware to thesouth, host the largest spawning aggregations of thespecies in the world. Horseshoe crabs are
economically and ecologically important; theirblood is highly valuable in the medical industry andmigratory shorebirds including the red knot
(Calidris canutus rufa), a threatened migratory
shorebird, feed on crab eggs when stopping in
Delaware Bay.

INTRODUCTION
For the first time in history, global seafood supply
from aquaculture exceeded that of wild fisheries(FAO, 2018). As aquaculture continues to develop
globally (FAO, 2018), as well as domestically in the
United States (National Marine Fisheries Service,2018), the industry faces key challenges assuringecological and social sustainability (Billing 2018).
Understanding the ways shellfish farms interact
with coastal wildlife, particularly species of special
concern, is among those challenges (Barrett et al.,2018; Collier et al., 2018). The ways in whichmolluscan aquaculture interacts with fundamental

Limited studies have been done to examine the89ecosystem processes such as particle depletion,
nutrient cycling, and benthic-pelagic coupling has
been relatively well studied (Newell 2004,
Dumbauld et al. 2009, Rose et al. 2015). Likewise,
attractiveness of farms in marine habitats, such as
fish net pens and shellfish farm structures, to mobile
fish and crustaceans is well documented (Callier etal, 2017). However, central to sustainability isappreciating the nature of the interaction among
farms and wildlife that may use habitat near to or
occupied by farms (Price et al., 2017; Barrett et al.,2018), interactions that are as of yet poorly studied.

the spring northward bird migration (Castro &Myers, 1993). The eggs become available to thetransitory bird flocks as they are exhumed fromnests by sediment disturbance such as crab
burrowing and wave action (Kraeuter & Fegley,
1994; Smith, 2007) and become concentrated in theupper intertidal zone as the tide rises (Nordstrom etal. 2006).
A small portion of the total Delaware Bay shoreline
is collectively used by crabs for spawning. This is a
nexus of shorebird migratory stopover habitat, andis home to oyster farming. The overlap in both timeand space of these two iconic and ecologicallyimportant species, and oyster farm activities,
presents a unique opportunity to examine potential
ecological interaction among wild species/stocks

Shellfish aquaculture in the Delaware Bay, Cape
Shore region of New Jersey has a long history as a
low impact, sustainable food production system(Hilborn et al., 2018; van der Schatte Olivier et al.
2018). Oyster farms currently occupy
approximately 10 acres in this area of New Jersey
and produce over 1.8 million market-sized oysters
annually (Calvo, 2016); however, during the firsthalf of the 20th century, oyster farming was much
more expansive, using large wooden intertidal racks
to cultivate oysters over wide stretches of thisregion (Ford and Haskin 1982). The vast intertidal
mudflats of the lower bay, with rich oyster foodresources, make this region a desirable location to
grow high quality oysters; it is also a region used byimportant wildlife species.
Horseshoe crabs (Limulus polyphemus) are an
economically, medically (Novitski, 2009) andecologically important species. Known to be thelargest spawning aggregations in the world (Shuster
and Botton, 1985), hundreds of thousands comeashore during the spring to mate and lay eggs along
sandy beaches of the Delaware Bay (Smith et al.,2002). The rufa subspecies of the red knot (Calidris
canutus rufa), a migratory shorebird that uses
stopover areas along the Atlantic coast of the U.S.,
including the Delaware Bay, was designated‘threatened’ in 2015 under the Endangered Species
Act in the U.S. by the U.S. Fish and Wildlife
Service (50 C.F.R. § 17 2014). An important foodsource for red knots are lipid-rich horseshoe crab
eggs, deposited on beaches by mating crabs during

Figure 1: Photo of a typical oyster rack with bag on top at a Cape
Shore farm. Horseshoe crabs can be seen among the racks and in theflooding tide waters. Crab spawning habitat (sandy beach slope) canbe seen in the background.

90and shellfish farms. Little data has been collected
specifically addressing the ability of horseshoe
crabs to traverse intertidal rack-and-bag oyster
farms to reach their spawning habitat (Figure 1). If
crab spawning migrations are impeded by farms, it
is possible that crab populations themselves may
suffer, and in turn shorebird foraging patterns andopportunities may be altered. In this case study, ourprimary goal is to characterize the ways thathorseshoe crabs interact with farm structures while
passing through farms en route to inshore spawning
habitat, and determine if the farms themselvespresent a barrier to crabs reaching beaches to lay
eggs. Ultimately, this information is important for
both wildlife species conservation and sustainable
farm management.

each experimental trial. Before being placed into thetank, each crab was measured (widest distanceacross the prosoma) and sexed, then assigned an
identification number that was written on both sidesof the shell in yellow waterproof crayon (this markwas removable and was wiped off before returningthese crabs to the flats after the experiment). The
size, sex, and id number of each crab was recorded
and marked animals were placed into the tank andallowed to acclimate for 15 minutes before theexperimental treatment was placed in the tank withthem. During this acclimation period, many of thefemales in the tank were attached to by males,
forming amplexus pairs.
A total of eleven treatments were tested. Treatments
included a control, in which the footprint of anoyster rack was drawn on the tank bottom but nophysical structure was put in the tank, and 10 farm
gear treatments of varying heights andconfigurations. The suite of treatment types
included three rack heights (3”, 5” and 8” above thebottom) with and without oyster bags attached, anoyster bag on the bottom of the tank (no rack), a
floating oyster bag tethered to the bottom with ¼”
braided sinking line, oyster bags leaning on the sideof a rack and a rack on its side (no bag). All racks
were rebar racks that are used by farmers, hadelastic cords with metal hooks attached (farmers use
these to hold bags onto the racks), and bags
contained oyster shells to mimic adult oysters. The
designated treatment was placed in the tank with 20
crabs and a timer started. The crabs were observed
continuously for 15 minutes as they moved about
the tank and interacted with the treatment structure.
A note was recorded for every crab each time that
individual crab passed to the side, beneath, or over a
structure. This 15 minute observation period wasconsidered one replicate for the given treatment.
Each treatment was replicated a minimum of nine,and maximum of 14 times, for a total of 139 trials.

METHODSAll experiments and surveys were carried out
during the horseshoe crab spawning season (Smith
and Michels 2006; Shuster and Botton 1985), from
May through July of 2018, at the Rutgers University
Cape Shore Laboratory and nearby oyster farms
located along the lower Delaware Bay. First, an
experiment was conducted in which crabs wereplaced in a tank, along with oyster farm equipment(rack-and-bag) to observe the ways that crabs are,or are not, able to move past this equipment.
Second, surveys were conducted on the flats during
low and high tide conditions to assess crab
presence/absence relative to farm footprints, and toevaluate whether crabs use intertidal habitatsdifferently when farm gear is present.
For the observation of crab movement around racks,
a large fiberglass tank (3.7 m long x 1.5 m wide x
0.51 m deep) was filled with filtered baywater to adepth of 30 cm. Mature horseshoe crabs werecollected by hand from the adjacent Cape Shore
mudflats. Experiments began May 9, 2018, andcontinued through June 21, 2018, during the
spawning period for horseshoe crabs; thus, allanimals used in the experiment were actively
coming ashore to spawn and assumed mature.
Twenty crabs, collected at random, were used for

Low tide surveys were conducted using paired
transects, laid out and marked on four intertidal
farms in the Cape Shore region. At each farm,paired transects (1m wide) were oriented
91

perpendicular to the shore with one transect
intersecting a farm, and a parallel control transect
passing through adjacent un- farmed intertidal
habitat following methods described in Munroe etal. (2017). During daytime low tides, starting on
May 8, 2018, and continuing through May 25, 2018,transects on all four farms were walked and all
crabs encountered along the transect were
documented, and their location (inshore of or withinfarm) was noted. Walks were repeated a minimumof weekly, for a total of nine repeat surveys during
the 2018 spawning season. A Kruskal-Wallis test
was used to test if differences were observed in thenumber of crabs observed inshore, and within farm
gear among paired farm:control transects.

In zero of the 128 trials involving oyster racks
and/or bags (i.e. all non-control treatments) was acrab observed to be stuck or impeded from moving
past or through the oyster gear.

Waters in the Delaware Bay are sufficiently turbid,that even in shallow water and over short distances,visual observation of crabs on the bottom duringhigh tide is not possible. Therefore, specialized
sonar (DIDSON sonar) mounted to the front of asmall aluminum boat was used to observe crabs on
the bottom during high tide. Sonar video wasrecorded as the boat motored slowly along paralleltransects set in oyster farm and non-farm habitats,laid out as described for the low tide surveys. In
addition to the transect videos, sonar video wastaken at two paired mooring locations for durationsof 10-15 minutes. One mooring point allowed
observation within a farm; the other in a comparable
control location with no farm gear. A total of nine
paired mooring videos were taken.

Figure 2: Crab counts along transects made during low tide. Upper
panel shows crabs observed within the outer intertidal region, among
the farm gear. Lower panel shows crabs observed inshore of the farm
gear. Note that the length of transects varies among farms, anddiffers between outer and inshore habitats. Line colors depict each of
the four farms, and dashed lines are counts along the transect
bisecting the farm, solid lines show counts made along the control
transect.

Numbers of crabs observed during the low tidesurveys varied through the season, and among farms(Figure 2). Counts ranged from a low of zero at the
beginning and end of the observation period, to ahigh of 135 per transect during the observation
made on May 21st, 2018; this period fell between
the New and Full moon in late May. In total, over
all transects and across the entire observation
period, 1,176 crabs were observed on the four
farms, zero of which were impinged in farm gear.
No difference exists among the number of crabs
counted at habitat inshore of farm gear compared tocontrols (Kruskall-Wallace test, p=0.73), nor athabitat within the farm footprint compared tocontrols (Kruskall-Wallace test, p=0.45). The
numbers of crabs counted during low tide differedsignificantly among farm sites (Kruskall- Wallace

RESULTS
In total, 330 male and 129 female crabs were usedin the experimental trials, ranging in size from 16 to
28 cm prosoma width for males (mean=20.1 cm),and from 21 to 30.5 cm for females (mean = 25.8cm). Across all eleven rack treatments tested, allcategories of crabs (single male, single female andamplexus pairs) were observed moving around andunder/over/through the farm gear treatment.
Interestingly, this includes single female crabs andcrabs engaged in amplexus successfully passing
both under and over the two shortest rack heights.
92

test, p<<0.001 inshore habitat, p=0.004 within farm
habitat).

amplexus pairs were observed at the farm mooring(Wilcoxon signed rank, p<0.05).

DISCUSSION
Experiments and surveys conducted during thehorseshoe crab spawning season in 2018 tested theability of horseshoe crabs to move among andthrough oyster farm gear and evaluated the waysthat crabs use habitat around oyster farms in the
Delaware Bay region. Across all of the surveys andexperiments, our results indicate that crabs can
successfully traverse farms and reach spawning
beaches, and that crabs do not avoid farm gear whenaccessing spawning beaches. These results provide
important context for developing frameworks for
ecological interactions among farms and wildlife
species of concern, specifically with respect toconcerns about the potential for changes inabundance of horseshoe crabs or their behavior due
to the presence of oyster farm gear.

Figure 3: Single and amplexus crab counts made using sonar fromall paired mooring events. Gray points represent the mean ± standard
deviation. Colored points show data per observation event, with each
event colored the same.

Sonar video surveys of crabs during high tide
showed large aggregations of crabs in sloughs(muddy depressions), and sparsely distributed crabs
moving independent of other crabs in seemingly
random directions outside of sloughs. On manyoccasions, crabs were observed to move under andout from farm gear unimpeded. The number ofcrabs observed during high tide along transects
varied depending on the day of observation, thefarm, and whether counts were inshore of the farm
gear versus in the area of the farm gear. For theregion inshore of the farm gear, no significant
difference was detected among control and farmcounts for single crabs (p=0.54) nor amplexus pairs(p=0.53). In the intertidal area within the footprintof the farm gear (outer intertidal), no significant
difference was detected among control and farmcounts made for single or amplexus crabs, (all pvalues>0.13). Counts of crabs made during mooring
events, standardized by effort to crabs/minute,
ranged from zero to nearly two crabs per minute(Figure 3). No significant difference was detectedamong paired control and farm counts for single
crabs (paired T-test, p=0.37), nor amplexus pairs(Wilcoxon signed rank, p=0.33). When counts werecorrected for view obstruction by farm gear, nosignificant difference among single crab counts at
control vs. farm was detected (Wilcoxon signed
rank, p=0.16); however, significantly more

Mature crabs ranging in size and including single
male and female crabs, as well as amplexus pairs,
were observed to move beneath, over and aroundoyster racks ranging in height from three to eight
inches off the bottom when underwater. Likewise,sonar data collected during high tide at farm andcontrol locations on the Cape Shore intertidal flats
documented crabs (both single crabs and amplexus
pairs) moving unobstructed among farm gear in
daylight and moonlight conditions. Crabs were able
to access habitat inshore of farms at the same rate as
those inshore of comparable control sites,suggesting that the presence of farms does not
impede crabs from accessing upper intertidal
spawning sites; consequently, the reproductive
ability of the crabs is not impacted by oyster farms.
When sonar counts of crabs at farm sites werecorrected for obstruction of the view of the bottom,
there in fact appears to be more crabs moving
within farms at high tide compared to control sites
without gear. Attractiveness of farm structures inmarine habitats, such as fish net pens and shellfish
gear, to mobile fish is well documented (Callier et
al. 2017). It is possible that horseshoe crabs also93

find oyster farm gear attractive due to increased
foraging opportunities, shelter or other cues; future
research may address this possibility.
Concepts of conservation aquaculture highlight theimportant contributions shellfish farms and otherforms of aquaculture can make in achieving local
and global conservation goals (Froehlich et al2017). When evaluating aquaculture impacts andinteractions with wildlife, it is important to (1)establish appropriate reference sites at which tocompare wildlife abundance to farms, and (2)collect data on potential differences in behavior andreproductive capacity for wildlife at farm sites(Barrett et al, 2018). In the studies documented inthis report, appropriate controls were used in aneffort to best illuminate changes, if any exist, inabundance of horseshoe crabs or their behavior due
to the presence of oyster farm gear, with theintention to serve the needs of an adaptive
management process aimed at balancing wildlife
species conservation and sustainable farmmanagement. Adaptive approaches that allow for
inclusion of new data are critical to coastalmanagement, and particularly so when considering
appropriate ecosystem-based decisions that ensure
protection of wildlife and viability of important
coastal industries.

recommendations are those of the author and do notnecessarily reflect the views of the NJSGC or the
U.S. Department of Commerce. NJSG-19-964.
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ACKNOWLEDGEMENTS
I am indebted to the fearless field crew that helped
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for the continued cooperation of the oyster farmers
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by Cape May County. This white paper is the resultof research sponsored by the New Jersey Sea Grant
Consortium (NJSGC) with funds from the National
Oceanic and Atmospheric Administration (NOAA)
Office of Sea Grant, U.S. Department ofCommerce, under NOAA grant Award #NA10OAR4170085 and the NJSGC. The
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contribution to the population biology ofhorseshoe crabs, Limulus polyphemus (L.), in
Delaware Bay. Estuaries. 3: 363-372.

Smith, D. R. 2007. Effect of horseshoe crab
spawning density on nest disturbance andexhumation of eggs: a simulation study.
Estuaries and Coasts. 30(2): 287-295.
Smith, D. R., P. S. Pooler, R. E. Loveland, M. L.
Botton, S. F. Michels, R. G. Weber, and D. B.
Carter. 2002. Horseshoe Crab (Limulns
polyphemus) Reproductive Activity on
Delaware Bay Beaches: Interactions with
Beach Characteristics. Journal of Coastal
Research. 18(4): 730-740.
Smith, D. R. and S.F. Michels. 2006. Seeing theelephant: Importance of spatial and temporal
coverage in a large- scale volunteer-based
program to monitor horseshoe crabs.
Fisheries. 31(10): 485-491.
van der Schatte, Olivier A., L. Jones, L. L. Vay, M.
Christie, J. Wilson, and S. K. Malham. 2018.
A global review of the ecosystem services
provided by bivalve aquaculture. Rev
Aquacult. 1-23 doi: 10.1111/raq.12301
National Marine Fisheries Service (2018) Fisheriesof the United States, 2017. U.S. Department ofCommerce, NOAA Current Fishery Statistics
No. 2017 Available at:https://www.fisheries.noaa.gov/resource/docu
ment/fisheries-united-states-2017-report
ANNOTATED BIBLIOGRAPHY OF KEY
WORKSIn this annotated bibliography, I have selected
recent works that focus on interactions among
wildlife - in particular horseshoe crabs or
kabutogani - and shellfish farms. Limited research
has focused on this issue; therefore, I have included
two experimental studies, and two broader reviews.
Horseshoe crabs are an important and iconic species
along the eastern United States, as well as in Japan.
Barrett, Luke T., Stephen E. Swearer, and Tim
Dempster. 2018. Impacts of marine andfreshwater aquaculture on wildlife: a global
meta-analysis. Reviews in Aquaculture. 1-23.
doi: 10.1111/raq.12277

95

In this review, authors from the University of
Melbourne in Australia demonstrate evidence that
supports higher wildlife biomass and diversity
found around aquaculture farms. They argue that toproperly understand the possible impacts of farms
on wildlife, studies have thus far failed to address
issues of impacts on behavior, reproduction or
fitness. If animals that tend to aggregate aroundfarms suffer poor fitness relative to those occupying
non-farm habitats, the farm becomes an ‘ecologicaltrap’.

Cheung. 2018. Habitat use of globallythreatened juvenile Chinese horseshoe crab,
Tachypleus tridentatus under the influence of
simulated intertidal oyster culture structures inHong Kong. Aquatic Conservation: Marine
and Freshwater Ecosystems. 28: 124-132.
The authors conducted this study to test if juvenile
Chinese horseshoe crabs differ in abundance and if
their feeding trails differ at oyster farm spatcollection locations. They performed their test at ashallow mudflat in Hong Kong which is home to alarge population of juvenile horseshoe crabs.
Addition of oyster spat collectors (bricks on the
mudflat) lead to a significant reduction in thenumber of juvenile horseshoe crabs, and a reductionin the length of feeding trails.

Callier, M.D., C.J. Byron, D.A. Bengtson, P.J.
Cranford, S.F. Cross, U. Focken, H.M. Jansen,
P. Kamermans, A. Kiessling, T. Landry, and F.
O'beirn. 2017. Attraction and repulsion ofmobile wild organisms to finfish and shellfish
aquaculture: a review. Reviews in
Aquaculture.

Munroe, D., D. Bushek, P. Woodruff, and L. Calvo.
2017. Intertidal rack-and-bag oyster farms
have limited interaction with Horseshoe Crab
activity in New Jersey, USA. Aquaculture
Environment Interactions. 9: 205-211.
https://doi.org/10.3354/aei00227

The ways in which finfish and shellfish farms act as
attractant or repulsive areas for mobile marine
species is reviewed. At finfish farms, food inputs
and physical structure of the farm tend to drive
aggregating effects. Likewise, at shellfish farms, thefarm structure (both farm gear and shellfish
themselves) and increased feeding opportunities via
biodeposition, the shellfish crop or foulingorganisms tend to attract wildlife to farms. In
general, the authors note that a great deal ofvariability in attractiveness or repulsiveness exists
when considering the suites of habitats, farms, andmobile fauna that have been studied thus far.

In this study, the authors sought to assess whether
intertidal oyster farms altered the distribution of
mature horseshoe crabs as they came ashore to
spawn in the lower Delaware Bay. In total, theyused a combination of field surveys of crabs on
mudflats and stranded along the upper shore, andcontrolled tests to determine if crabs can move pastfarm gear. Results show no evidence that horseshoe
crab distribution within this area is altered by thepresence of oyster farms.

Kwan, Billy K.Y., Chan Hoi Kin, and Siu Gin

96

Use of Point-of-View Video Cameras to Assess Fish Interactions with Oyster
Aquaculture Cages
Gillian Phillips1, 2*, Renee Mercaldo-Allen1, Peter J. Auster3, Paul Clark1, Erick Estela1, Yuan Liu1, 2, Lisa
Milke1, Dylan Redman1, and Julie Rose1
1

NOAA Fisheries, Northeast Fisheries Science Center, Milford Laboratory, 212 Rogers Avenue, Milford, CT
06460 USA
2
Integrated Statistics, 16 Sumner Street, Woods Hole, Massachusetts 02543 USA
3Department of Marine Sciences & Mystic Aquarium, University of Connecticut, 1080 Shennecossett Road,
Groton, CT 06340 USA
Corresponding author: gillian.phillips@noaa.gov
Keywords: Aquaculture, Shellfish, Habitat, Ecosystem Services, eDNA

ABSTRACTwith documentation of behavioral interactions
between animals and these habitats. Analysis of
eDNA detected significant differences in fish
community composition across the three months
sampled, and significant differences between sites
in two of the three months sampled.

Oyster bottom cages are an increasingly popular
style of aquaculture gear in the Northeastern UnitedStates. Cages are used to contain and protect
shellfish during grow-out but also likely provide
three- dimensional structure that may benefit the
local wild fish community. Understanding theecosystem services provided by aquaculture gear
may help to inform regulatory decisions and informa broader public discussion of shellfish farming andhow farms interact with the local environment.

*The Federal Government does not endorse the useof GoPro™ cameras

INTRODUCTION
Bottom cages are an increasingly common type ofaquaculture gear for cultivating oysters in the
Northeastern United States (Archer et al, 2014;
Flimlin et al, 2010). Cages are used to contain andprotect shellfish during grow-out but also provide
three-dimensional structure that may attract the
local wild fish community and provide habitat
services (Dumbauld et. al, 2009). A trapping andmark- recapture study in Narragansett Bay, Rhode
Island, found the seafloor with oyster grow-out
cages provided valuable habitat for juvenile andadult temperate reef fish (Tallman and Forrester,2011). Habitat offered by oyster aquaculture gear
may provide food resources, shelter, refuge fromcurrents, and protection from predation forcommercially and recreationally important species

A low-cost camera mounting system was developed
and tested to allow visual assessment of fish activity
near an oyster cage farm and in a surrounding low
seafloor structure environment. Minimal-structure tplatform stands were created to mount cameras
adjacent to boulders, enabling comparison of fishinteractions with cages to fish activity observed onnatural structured rock reef habitat. Seawater
samples were collected monthly during pilot camera
deployments for environmental DNA (eDNA)
analysis to detect fish species that may be present
but not observed in video.
Video collected by cameras attached to oyster cages
and near boulders facilitated visualization andquantification of the wild fish communities along97

of fish. Documenting and quantifying the ecosystemservices provided by aquaculture gear may help
inform regulatory decisions and inform a broader
public discussion about shellfish farming and how
farms interact with the local environment.

compare how the habitat services provided byoyster cages compare with those provided bynatural structured habitats, and to pilot the use of
eDNA metabarcoding to characterize finfish
communities associated with our study habitats overtime and space.

Several structure-oriented species common to Long
Island Sound are known to support important
recreational and commercial fisheries in the
Northeast. Demersal species including Black Sea
Bass and Scup are federally managed under the“Summer Flounder, Scup, and Black Sea BassManagement Plan” for New England stocks(Atlantic States Marine Fisheries Commission,2018). Tautog is a temperate reef fish also highly
valued by fishermen, with 90% of landings
contributed from recreational harvest (Atlantic
States Marine Fisheries Commission, 2015). An
understanding of habitat preferences and use,
particularly among species of commercial andrecreational interest, may aid in regional
management decisions related to aquaculture
leasing.

METHODS
We studied a style of commercial oyster bottom
cage commonly used in the Northeast (Figure 1).
Cages measure 4 x 3 x 2 ft (1.2 x 0.9 x 0.6 m) andconsisted of three shelves, with two bags of oysters
per shelf. We stocked each bag with 150 seedoysters (~1 in; 2.5 cm) based on information fromindustry partners. Bricks were added to the base of
the cage for ballast. Cages were deployed from
May-September, 2017, at two nearshore sites off
Milford, Connecticut; one adjacent to a high-density
cage farm, and one in a low-structure environment.
Water depths at the study sites ranged from 15-20
feet at high tide.

Small self-contained point-of-view video cameras
can enable fine spatial-scale studies to quantify theinteractions between fish and oyster aquaculture
gear. Here we report on a pilot study using thesecameras and other commercially-available
accessories to create an optimized and transferable
system for the deployment of underwater video
cameras across multiple sampling periods andgeographic study locations. The information gained
from this pilot program is presented in this report.
The ultimate goal of our research program is toutilize this system to gain information on species
composition and abundance of fish interacting withoyster cages, to quantify and document details of
how fish species interact with oyster cages, toFigure 1. Oyster cage used in this study. Full view showing three
shelves, each with two bags of oysters. Feet of cages show added
bricks for ballast.

98

Figure 2. On bottom cage with camera views. (A) Close-up of cage
fitted with cameras, showing side view in the front of the photo andtop camera at the rear. (B) Close-up of top camera looking across thehorizontal surface of the cage. (C) The resulting view of the top
camera. (D) View of the side camera looking at two sides of the cage
and where the cage meets the sea floor. (E) Resulting view of the sidecamera.

positioned in a periscope like mount, which wasable to capture the top horizontal surface of thecage, including viewing the top of the oyster bags
proximal to the camera. (Figure 2).

We experimented with several methods for
attaching point-of-view (GoPro®) cameras to oyster
cages. Initially a spring attachment was placed at acorner of the cage to provide flexibility to protect
cameras during deployment and retrieval, but
unfortunately resulted in too much camera motion
during deployment and retrieval of cages. Camera
mounts were instead constructed with flexible
marine hose and ¼ inch PVC, which provided
stability during video recording but allowed someflexibility for camera protection. We found a
combination of two time-synced cameras to provide
adequate visual coverage of most of the cage andimmediate surroundings. The first camera was
affixed to a cage corner by ~2 feet (0.6 m) of 7/8’
flex marine wet exhaust and water hose, allowing
the camera to hang off the side of the cage, enablinga view of two cage sides as well as the area where
the cage feet met the seafloor. We initiallyexperimented with a second camera positioned tolook down at the top of the cage, but the poor water
clarity prevented viewing the entire top of the cage.
The final configuration had the second camera

Figure 3. T-platform construction to mimic camera views on natural
structured habitat. (A) Full view of t-platform with cameras attached.
(B) Image of t-platform deployed on a natural rock reef adjacent toboulder. (C) Resulting top view of the boulder. (D) Resulting sideview of the boulder which also captures where the boulder and sea
floor meet.

To compare fish interactions with cages tointeractions with natural structured rock reef
habitat, “t-platform” stands were devised to mountcameras adjacent to boulders while adding minimal
additional structure (Figure 3). T-platforms were99

built using threaded metal pipe and were designed
to provide a field of view similar to that on the cage,
capturing the top of the boulder and one side where
the boulder meets the seafloor. The platforms areintended to be deployed at the beginning of the fieldseason and left in place, using divers to attach andremove cameras. This should minimize thedisturbance associated with camera
deployment/retrieval, and ensure consistent fields ofview across multiple deployments.

conducted on PCR amplicons, followed bybioinformatics analysis.
Pilot-scale deployments over the May-September
2017 field season yielded 20+ hours of video on
weeks with the cage density comparisons. During
weeks where t-platforms were deployed, 18+ hoursof video was recorded on a continuous recording
scheme. Video was downloaded immediately
following trips and duplicate copies stored in two
separate locations for data management. Each file
was renamed with a unique identification to ensure
proper organization. Observer XT software (v14.0;
Noldus Information Technology, Wageningen,
Netherlands) was used to analyze videos.
Advantages of this software platform included
multi-video playback, inter-coder analysis andflexible coding scheme, making it an ideal tool forour analysis.

Intervalometer Blink® timers (CamDo, Vancouver,
Canada) were used with GoPro cameras to delay thestart of video recording by 24-hours, to minimize
any deployment-related disturbance prior torecording. Video was recorded for eight minutes
every hour from 7AM to 7PM to cover a complete
tidal cycle and most daylight hours. A Polar Pro®
magenta filter, with a 0.5 stop reduction in
exposure, was affixed to each camera lens to reducegreen coloration of video footage caused by thenatural attenuation of light in water, as well as
phytoplankton growth in Long Island Sound. The
addition of any filter reduces total light capture by
the cameras, which limited the depth of our
deployments, but the importance of this deployment
restriction was outweighed by the large reduction intime required for video post-processing. One
TCM-1 tilt current meter (Lowell Instruments LLC,
North Falmouth, MA, USA) was deployed at eachsite to measure current speed and direction during
trials. HOBO pendant temperature and light meters(Onset Computer Corporation, Bourne, MA, USA)
were also deployed to document light penetrationand seawater temperature.

Video analysis is currently underway. Fishabundance will be reported using the MaxN metric(Watson et al. 2005). The likelihood of doublecounting the same fish within each interval of video
recording was increased by the typical behavior offish associated with a structured environment. The
MaxN metric avoids double-counting by limiting
abundance to the maximum number of fishobserved at any one time, and is thus likely aconservative estimate of total fish abundanceassociated with a structure. For our study, MaxN
was defined as the maximum number of individuals
of a given species present in a single frame withineach 1-minute segment of video (Watson et al.
2005). MaxN was calculated separately for eachspecies observed. A behavior matrix underdevelopment will quantify and describe specificbehaviors observed in video records. Precise
definitions allow distinct behaviors to be identified
and reduce the subjectivity in behavior analysis andidentification among coders. For example, thecurrent working definition for “station keeping
above the cage” is “small fin movements tomaintain a position both in a period of foraging andwhile in the water column or above cage.”

Environmental DNA (eDNA) analysis was used todetect fish species that may have been present but
not observed on video. Following camera retrieval,seawater was collected near the seafloor using a
Niskin Bottle during one deployment each in themonths of June, July, and August 2017. Samples
were kept on ice until filtration (0.45 µm) and thenfrozen (-20 °C) until analysis. DNA was extracted
and PCR was performed on the 12S mitochondrial
rDNA region. Next generation sequencing was then100

RESULTS AND DISCUSSION(73.4%) among samples while the axis associatedwith habitat type represented minor variation(15.6%) among samples.

Video analysis is ongoing, so only initialobservations are reported here. The four mostfrequently observed fish species thus far were:
cunner (Tautogolabrus adspersus), black sea bass
(Centropristis striata), tautog (Tautoga onitis) andscup (Stenotomus chrysops), which are allcommonly found in and around structurally
complex habitat in Long Island Sound. These four
species have been observed both on boulders and on
oyster cages. Other species of fish captured onvideo include: butterfish (Peprilus triacanthus),
banded rudderfish (Seriola zonata), hake
(Urophycis chuss), sea robin (Prionotus carolinus),
striped bass (Morone saxatilis), summer flounder(Paralichthys dentatus), windowpane flounder(Scophthalmus aquosus), smallmouth flounder(Etropus microstomus) and yellow jack(Carangoides bartholomaei). Animals observed
inside cages during camera deployment or retrieval,but not in video, include: naked goby (Gobiosoma
bosci), oyster toadfish (Opsanus tau), conger eel(Conger sp.), and rock gunnel (Pholis gunnellus).
Some of these species are more active at night,
which may account for their absence from video todate. Invertebrates associated with cages (either invideo or observed during deployment/retrievals)
include oyster predators such as oyster drill and sea
star, as well as prey species for fish such as
black-fingered mud crab. Other invertebrate species
noted adjacent to a cage in video include: spider
crab, blue mussel, slipper shell, horseshoe crab,
blue crab, and channel whelk.

Outreach is an important component of this project.
Public perception and understanding of aquaculture
practices can be enhanced by news articles andother types of outreach (Froehlich et al. 2017).
Heightening awareness of aquaculture practices andpositive outcomes associated with aquaculture gearcan increase support for aquaculture in the scope ofthe growing demand for shellfish. Our methodology
was based on low-cost, readily-available cameras
and mounts. An advantage of this design is that it
can easily be replicated by aquaculture industry
members, extension specialists, and citizen
scientists. We have provided our methods in detail,with photos, within a .pdf document that is publicly
available online(https://www.fisheries.noaa.gov/webdam/download/
89562100). Our intention is to broaden video
collection to other geographic locations, shellfish
aquaculture industry practices, and seasons, toultimately enable a more comprehensive
understanding of the habitat services provided byshellfish aquaculture. Selected video clips collectedfrom oyster cages are posted on our project website(https://www.fisheries.noaa.gov/aquaculture-habitat-NE). There has also been traditional news andsocial media coverage of this project via local news
outlets as well as Twitter and Facebook coverage
from both local and national NOAA Fisheries
accounts.
This project has yielded a low-cost, easily replicatedsystem for observing fish activity associated withoyster aquaculture gear and natural structured
habitat. Video analysis generated during pilot-scale
deployments will provide data on fish abundanceand community composition in these two habitats,as well as insights into fish behavior. The continued
investigation of fish interactions with aquaculture
gear may help inform regulators, policy makers andfishery managers who make decisions aboutaquaculture practices. Video generated by this study
could additionally contribute to a broader public

The results of eDNA metabarcoding efforts yielded
17 major and 20 rare finfish species from samples
collected monthly during June, July, and August
deployments in 2017. Fish communities obtained by
eDNA metabarcoding were significantly different
across the three months, and communities betweensites were different during the months of July andAugust (Liu et al. 2019). eDNA was detected for allof the fish species observed thus far in the videos.
Using ordination analysis, the axis associated withsampling month represented the highest variation
101

understanding of interactions between shellfish
aquaculture and the local environment.

One 12 (1): e0169281.
Liu, Y., G.H. Wikfors, J.M. Rose, R.S. McBride, L.
Milke, and R. Mercaldo-Allen. 2019.
Application of Environmental DNA
Metabarcoding to Spatiotemporal Finfish
Community Assessment in a Temperate
Embayment. Frontiers in Marine Science 6:
674.
Reid, R.N., F.P. Almeida, and C.A. Zetlin. 1999.
Essential fish habitat source document:
fishery- independent surveys, data sources,
and methods. NOAA Technical meme NMFS
NE 122.
State of Connecticut Department of Environment
Protection. 2007. A study of marine recreation
fisheries in Connecticut. F-54-R-27.
State of Connecticut Department of Environment
Protection. 2017. A study of marine recreation
fisheries in Connecticut. F-54-R-36.
Tallman J.C. and G.E. Forrester. 2007. Oyster
grow-out cages function as artificial reefs fortemperate fishes. Transactions of theAmerican Fisheries Society 136 (3):790-799.
Watson D.L., E.S. Harvey, M.J. Anderson, and G.A.
Kendrick. 2005. A comparison of temperate
reef fish assemblages recorded by three
underwater stereo-video techniques. Marine
Biology 144: 415-425.

REFERENCES
Archer A., J. Reitsma, and D. Murphy. 2014. A
Comparison of Bottom and Floating Gear for
growing American Oysters (Crassostrea
virginica) in Southeastern Massachusetts.
Woods Hole Sea Grant and Cape Cod
Cooperative Extension. Retrieved fromhttps://ecsga.org/wp-content/uploads/2019/02/
Oyster_Grow_FINA L_185504.pdf
Atlantic States Marine Fisheries Commission. 2018.
Addendum XXXI to the Summer Flounder,
Scup, and Black Sea Bass fishery management
plan.
Atlantic States Marine Fisheries Commission. 2015.
ASMFC Stock Assessment Overview: Tautog.
Retrieved fromhttp://www.asmfc.org/files/commissionerMan
ual/ISFMP/28j_2015TautogAssessmentOvervi
ew_Feb2015.pdf
Dealteris J.T., B.D. Kilpatrick, and R.B. Rheault.
2004. A comparative evaluation of the habitat
value of shellfish aquaculture gear, submerged
aquatic vegetation and a non-vegetatedseabed. Journal of Shellfish Research 23(3):867-874.
Dumbauld, B.R., J.L. Ruesink, and S.S. Rumill.
2009. The ecological role of bivalve shellfish
aquaculture in the estuarine environment: A
review with application to oyster and clam
culture in West Coast (USA) estuaries.
Aquaculture 290:196-223.
Flimlin G, S. Macfarlane, E. Rhodes, and K.
Rhodes. 2010. Best management practices for
the East Coast shellfish aquaculture industry.
East Coast Shellfish Growers Association.
Retrieved fromhttps://ecsga.org/wp-content/uploads/2018/01/
BMPmanual.pdf
Froehlich H.E., R.R. Gentry, M.B. Rust, D. Grimm,
and B.S. Halpern. 2017. Public perceptions ofaquaculture: evaluating spatiotemporal
patterns of sentiment around the world. PloS

ANNOTATED BIBLIOGRAPHY OF KEY
WORKSWatson D.L., E.S. Harvey, M.J. Anderson, and G.A.
Kendrick. 2005. A comparison of temperate
reef fish assemblages recorded by three
underwater stereo-video techniques. Marine
Biology 144: 415-425.
The authors deployed underwater stereo-video
cameras to sample the relative density and species
richness of temperate reef fishes in Southwest
Australia. This study compared diver deploy, unbaited remote and baited remote cameras. They
defined MaxN as “species presence and the
maximum number of individuals belonging to eachspecies in the field of view at one time (MaxN).”
102

This metric avoids repeated counting of fish withina given time frame and gives a conservative
estimate of relative density relating to the area ofsurvey.

compared three oyster grow-out sites, six naturalrock reefs, and one artificial reef built for fish
habitat, looking to identify patterns in fish density,
growth and disappearance rates. Their traps were
designed to sample both juvenile and adult fish andwere deployed in the summer and fall. The study
showed that oyster cages provide habitat for fish
associated with hard bottom habitats including scup,tautog, and black sea bass. Dumbauld, B.R., J.L.
Ruesink, and S.S. Rumill. 2009. The ecological roleof bivalve shellfish aquaculture in the estuarine
environment: A review with application to oyster
and clam culture in West Coast (USA) estuaries.
Aquaculture 290:196-223.

DeAlteris J.T., B.D. Kilpatrick, and R.B. Rheault.
2004. A comparative evaluation of the habitat
value of shellfish aquaculture gear, submerged
aquatic vegetation and a non-vegetatedseabed. Journal of Shellfish Research 23(3):867-874.
The authors set out to evaluate habitat value ofshellfish aquaculture gear (SAG), submerged
aquatic vegetation (SAV), and shallow
non-vegetated seabed (NVSB) over one year in Port
Judith, RI, USA. Oyster cages were sampled using
lift nets that were diver deployed under oyster cages
and left to soak for two weeks prior to retrieval.
Submerged aquatic vegetation and non-vegetatedseabed sites were sampled with quadrats, drop nets,
and a venturi- driven suction dredge deployed froma skiff. Sessile invertebrate growth was documented
on eelgrasses and on oysters and oyster cages. Allfish >5mm were collected. The authors documented
that the physical habitat of shellfish aquaculture
gear had greater surface area compared with theother two habitat types as well as a significantly
higher abundance and species richness of organisms
per meter squared throughout the year. The authors
concluded that shellfish aquaculture gear had
greater habitat value compared to their control sites.

This review article focuses on bivalve shellfish
aquaculture on the US West Coast. Much likenatural rock reefs, cages become colonized by
epibenthic, emergent, and encrusting organisms andmay provide sheltering fish with camouflage, refuge
from predation, respite from high current flow, anda source of food. Multi-tiered off-bottom cages, anincreasingly popular method for growing oysters ona smaller footprint, offer vertical three-dimensional
structure and surface area similar to that afforded bynatural reefs. In traditional on-bottom shellfish
culture, where live oysters and shell cultch are
distributed on sediments for grow-out and spatcollection, oysters act as ecosystem engineers
creating hard bottom irregular substrate onotherwise featureless seafloor. Evaluation of habitat
services provided by oyster aquaculture can bechallenging. Shellfish farms vary in spatial size andbottom area covered, variety and quantity of
aquaculture gear, and have a seasonally variable
footprint as gear is relocated or harvested.
Aquaculture of oysters, whether in cages or grownon the seafloor, may represent valuable estuarine
habitat.

Tallman J.C. and G.E. Forrester. 2007. Oyster
grow-out cages function as artificial reefs fortemperate fishes. Transactions of theAmerican Fisheries Society 136 (3): 790-799.
The authors compared fish habitats within
Narragansett Bay, RI, USA using trap surveys. They103

Vibrio coralliilyticus Induction of Virulence Toward Larval Oysters and Corals at
Elevated Seawater Temperatures and Potential Mitigation Strategies
Gary P. Richards1* and Blake Ushijima2
1

United States Department of Agriculture, Agricultural Research Service, Dover, Delaware USA
Current Address: University of North Carolina Wilmington, Dept. of Biology and Marine Biology,
Wilmington, North Carolina USA

2

Corresponding author: gary.richards@usda.gov
Keywords: Vibrio coralliilyticus, oyster, larvae, coral, mortalities

ABSTRACTtemperatures ≥ 27°C. In our studies, multiple strains
of V. coralliilyticus produced high larval oyster
mortalities with LD50’s ranging from 3.8 × 103 to 4
× 104 CFU/ml of seawater, depending on the V.
coralliilyticus strain and the oyster species (Eastern
vs. Pacific oysters). Studies with knockout
mutations demonstrated that the transcriptional
regulator ToxR and the outer membrane protein
OmpU were important in larval oyster (and coral)
infections caused by V. coralliilyticus. Grosspathological changes occur in V.
coralliilyticus-infected larvae, initially in the velum
and cilia and then in the internal organs, whichbecome liquefied. Infection of larval oysters is
likely induced by stress and a lowering of resistanceto these pathogens. Stressors, particularly inhatcheries, likely include incorrectly adjusted
seawater temperatures, salinities, pHs, anddissolved oxygen levels or inadequate nutrition,over stocking, high overall bacterial loads, etc.

Recent research has advanced our understanding of
the role pathogenic vibrios play in disease ofvarious aquaculture species. Vibrio coralliilyticus,
formerly thought to infect just corals, is now known
to infect larval oysters, causing major losses inhatchery settings. The negative effect of V.
coralliilyticus on wild-type oyster production
remains uncertain, but circumstantial evidence
suggests it may be substantial. We identified eightstrains of V. coralliilyticus that infect both Easternoyster (Crassostrea virginica) larvae and Pacificoyster (Crassostrea gigas) larvae in U.S. East andPacific coast hatcheries. West Coast hatcheries haveexperienced major production losses over the years
due to the presence of V. coralliilyticus. Some of thelargest hatcheries have reported losses of up to
80%, resulting in shortages in seed oysters needed
for commercial oyster production. Losses canexceed 10 billion larvae per year in the larger
hatcheries. Most U.S. East Coast hatcheries are
considerably smaller, but also experience losses in
Eastern oysters, possibly from V. coralliilyticus, butalso from the known shellfish pathogen V.
tubiashii. Interestingly, V. tubiashii only infected
Eastern oyster larvae, unlike V. coralliilyticus whichcauses acute mortalities with Eastern and Pacificspecies.

To begin to mitigate vibrios in hatcheries, we
identified a variety of predatory bacteria which innature help to modulate Vibrio levels in seawater
and/or shellfish. They include several predatory
bacteria such as Pseudoalteromonas piscicida,
which secrete digestive enzymes that inhibit and kill
many kinds of bacteria including vibrios; andHalobacteriovorax, a Bdellovibrio and Like
Organism (BALO), which infect Gram-negative
bacteria, like vibrios, and replicate within them,killing the vibrios in the process. Together these

The infection and death of corals by V.
coralliilyticus is known to be enhanced at seawater104

bacteria may serve as probiotics in reducing
hatchery mortalities and in killing vibrios and otherbacterial pathogens in a variety of other aquaculture
settings. Bacteriophages (phages) are another means
to inactivate many undesirable pathogens in theenvironment, and are increasingly being used inaquaculture. To date, we isolated multiple phages
against V. coralliilyticus and V. tubiashii andshowed the practical application of phage therapy to
reduce or eliminate mortalities from V.
coralliilyticus and V. tubiashii in larval oysters.

addition, bacteriophages (phages) against both V.
coralliilyticus and V. tubiashii offer some promise
as a therapeutic treatment for reducing
hatchery-associated larval mortalities. Phage
therapy is increasingly being used to combat
bacterial disease in aquaculture (reviewed by
Richards 2014).

INTRODUCTION
Naturally-occurring Vibrio coralliilyticus is a wellrecognized pathogen of some corals. More recently,
it has been found to be highly pathogenic towardlarval Pacific oysters (Crassostrea gigas) andEastern oysters (Crassostrea virginica) (Richards etal., 2015; Ushijima et al., 2018). These two oyster
species are the primary commercially-harvested
oysters in the United States. Another pathogen oflarval Eastern oysters is Vibrio tubiashii, which is
better known for disease outbreaks in East Coast
hatcheries. Although literature has commonlyreferred to V. tubiashii as a pathogen in West Coast
shellfish hatcheries, some of the more prevalentstrains associated with the mortalities weremisidentified and were confirmed by genomic
sequencing to be V. coralliilyticus (Richards et al.,2014, 2018). Together, these two vibrios havecontributed to episodic losses in hatcheryproduction and shortages in seed oysters needed forcommercial shellfish aquaculture. Interestingly, V.
coralliilyticus and V. tubiashii are only known toinfect and kill oysters while in the larval stage,
which is only two to–three weeks in most cases.
Once the larvae undergo metamorphosis, they
appear resistant to infection by these vibrios.

Figure 1. Normal and diseased Pacific oyster larvae. A) Normal,
healthy larva and (B-D), larvae infected with V. coralliilyticus. B)
Early-stage infected larva with tangled cilia and lumpy velum. C)
Later stage infection with loss of cilia and velar cells. D) Final stage
showing dead larva and release of digested/liquified tissues fromwithin the valves.

We conducted studies of larval oysters to compare
disease pathologies associated with both V.
coralliilyticus and V. tubiashii (Ushijima et al.,2018). We identified the infectious doses of thesepathogens in larval oysters. It is well known thatelevated seawater temperatures enhance coraldisease from V. coralliilyticus, so we also
determined if similar high temperatures enhanced
larval oyster mortalities. We also explored genes
potentially responsible for the virulence of V.
coralliilyticus in knock-out experiments (Ushijima
et al., 2018) and are currently investigating
mitigation strategies to reduce larval oyster
mortalities using bacteriophages and predatory
bacteria.

Methods are needed to mitigate larval oyster
diseases in shellfish hatcheries. Several potential
probiotic approaches include the use of predatory
bacteria, like Halobacteriovorax (Richards et al.,2012, 2013, 2016) and Pseudoalteromonas
piscicida (Richards et al., 2017a, 2017b). In
105

seawater. In the case of V. tubiashii, LD50’s forstrains ATCC 19106 and ATCC 19109 were 3.8 ×
103 and 1.2 × 104 CFU/ml, respectively, in Easternoysters; however, these strains were not infectious
to Pacific oysters. These ATCC strains are obtained
from the American Type Culture Collection,
Manassas, Virginia (USA).

RESULTS AND DISCUSSION
Disease Pathology
Normal, uninfected, Pacific oyster larvae had asmooth velum and ordered cilia (Fig. 1A). They
remained actively motile. Larvae infected with V.
coralliilyticus showed tangled cilia and a lumpylooking velum within a day or two post infection(Fig. 2B), which greatly restricted their motility.
Shortly thereafter, the cilia were either digested orreleased into the surrounding milieu while
individual velar cells were also released into themilieu. Usually within a day, the tissues within thevalves appeared to liquefy by microbial digestion
and were released from the valves (Fig. 1C).
Eastern oysters exhibited the same pathology fromV. coralliilyticus. Pathological signs of V. tubiashii
infection were the same as for V. coralliilyticus
infection in Eastern oysters, but V. tubiashii did notinfect larval Pacific oysters in our studies.

Effects of Elevated Seawater Temperatureon Larval Mortalities
We also evaluated and determined that elevatedseawater temperatures (27°C) increased larval
oyster mortalities by 15.4% to 42.8%, depending on
the V. coralliilyticus strain, compared to larval
oysters maintained at 23°C (Table 1).
Virulence Genes in V. coralliilyticus
We also evaluated the ToxR transcriptional
regulator and the outer membrane protein OmpU to
determine if they were virulence factors in V.
coralliilyticus infection of larval Pacific oysters.
Studies with knockout mutations demonstrated that
ToxR and OmpU in V. coralliilyticus were
important in larval oyster (and coral) infections(Table 2). Mutants lacking ToxR had significantly
reduced virulence compared to the wild type strain,
while the mutant lacking OmpU had completely
attenuated virulence at doses <106 CFU/ml. Larvae
exposed to the OmpU mutant did not experiencesignificantly more mortalities compared toreplicates exposed to filtered sea water or a
non-pathogenic bacterium, suggesting OmpU isessential for V. coralliilyticus infection of Pacificoyster larvae. Furthermore, both the ToxR and
OmpU mutant are avirulent towards coral,suggesting these proteins play similar roles for
infections of multiple hosts (Ushijima et al. 2016,2018). In contrast, the mannose-sensitive
hemagglutinin (MSHA) type IV pili that are
required for coral infection (Ushijima et al. 2016)
were not required for larval oyster infection(Ushijima et al. 2018).

Table 1. Increased larval oyster mortality from four V. coralliilyticus
strains due to elevated seawater temperatures

Vibriocoralliilyticus
strainPercent
mortality
at 23°C

Percent
mortality
at 27°C

Increasedmortality
due tohighertemperature

ATCC BAA450

23.4

38.8

15.4%

OCN008

54.9

78.8

23.9%

OCN014

50.9

93.7

42.8%

RE98

53.3

79.2

25.9%

Infectious Dose of V. coralliilyticus and V.
tubiashii in Eastern and Pacific Oysters
Our studies showed that the dose of pathogens
causing 50% mortality (the LD50) for both Easternand Pacific oysters varied somewhat based on thestrain of V. coralliilyticus used. In general, LD50’s
ranged from approx. 1 × 103 to 1 × 104 CFU/ml of

The protein ToxR is believed to positively respondto various environmental signals, including elevatedwater temperatures, which is then involved with106

upregulating the expression of various genes,
including the gene encoding OmpU. Therefore,
ToxR is not believed to be directly involved withshellfish mortalities, however, represents a“molecular messenger” that translates
environmental signals, most of which have yet to be
identified, to physiological responses. Hence,
understanding the conditions conducive to increased
V. coralliilyticus virulence (i.e. the regulators ofvirulence) would complement, and potentiallyenhance, the various mitigation efforts described
here.

that these phages can be combined in a cocktail toprovide a safe treatment for V. coralliilyticus in
hatcheries. Testing of a preliminary phage cocktail
at a West coast hatchery at Oregon State University
showed nearly a 100% reduction in mortalities after
challenging larval oysters with a lethal dose of V.
coralliilyticus. East coast hatchery trials are plannedin the spring. The goal is to market a phage cocktail
for use in hatcheries world-wide. Probiotic means toeliminate V. coralliilyticus, V. tubiashii and otherpathogens in hatcheries are also being exploredusing the predatory bacteria Halobacteriovorax (agenus of Bdellovibrio and Like Organisms[BALO’s]) and Pseudoalteromonas piscicida. Bothpredators have broad host specificity, with theHalobacteriovorax targeting Gram-negative
bacteria and P. piscicida targeting both
Gram-positive and Gram-negative bacteria.

Table 2. Larval mortalities after deletion (Δ) mutation of various
genes believed to be important for V. coralliilyticus virulence.
Vibriocoralliilyticus
strainPercent
mortality
at 23°C

Percent
mortality
at 27°C

Statisticallydifferent
from wild
type?

Statisticallydifferent
from thenegative
control?

Table 3. Eight strains of V. coralliilyticus killed by phage treatment.

Wild type

54.9

78.8

--

Yes

ΔtoxR
mutant

15.8

29.4

Yes

Yes

ΔompU
mutant

4.5

6.1

Yes

No

ΔMSHA
mutant

44.1

72.4

No

Yes

Mitigation Hatcheries Strategies for V.
coralliilyticus in Hatcheries
We have conducted considerable research over the
past 10 years to isolate, characterize and test theefficacy of phages against V. coralliilyticus and V.
tubiashii strains in Eastern and Pacific oyster larvae.
From over 20 phages isolated and characterized,
several have been shown to be lytic phages (basedon genomic sequencing). Such lytic phages aredesirable for use in mitigation efforts because theirreplication cycle consistently results in host death.
Currently, we have phages against eight strains of V.
coralliilyticus, including the principal strains
associated with hatchery outbreaks on the West
Coast (Table 3), and against V. tubiashii strain
ATCC 19106. Proof-of-principle testing has shown

Vibriocoralliilyticus
strainVibriocoralliilyticus
strainatccATCC
BAA-450*

RE98

ATCC 19105*

RE09-105-8

RE22

OCN008RE90

OCN014*Indicates strains known to infect both corals andoyster larvae

CONCLUSION
There are many obstacles to successful hatcheryoperations. Since V. coralliilyticus and V. tubiashii
appear to be opportunistic pathogens, anything thatstresses larval shellfish makes them more
susceptible to infection. High seawater temperature
is one factor that enhances V. coralliilyticus
virulence and may be managed by maintaining
temperatures under 27°C in the hatcheries.
107

Likewise, close monitoring of other hatcheryparameters (like salinity, dissolved oxygen, pH,
food supply, larval densities, etc.) and adequate
hatchery sanitation will reduce larval stresses toenhance productivity. With new treatments on thehorizon to combat vibrios, the future is looking
brighter for hatcheries and the commercial oyster
industry as a whole.

bacterial pathogens in aquaculture: a review of
the technology. Bacteriophage
doi:10.4161/21597081.2014.975540
Richards, G.P., J.L. Bono, M.A. Watson, and D.S.
Needleman. 2014. Complete genome
sequence for the shellfish pathogen Vibrio
coralliilyticus RE98 isolated from a shellfish
hatchery. Genome Announc. 2(6),doi:10.1128/genomeA.01253-14
Richards, G.P., M.A. Watson, D.S. Needleman,
K.M. Church, and C.C. Häse. 2015.
Mortalities of Eastern and Pacific oyster
larvae by the pathogens Vibrio coralliilyticus
and Vibrio tubiashii. Appl. Environ.
Microbiol. 81:292-297.
Richards, G.P., J.P. Fay, J. Uknalis, O.M. Olanya,
and Watson. 2016. Purification and hostspecificity of predatory Halobacteriovorax
isolated from seawater. Appl. Environ.
Microbiol. 82:922-927.
Ushijima, B., P. Videau, D. Poscablo, J.W. Stengel,
S. Beurmann, A.H. Burger, G.S. Aeby, and
S.M. Callahan. 2016. Mutation of the toxR or
mshA genes from Vibrio coralliilyticus strain
OCN014 reduces infection of the coral
Acropora cytherea. Environ. Microbiol.
18:4055–4067.
Richards, G.P., M.A. Watson, D.S. Needleman, J.
Uknalis, E.F. Boyd, and J.P. Fay. 2017a.
Mechanisms for Pseudoalteromonas
piscicida- induced killing of vibrios and otherbacterial pathogens. Appl. Environ. Microbiol.
83:11. doi:10.1128/AEM.00175-17.
Richards, G.P., D.S. Needleman, and M.A. Watson.
2017b. Complete genome sequence of
Pesudoalteromonas piscicida strain DE2-B, abacterium with broad inhibitory activity
toward human and fish pathogens. Genome
Announc. 5:33.
doi:10.1128/genomeA.00752-17
Richards, G.P., B.F. Kingham, O. Shevchenko,
M.A. Watson, and D.S. Needleman. 2018.
Complete genome sequence of Vibrio
coralliilyticus RE22, a marine bacterium
pathogenic toward larval shellfish. Microbiol.

ACKNOWLEDGEMENTS
The authors thank Ralph Elston, Aquatechnics,
Sequim, WA, for the RE strains of V. coralliilyticus;
Chris Langdon and David Madison, Oregon State
University, Hatfield Marine Science Center,
Newport, OR, for phage efficacy testing; Alexander
Sulakvelidze and Jacob Chamblee, Intralytix Inc.,
Baltimore, MD, for upscaling and preparing thephage cocktail; the staff of the Aquaculture
Innovation Center, Rutgers University, Cape May,
NJ, for larval Eastern oysters; Joan Hendricks,
Taylor Shellfish Farms, Quilcene, WA, for larval
Pacific oysters; Keith Olson, Natural Energy
Laboratory of Hawaii Authority, Kailua-Kona, HI,
for seawater; Ronald Lau, Kona Coast Shellfish
LLC, Kailua-Kona, HI, for seawater and larvae;
Claudia Häse, Oregon State University, Corvallis,
OR, for technical assistance, and Michael Watson,
USDA, ARS, Dover, DE, for conducting many of
the assays.

REFERENCES
Richards, G. P., J. P. Fay, K. A. Dickens, M. A.
Parent, D.S. Soroka, and E. F. Boyd. 2012.
Predatory bacteria as natural modulators ofVibrio parahaemolyticus and Vibrio vulnificus
in seawater and oysters. Appl. Environ.
Microbiol. 78:7455-7466.
Richards, G.P., M.A. Watson, E.F. Boyd, W.
Burkhardt III, R. Lau, J. Uknalis, and J.P. Fay.
2013. Seasonal levels of the Vibrio predator
Bacteriovorax in Atlantic, Pacific and Gulf
Coast seawater. Intl. J. Microbiol.
doi:10.1155/2013/375371
Richards, G.P. 2014. Bacteriophage remediation of108

Resour. Announc. doi:10.1128/MRA.
01332- 18.
Ushijima, B., G.P. Richards, M.A. Watson, C.B.
Schubiger, and C. Hase. 2018. Factors
affecting infection of corals and larval oysters
by Vibrio coralliilyticus. PLoS One.
doi:10.1371/journal.pone.0199475.

larvae by the pathogens Vibrio coralliilyticus
and Vibrio tubiashii. Appl. Environ.
Microbiol. 81:292−297.
Vibrio tubiashii has been a well-known pathogen in
Eastern oyster (Crassostrea virginica) larvae on the
United States (U.S.) East Coast for many years andhas also been associated with Pacific oyster(Crassostrea gigas) larvae on the U.S. West Coast.
Vibrio coralliilyticus, a well-known coral pathogen
and a cause of coral bleaching, has also been
associated with disease of Pacific oyster larvae,
particularly in oyster hatcheries. It became evident
that some of the outbreaks on the West Coast thatwere thought to be from V. tubiashii were actuallycaused by V. coralliilyticus. In this study, weevaluated two strains of V. tubiashii and four strains
of V. coralliilyticus in both Eastern and Pacificoyster larvae to determine their ability to cause
larval mortalities. We showed that V. tubiashii
strains infected primarily the Eastern oyster larvae
while all four of the V. coralliilyticus strains
infected the Eastern and Pacific oyster larvae. The
LD50 values for the V. coralliilyticus strains ranged
from 1.1 × 104 to 3 × 104 CFU/ml of seawater in
Eastern oyster larvae. In Pacific oyster larvae, theLD50 range was 1.2 × 104 to 4 × 104 CFU/ml ofseawater. Together, these studies defined the hostspecificity of these Vibrio strains with regard to
Eastern and Pacific oyster larvae and demonstrated
the ability of coral- associated pathogens to also
infect and kill larval oysters. Subsequent research(Ushijima et al., 2018, see abstract below) showedsimilar results with other strains of V. coralliilyticus
and an up-regulation of mortalities at elevatedseawater temperatures.

ANNOTATED BIBLIOGRAPHY OF KEY
WORKSRichards, G.P., J.P. Fay, K.A. Dickens, M.A. Parent,
D.S. Soroka, and E.F. Boyd. 2012. Predatory
bacteria as natural modulators of Vibrio
parahaemolyticus and Vibrio vulnificus inseawater and shellfish. Appl. Environ.
Microbiol. 78:7455−7466.
Levels of the human pathogenic Vibrio
parahaemolyticus and Vibrio vulnificus are
increasing in shellfish harvesting areas in many
parts of the world due, in part, to elevated seawatertemperatures. A means to reduce these vibrios inshellfish is needed. When seawater and oysters werespiked with V. parahaemolyticus or V. vulnificus, theVibrio levels rapidly diminished over a 3-day period
when natural seawater was used, but counts quickly
climbed when sterile seawater was used. In an effort
to determine what was inhibiting the vibrios innatural seawater, tests showed the presence ofVibrio predatory bacteria, later classified as
Halobacteriovorax species. The Halobacteriovorax
in natural seawater readily eliminated vibrios thatwere added to the seawater. In contrast, oysters
spiked with vibrios showed some initial increase in
Vibrio counts within the first 24 h, followed by a
rapid decline to baseline levels. Thus, it appears that
Halobacteriovorax are one of nature’s tools tomodulate bacterial levels in shellfish. Harnessing
this tool may provide a means to reduce or
eliminate pathogens in aquaculture operations andin the seafood industry.

Richards, G.P., J.P. Fay, J. Uknalis, M. Olanya and
M.A. Watson. 2016. Purification and hostspecificity of predatory Halobacteriovorax
isolates from seawater. Appl. Environ.
Microbiol. 82: 922−927.
Halobacteriovorax are small predatory bacteria
found in the marine environment and may serve as
biocontrol agents against human, fish and shellfish
pathogens. They are within a group of predatory

Richards, G.P., M.A. Watson, D.S. Needleman,
K.M. Church, and C.C. Häse. 2015.
Mortalities of Eastern and Pacific oyster109

bacteria known as the Bdellovibrio and Like
Organisms (BALO). Halobacteriovorax wereisolated from seawater and had broad specificity
toward five strains of Vibrio parahaemolyticus,
while two additional Halobacteriovorax strains
isolated from low salinity seawater readily infected
and killed the human pathogens E. coli O157:H7
and Salmonella Typhimurium DT104. The
replication rate of Halobacteriovorax against E. coliand Salmonella increased as salinities decreased. It
is likely that Halobacteriovorax could readily beisolated against a broad range of Gram-negative
seafood pathogens. Improved methods were also
developed to purify infectious Halobacteriovorax
from their host cells. Overall, the use of
Halobacteriovorax in various aquaculture
applications may serve as an environmentallyfriendly, non-antibiotic treatment to reduce
pathogens in fish and shellfish.

identified were aminopeptidase B, a trypsin-like
serine protease, a chymotrypsin-like serine protease,and a cysteine protease. Pseudoalteromonas
piscicida inhibited and killed the pathogens Vibrio
parahaemolyticus, V. vulnificus, V. cholerae,
Photobacterium damselae, Shewanella algae, andStaphylococcus aureus. Together, this data indicatesthat Pseudoalteromonas piscicida produce
important antibacterial compounds that have apotential role in the probiotic treatment ofaquaculture products and in reducing biofilm
formation.
Ushijima, B., G.P. Richards, M.A. Watson, C.B.
Schubiger, and C.C. Häse. 2018. Factors
affecting infection of corals and larval oysters
by Vibrio coralliilyticus. PLoS One.
https://doi:10.1371/journal.pone.0199475
Vibrio coralliilyticus is a naturally occurring marine
bacterium which infects and kills corals and larvalshellfish. It is best known as the cause of coralbleaching, which has contributed to the loss ordamage to coral reefs worldwide. We determinedthat V. coralliilyticus strains also infect and killlarval oysters and other shellfish, particularly inshellfish hatcheries, causing shortages of seedoysters needed for commercial shellfish operations.
We determined that larval oyster mortalities weresignificantly higher at an elevated seawatertemperature (27°C) compared to a lower
temperature (23°C). Thus, multiple coral pathogens
were found to infect larval oysters in a temperatureand dose-dependent manner. Also identified werevirulence factors that promote the infection of bothcoral and oyster larvae. This work demonstrates forthe first time that elevated seawater temperatures
enable V. coralliilyticus to more readily infect oyster
larvae. It also serves to warn hatchery operators tomaintain seawater temperatures below 27°C.

Richards, G.P., M.A. Watson, D.S. Needleman, J.
Uknalis, E.F. Boyd, and J.P. Fay. 2017.
Mechanisms for Pseudoalteromonas
piscicida- induced killing of vibrios and otherbacterial pathogens. Appl. Environ. Microbiol.
83:e00175-17,
https://doi:10.1128/AEM.00175-17
Pseudoalteromonas are marine bacteria that are
known to secrete antimicrobial compounds which
inhibit competing bacteria in the marine
environment. We identified a second method bywhich some Pseudoalteromonas (P. piscicida) killcompeting bacteria. It involves the direct transfer ofdigestive vesicles from the surface of thePseudoalteromonas to the surface of competitors,
digestion of holes in the competitor’s cell wall byproteolytic enzymes associated with the vesicles,
and apparent feeding of the Pseudoalteromonas off
the nutrients released by the digested bacterium in a
predatory fashion. Among the P. piscicida enzymes

110

Cyst Distribution Patterns of the Paralytic Shellfish Poisoning Plankton Species
Alexandrium catenella and A. pacificum off the Pacific Coast of Eastern Japan
Tomoko Sakami1*, Tetsuroh Ishikawa2, and Toru Udagawa3
1

National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 422-1
Nakatsuhamaura, Minami-ise, Mie, Japan 516-0193
2
Miyagi Prefectural Government, 3-6-16, Honcho, Aoba-ku, Sendai, Miyagi, Japan 980-0014
3
National Research Institute of Fisheries Engineering, Japan Fisheries Research and Education Agency, 7620-7,
Hasaki, Kamisu, Ibaraki, Japan 314-0408
Corresponding author: sakamisiro3@gmail.com
Keywords: paralytic shellfish poisoning, cyst, Alexandrium catenella, Alexandrium pacificum

ABSTRACT

from their products. PSP occurs in the north-east
Pacific coast and western part of Japan, Seto inlandsea, and Kyushu area. However, PSP does not occur
in the middle parts of the Pacific coastal area andJapan sea coastal area except in a few inner bays.
Moreover, PSP occurrence has been shown toincrease suddenly, for example, after the Great East
Japan Earthquake in 2011 (Ishikawa et al., 2015). It
is, therefore, essential to study the risks of PSP
outbreaks in shellfish aquaculture areas without
previous reports. One of the important precautions
is monitoring of the toxin producing organisms.
There are four PSP producing phytoplankton
species in Japan. Alexandrium catenella and A.
pacificum are the major causative organisms. These
phytoplankton species used to be called A.
tamarense and A. catenella, respectively; however,recent molecular work has indicated a newnomenclature for species within the Alexandriumtamarense species complex (Litaker et al., 2018). A.
catenella prefers cold water and often causes PSP in
the northern areas of Japan. On the other hand, A.
pacificum prefers warm water. It is found mainly inthe western part of Japan, and also in north-easternJapan during summer. The toxicity level of A.
pacificum is relatively low compared to A. catenella
and is not generally considered to cause PSP in thenorth-eastern Japan areas.

Mainly two dinoflagellate plankton species,
Alexandrium catenella and A. pacificum, cause
paralytic shellfish poisoning in Japan. To examine
how the two Alexandrium species’ cysts distribute
off the Pacific coast of eastern Japan, we measured
cyst abundance of the two species by quantifying
their specific gene abundance in DNA extracted
from marine sediments. Both species were detectedin some ria-type inner bays in the Sanriku region.
However, only A. catenella was detected from the
Joban area, which is located to the south of Sanriku
region, opening widely to the Pacific Ocean. In
addition, only A. pacificum was detected in the
Sotobo area, which is located to the south of the
Joban area and is under the strong influence of thewarm Kuroshio current. These results suggest thatthe distribution patterns of the two toxic
phytoplankters differ depending on topography andthe ocean current system.

INTRODUCTION
Paralytic shellfish poisoning (PSP) is caused byeating bivalves contaminated with a marine biotoxinthat is produced by microalgae. In Japan, it is aserious problem for fishermen because manybivalves cause PSP, and a voluntary shipment
regulation is carried out when a PSP is detected

These phytoplankton produce a cyst after blooming
111

METHODS

stimulated by nutrient depletion. The germinatingplankton become a seed population in the year of
PSP outbreaks. To assess risks of PSP outbreaks,
cyst abundances are often investigated in the area.
The fluorescent dye method is commonly used todetermine Alexandrium spp. cyst abundance(Yamaguchi et al., 1995). However, it is difficult todetermine whether the cysts belong to A. catenellaor A. pacificum species (Nagai et al., 2012). There
are some reports where molecular techniques havebeen used for identification of A. catenella and A.
pacificum cysts (Kamikawa et al., 2007; Erdner etal., 2010). In this study, we measured cyst
abundances of A. catenella and A. pacificum by
quantifying their specific gene abundance in the
DNA extracted from marine sediments to examine
how the cysts of the two Alexandrium species are
distributed off the Pacific coast of eastern Japan.

Figure 1. Sample collection points along the Pacific Coast of easternJapan in 2015, located in the Sanriku rias coastal area, northern partof the Joban area, southern part of the Joban area, and the Sotobo
area.

Sediment samples were collected from the Sanriku
area, northern and southern parts of the Joban area,
and the Sotobo area (Fig. 1). The Sanriku area is arias coast where mountains approach the sea and thecoast line is very complex. The Joban and Sotobo
areas are geographically flat and are widely open to
the Pacific. The Sanriku and Joban areas are regions
where the cold Oyashio current mixes with thewarm Kuroshio current. The Sotobo area is mainly
under the influence of the Kuroshio current. PSP
generally occurs in the Sanriku and Joban areas but
not in the Sotobo area.

112

Sediment samples were collected using a grab orcore type bottom sampler during the summer of2015 when Alexandrium vegetative cells were notobserved. In the Sanriku area, samples werecollected at 3-12 points for each examined bay. A
surface layer sediment of approximately 2 cm depth
was taken and kept in cold and dark conditions
immediately after collection. Samples were
preserved at -30 ℃ until DNA extraction.
DNA was extracted from marine sediments using acommercial kit, FAST DNA-SPIN kit for soil (MPBiomedicals). DNAs of A. catenella and A.
pacificum were amplified by qPCR from thesediment DNA using specific primer sets reported
by Nagai (2011). PCR reactions were performed
using SYBR Premix Ex Taq II (Tli RNaseH Plus)(Takara Bio) with ABI StepOne™ System (Applied
Biosystems). Plasmid DNA (pTaq2, DynaExpress
co. ltd) into which the PCR target fragment wasinserted was used as a standard DNA. To estimate
the density of cysts, one cyst was picked under the
microscope, its DNA extracted, and the copy
number determined using qPCR.

Figure 3. Cyst abundances of Alexandrium catenella and A.
pacificum determined by qPCR in sediments collected in the northernpart of Joban area.

RESULTS

Figure 4: Cyst abundances of Alexandrium catenella and A.
pacificum determined by qPCR in sediments collected in the southernpart of Joban area and the Sotobo area.

In the Sanriku rias coastal area, both A. catenella
and A. pacificum cysts were detected in thesediment samples, except in one small bay locatedFigure 2. Cyst abundances of Alexandrium catenella and A.
pacificum determined by qPCR in sediments collected in the Sanriku
rias coastal area

113

part is sand, in which cyst densities are generally
low (Yamaguchi et al., 2002; Shimada andMiyazono, 2005; Yamamoto et al., 2009). In thepast fifteen years, PSP occurred in the northern andsouthern part in thirteen years and seven years,
respectively. Therefore, the difference in cyst
abundance seems to indicate the difference in PSP
outbreak risk in these areas.

at the southernmost part of the area (Fig. 2). The
proportion of A. catenella cysts was found toincrease with the total cyst abundance. In thenorthern Joban area, only A. catenella cysts weredetected (Fig. 3). Cyst abundance was high in thisarea compared to the rias coastal area. Also in thesouthern part of the Joban area, only A. catenella
cysts were detected, although the density observed
was very low (Fig. 4). In the Sotobo area, only A.
pacificum cysts were detected and the density was
as small as that observed for A. catenella cysts inthe southern Joban area.

In the Sotobo area, cysts of only A. pacificum weredetected. The Sotobo area, located south to Cape
Inubo, is under the strong influence of the strong
and warm Kuroshio current which runs along
Japan’s south coast. The lowest water temperature is12-13℃ to the north of Cape Inubo, and 14-15℃ in
the Sotobo area. The water temperature may be toohigh for A. catenella in the Sotobo area. There is areport that A. catenella cysts seldomly germinate at
temperatures higher than 15℃ (Yamamoto et al.,2009). In fact, the lowest water temperatures arearound 10-13℃ in Hiroshima and Osaka Bays in
the western part of Japan, where A. catenella causes
PSP. In addition, the river mouth of the Tone River,the largest river in Japan, opens to the north of Cape
Inubo. The large freshwater discharge can be abarrier for the distribution of the phytoplankton.
Considering that PSP occurrence has not beenreported in the Sotobo area, it seems that
distribution of A. catenella is limited to the south ofCape Inubo.

DISCUSSION
Both Alexandrium species cysts were detected in
the Sanriku rias coastal area, whereas only A.
catenella was detected in the Joban area. It is
presumed that A. pacificum can survive in the
Sanriku area because of high water temperature,
which allows it to grow in summer in the small
shallow bays. Although A. pacificum is notconsidered to cause PSP, recently PSP is found tooccur during high temperature periods in the
Sanriku area. The relationship between PSP
occurrence and population dynamics of the two
Alexandrium plankton should be studied, as these
species concomitantly occur in some areas (Sakami
unpublished data) and their vegetative cells havevery similar forms. Therefore, it is important tomonitor both species of PSP-causing phytoplankton
to assess PSP outbreak risks in the Sanriku Rias
areas, where cysts of both species are present.

In conclusion, we determined cyst abundances of
two PSP-causing Alexandrium species by using aqPCR method from samples collected off the
Pacific coast of eastern Japan. We observed that: 1)
both A. catenella and A. pacificum cysts are foundin the Sanriku rias coastal area, 2) only A. catenella
cysts are found in the Joban flat coastal area,
extending to the southern tip area where the warm
Kuroshio current mixes with the cold Oyashio
current and 3) only A. pacificum cysts are found in
the Sotobo area under the strong influence of theKuroshio current. These results suggest that the cyst
distributions of the two species are influenced by
the coastal geographic features, the watertemperature and/or the current systems in different

In the Joban area, only cysts of A. catenella weredetected. A high cyst density of more than one
hundred per square centimeter was observed at theoffshore area, more than 50 km away from thecoastline with a water depth greater than 100 m.
This result suggests that not only coastal areas, but
offshore areas should be investigated for thepresence of PSP-causing plankton. The cyst density
was found to be much higher in the northern part
than in the southern part of the Joban area. One of
the reasons for the lower cyst density in thesouthern part is that the sediment in the southern114

ways. Although more data are required to clarify
relationships between cyst distribution andenvironmental factors, our study seems to indicatethat cyst abundances and/or fluctuation patterns areuseful parameters for forecasting PSP outbreak risks
in shellfish aquaculture areas.

Alexandrium species (Dinophyceae). J Phycol.
47: 703–708.
Nagai, S., M. Yamaguchi, N. Hata, and S. Itakura.
2012. Study of DNA extraction methods foruse in loop-mediated isothermal amplification
detection of single resting cysts in the toxic
dinoflagellate Alexandrium tamarense and A.
catenella. Mar Genomics. 7: 51–56.
Shimada, H. and A. Miyazono. 2005. Horizontal
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Yamaguchi, M., S. Itakura, I. Imai, and Y. Ishida.
1995. A rapid and precise technique forenumeration of resting cysts of Alexandrium
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Kotani. 2002. Distribution and abundance ofresting cysts of the toxic Alexandrium spp.
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Seto Inland Sea, Japan. Fish Sci. 68:
1012–1019.
Yamamoto, K., Y. Nabeshima, M. Yamaguchi, and
S. Itakura. 2009. Distribution and abundanceof resting cysts of the toxic dinoflagellates
Alexandrium tamarense and A. catenella in2006 and 2007 in Osaka Bay. Bull Jpn Soc
Fish Oceanogr. 73(2): 57–66. (in Japanesewith English abstract)

ACKNOWLEDGEMENTS
This study was supported financially in part by agrant of regulatory research projects for food safety,
animal health and plant protection from the
Ministry of Agriculture, Forestry, and Fisheries ofJapan.
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Erdner, D. L., L. Percy, B. Keafer, J. Lewis, andD.M. Anderson. 2010. A quantitative
real-time PCR assay for the identification andenumeration of Alexandrium cysts in marine
sediments. Deep Sea Res Part 2 Top Stud
Oceanogr. 1: 57: 279–287.
Ishikawa, T, K. Kusaka, A. Oshino, G. Nishitani,
and T. Kamiyama. 2015. Distribution patternsof Alexandrium vegetative cells and restingcysts, and paralytic shellfish poisoning in
Kesennuma Bay, after the Great East Japan
Earthquake. Nippon Suisan Gakkaishi. 81(2):
256–266. (in Japanese with English abstract)
Kamikawa, R., S. Nagai, S. Hosoi-Tanabe, S.
Itakura, M. Yamaguchi, Y. Uchida, T. Baba,
and Y. Sako. 2007. Application of real-time
PCR assay for detection and quantification of
Alexandrium tamarense and Alexandrium
catenella cysts from marine sediments.
Harmful Algae. 6 (3): 413–420.
Litaker, R.W., M. Montresor, M. Brosnahan, M.
Hoppenrath, S. Murray, J. Wolny, J. U., N.
Sampedro, J. Larsen, and A. J. Calado. 2018.
A practical guide to new nomenclature for
species within the “Alexandrium tamarense
species complex”. Harmful Algae News. 61:
13–15.
Nagai, S. 2011. Development of a multiplex PCR
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zebrafish genome after fertilization to match
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approach identified dynamic and unique patterns of
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embryo, which ‘inherits’ the DNA methylation
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Gavery, M.R. and S.B. Roberts. 2013. Predominant
intragenic methylation is associated with gene
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This dataset, generated using methylation-enrichedhigh-throughput bisulfite sequencing, represents thefirst high-resolution methylome in any mollusc.
DNA methylation data were compared to gene
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levels was identified. These data suggest that DNA
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Shao, C., Q. Li, S. Chen, P. Zhang, J. Lian, et al.
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The half-smooth tongue sole (Cynoglossus
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and pseudomale fish (generated by exposing genetic
females to high temperature during a sensitive
developmental window), the authors showed that
sex-reversed genetic females exhibit methylation
patterns consistent with genetic males, both ofwhich differ from the methylome of normal
females. Furthermore, it was reported that global
methylation patterns are inherited by F1
pseudomale offspring generated by crosses between
temperature-induced sex-reversed pseudomales andnormal females, suggesting transgenerational
epigenetic inheritance of sex reversal in this species.

Navarro-Martín, L., J. Viñas, L. Ribas, N. Díaz, A.
Gutiérrez, et al. 2011. DNA methylation of the
gonadal aromatase (cyp19a) promoter isinvolved in temperature-dependent sex ratio
shifts in the European sea bass. PLoS Genetics
7: e1002447.
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(Dicentrarchus labrax), which exhibits temperaturedependent sex determination, exposure to hightemperature in early development was associatedwith increased DNA methylation in the promoter of
the aromatase gene (cyp19a1a) and a higher
proportion of phenotypic males. Furthermore, in
vitro methylation of the aromatase promoter was
sufficient to suppress transcription of the gene,
supporting a role for DNA methylation as amechanistic link between temperature and sex ratios
in species exhibiting temperature-dependent sex
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Gavery, M.R. and S.B. Roberts. 2014. A context
specific role for DNA methylation in bivalves.
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doi:10.1093/bfgp/elt054.
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sample sizes are very low, this work suggests DNA
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http://dx.doi.org/10.1101/012831

Roberts, S. 2015. Compilation of DNA Methylation
Genome Feature Tracks (Crassostrea gigas).
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Genome feature tracks and accompanying IGV
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the Pacific oyster (Crassostrea gigas).

117

Trends in Aquaculture Production in Japan
Satoshi Watanabe* and Tomoko Sakami
Fisheries Technology Institute, Japan Fisheries Research and Education Agency, 2-12-4 Fukuura, Kanazawa,
Yokohama, Kanagawa, 236-8648, Japan
Corresponding author: swat@affrc.go.jp
Keywords: demographic problems, earthquake, tsunami, declining industry

ABSTRACT

continuous decreasing trend. Teleost fish production
was not severely affected by the earthquake, except
for Coho Salmon (Oncorhynchus kisutch), whose
production level quickly recovered. Although the
production of some seaweeds such as kelps(Saccharina spp.), wakame (Undaria spp.) andlaver or nori (Pyropia spp.) was devastated by theearthquake, not only in the Tohoku region but inmany parts of Japan, the long-term decreasing trend
is not attributed to the earthquake. One of the causes
for the production decline is considered to be thereduced number of management bodies due to theaging of the operators and shortage of successors.

A wide variety of aquatic organisms arecommercially cultured in Japan. For marine species,
national statistical data is published by the Ministryof Agriculture, Forestry and Fisheries on theaquaculture production of seven species of teleostfish (Oncorhynchus kisutch, Seriola spp., Trachurusjaponicus, Pseudocaranx dentex, Pagrus major,
Paralichthys olivaceus, Tetraodontidae spp.,
Thunnus orientalis), two species of bivalves(Mizuhopecten yessoensis and Crassostrea spp.),one species of prawn (Marsupenaeus japonicus),
one species of tunicate (Halocynthia roretzi), andfour species of aquatic plants (Saccharina spp.,
Undaria spp., Pyropia spp. and Nemacystus spp.).
Four freshwater species of teleost fish
(Oncorhynchus mykiss, Plecoglossus altivelis,
Cyprinus carpio and Anguilla japonica) are also
included in the statistics. Despite the historicallystrong affinity of Japanese consumers to seafood,production of aquaculture and capture fisheries areon a long-term moderate decreasing trend largely
due to socio-economic factors in Japan. The total
marine aquaculture production decreased from 1.28
× 106 t in 1996 to 1.03 × 106 t in 2016. Bivalve
aquaculture production was relatively constantaround 4.5 × 105 t. The production of oysters(Crassostrea spp., mainly Pacific oyster) andJapanese scallop (M. yessoensis) was heavily
damaged by the Great East Japan Earthquake andsubsequent tsunamis in 2011, and it has notrecovered to the level prior to the earthquake.
Teleost fish and seaweed production are on a

The number of management bodies of nori culture,for instance, decreased from 51,354 in 1963 to
3,819 in 2013. Mergers of the management bodies
is one of the reasons for the decrease, but the
number of farmers is certainly decreasing. On theother hand, aquaculture production of teleost fish
and bivalves has increased in value in recent years.
The increase in teleost fish production is
attributable to the recent inclusion of Pacific bluefin
tuna (T. orientalis), which was added to the MAFF
statistics list in 2012. For bivalves, production of
the common scallop leaped from JPY 25.7×109 in2012 to JPY 62.4 ×109 in 2016. In freshwateraquaculture, the Japanese eel (A. japonica) has by
far the largest production in volume and value. The
seeds for eel aquaculture are all wild caught, and the
low availability of glass eel has been problematic in
recent years. Prompt establishment of mass
production technologies for eel seed is desired.

118

INTRODUCTION

research and development by Motosaku Fujinaga(1903-1973). Some salmonids cultured or that used
to be cultured in Japan were introduced from the
U.S.: rainbow trout (Oncorhynchus mykiss, from
Shasta, CA, in 1877), brook trout (Salvelinus
fontinalis, in 1901-1902) and brown trout (Salmo
trutta, accidentally introduced with rainbow trout).
In addition to these, various other aquatic organisms
are commercially cultured in Japan. Despite thehistorically strong affinity of the Japanese for
seafood and fish, aquaculture production is on along-term moderate decreasing trend largely due tosocio-economic factors in Japan. This report
describes the present status of aquaculture in Japan,
and some of the problems confronting Japan’s
aquaculture industry.

Being surrounded by the sea, fish and seafood havebeen staple foods for the Japanese since ancient
times. Japan has a long history of aquaculture,
which mythically dates back to the era of Emperor
Keiko’s (BC13 – AD103, archeologically estimatedto be in the 4th century) reign (Higurashi, 1912).
The Emperor had a recreational fishing pond towhich cultured fish were released for hisamusement. Goldfish were introduced to Osakafrom China in 1502, and they have been cultured inJapan ever since. Aquaculture of laver or nori
(Pyropia tenera) is thought to have started in
Shinagawa Bay, Tokyo, in the Bunki era (16841688) (Higurashi, 1912).
The history of aquaculture in Japan was
summarized in a textbook (Tanigawa et al., 1966).
For example, Gorozaemon Konishiya started pole(bouchot) aquaculture of oysters (Crassostrea spp)
in Hiroshima Bay during the Enpo era (1673 1681), and it had been commonly operated incoastal intertidal areas with appropriate
environmental conditions in Hiroshima Bay,
Matsushima Bay, and Ariake Sound until thesuspended culture method was established by Senoand Hori in 1923. The suspended culture grewsignificantly because of its high productivity andapplicability in various environmental conditions.
Japanese eel (Anguilla japonica) aquaculture wasestablished by Kurajiro Hattori in Tokyo in 1879,
and commercial scale culture was started in an
80,000 m2 pond in Shizuoka Prefecture in 1897.

MATERIALS
The data analyzed and presented in this report are
based on the national statistical data for aquaculture
production published by the Ministry ofAgriculture, Forestry and Fisheries of Japan(MAFF). The statistical data on the aquaculture
production by volume and value of major species
are available online in the Japanese language(http://maff.go.jp/j/tokei/kouhyou/kensaku/bunya6.
html).
For marine aquaculture, the MAFF statistical datacontain seven species of teleost fishes(Oncorhynchus kisutch, Seriola spp., Trachurusjaponicus, Pseudocaranx dentex, Pagrus major,
Paralichthys olivaceus, Tetraodontidae spp.,
Thunnus orientalis), two species of bivalves(Mizuhopecten yessoensis and Crassostrea spp.),one species of prawn (Marsupenaeus japonicus),
one species of tunicate (Halocynthia roretzi) and
four species of seaweeds (Saccharina spp., Undaria
spp., Pyropia spp. and Nemacystus spp.). Four
freshwater species of teleost fishes (Oncorhynchus
mykiss, Plecoglossus altivelis, Cyprinus carpio andAnguilla japonica) are also included in the MAFF
statistics.

Japanese eel has by far the largest production by
volume and value in inland aquaculture in Japan
today. Yellowtail (Seriola quinqueradiata)
aquaculture was started by Sakichi Amino in
Kagawa Prefecture in 1927. It drastically expanded
in western Japan after the end of World War II,
owing to the introduction of synthetic fibers for net
cages and the establishment of wild seed collection
methods. Kuruma prawn (Marsupenaeus japonicus)
aquaculture techniques are the fruit of 30-years of

There are also miscellaneous groups, such as other119Marine aquaculture

teleost fish, other molluscan shellfish and otherseaweeds; production of each species is relatively
small and pooled together for convenience. Some
freshwater species were dropped from the statisticallist after 2000 because of the relatively minor
importance to the industry and the budgetary
constraints of the related agencies. These species
include the Japanese silver crucian carp (Carassius
auratus langsdorfii), tilapia, Chinese soft-shelledturtle (Pelodiscus sinensis), and other miscellaneousgroups.
Statistical data of the Fisheries Census is available
online at the e-Stat website in Japanese and English
languages (https://www.e-stat.go.jp/en). The
statistical data are made available by the Minister ofInternal Affairs and Communications to reveal thesituation surrounding the fisheries industry in Japan,such as the production and employment structuresof fishing communities, and the distribution andprocessing of fishery products. The data in thecensus include the number of fishery cooperatives,
fisheries management entities, fisheries workers andfishing vessels.

Figure 1. Total marine aquaculture production in Japan by volume(a) and value (b).

120

kisutch), whose production level drasticallydecreased from 1.48 × 104 t in 2010 to 116 t in 2011
because it was conducted mostly along thenorth-east pacific coast close to the epicenter.
However, it quickly recovered to 9.73× 103 t in2012.
Yellowtail (Seriola quinqueradiata, S. dumerili andS. lalandi) and red seabream (P. major) are the two
major marine teleost species cultured in Japan (1.41
× 105 t and 6.70 × 104 t, respectively in 2016), andthey had relatively minor damage to production.
Yellowtail production was greater in 2011 (1.46×
105 t) than in 2010 (1.39× 105 t). Damage toyellowtail aquaculture was relatively minor since
the main production area is in western Japan distantfrom the epicenter. About 12% of the yellowtail
produced in Japan is exported, and the US is thelargest importer (85% by value). The production of
red seabream was on a long-term decreasing trend
before the earthquake, the extent of which made thedamage by the earthquake appear insignificant. The
production, however, began an increasing trend
after 2013.

Fig. 2. Aquaculture production of marine teleost fish in Japan byvolume (a) and value (b).

Contrary to production volume, production value of
teleost fish decreased from 1997 to 2005 (JPY 1.92
× 1011) and thereafter turned into an increasing
trend regardless of the earthquake (Fig. 2b).
Production value was JPY 2.43 × 1011 in 2016. The
recent increase in teleost fish production is in part
attributable to the new inclusion of Pacific bluefin
tuna (T. orientalis) to the MAFF statistics list in
2012. Kindai University established the complete
aquaculture technique of Pacific bluefin tuna in
2002 and started providing hatchery produced
juveniles to the private sector in 2007.

Total marine aquaculture production gradually andcontinuously decreased from 1996 (1.28 × 106 t,
metric tons) to 2016 (1.03 × 106 t), i.e. a 19.6%
decrease over the 20 years in Japan (Fig. 1a). It
showed a sharp drop in 2011 (8.69 × 105 t) due tothe damage caused by the Great East Japan
Earthquake and consequent tsunamis, and it went
back to the long-term decreasing track in 2012. The
total marine aquaculture production by value, on theother hand, continuously decreased until hitting thebottom in 2011. It has been on an increasing trend
since 2012 (Fig. 1b). The production value in 2016
was JPY 5.10 × 1011 (approx. US$ 4.72 × 109). A
similar trend is seen in the production of marine
teleost fish. The total aquaculture production
volume of teleost fish is on a long-term moderatedecreasing trend, with a drop in 2011 due to theearthquake and tsunamis (2.56 × 105 t and 2.48 ×
105 t in 1996 and 2016, respectively; Fig. 2a).
Teleost fish aquaculture was not severely affectedby the earthquake, except for Coho Salmon (O.
121

Oyster production, on the other hand, was on along-term decreasing trend before the earthquake(2.2 × 105 t and 2.0 × 105 t in 1996 and 2010,
respectively) and the trend persisted after theearthquake (1.66 × 105 t and 1.59 × 105 t in 2011
and 2016, respectively). The production value ofoysters also showed an increase after the earthquake
but to a lesser extent compared to that of the scallop(JPY 3.04 × 1010 t and 3.54 × 1010 in 2012 and 2016,
respectively).
Seaweeds are important commodities in Japan as
human food. Laver or nori (Pyropia spp,
recategorized from Porphyra), for instance, has thesecond largest aquaculture production value (JPY1.00 × 1011 in 2016) following yellowtail (JPY

Fig. 3. Aquaculture production of marine molluscan shellfish in
Japan by volume (a) and value (b).

The production of marine molluscan shellfish
aquaculture was relatively constant around 4.5 ×
105 t from 1996 to 2010 (Fig. 3a). However,production was severely impacted by the earthquake
in 2011 (2.85 × 105 t) and has not recovered to thelevel prior to the earthquake as of 2016 (3.74 × 105t).
The production value of marine molluscan shellfish,
however, showed a big leap after the earthquake(Fig. 3b) increasing by 1.73 times from 2012 (JPY5.69 × 1010) to 2016 (JPY 9.86 × 1010). The
production of oysters (mainly Pacific oyster, C.
gigas) and Japanese scallop (M. yessoensis) wereboth heavily affected by the earthquake. Japanesescallop production was around 2.3 × 105 t before it
declined to 1.18 × 105 t in 2011. It recovered to 2.48
× 105 t in 2015, exceeding the value in 2010 (2.20 ×
105 t). The production value of the scallop showed a
big leap after 2012 (JPY 2.57 × 1010), reaching JPY
6.24 × 1010 in 2016.

Fig. 4. Aquaculture production of seaweed in Japan by volume (a)and value (b).

1.18 × 1011) in Japan. The aquaculture production ofseaweeds is on a long-term decreasing trend both involume and value (Fig. 4). The production volume
122

and value of seaweed were 3.91 × 105 t and JPY
1.22 × 1011, respectively in 2016. These were 25.6%
and 12.3% less than those in 1993.
Aquaculture of kelps (Saccharina spp,) and wakame(Undaria spp.) are operated mainly in the Tohoku
and Hokkaido regions and were damaged by theearthquake in 2011. Production of wakame
decreased from 5.24 × 104 t in 2010 to 1.88 × 104 t
in 2011 (i.e. 64% decrease), and it quickly increased
to 4.83 × 104 t in 2012. Production of kelp was
reduced by 42% from 2010 (4.33 × 104 t) to 2011
(2.51 × 104 t). Damage to nori aquaculture wasrelatively minor since the main production area is inwestern Japan.
Inland aquaculture
Japan’s inland (freshwater) aquaculture production
is small compared to marine aquaculture, only about
3% and 20% by volume and value, respectively in2016. Japanese consumers generally choose seafoodin preference to freshwater fish. Japanese eel (A.
japonica) may be the only exception, although it iscatadromous and not a freshwater species in a strict
sense. It is not common to culture freshwatermolluscs, crustaceans and algae in Japan. Major
freshwater aquaculture species in the global market,
such as carp, catfish, and tilapia are not consumed
in large quantities in Japan.

Fig. 5. Total inland aquaculture production in Japan by volume (a)and value (b)The production volume of freshwater teleost fish
has been on a continuous decline (Fig. 5a). The
production decreased by 42% from 1998 (6.05 ×
104 t) to 2018 (3.50 × 104 t). The production valueof freshwater teleost fish decreased from 1993 (JPY9.20 × 1010) to 2002 (JPY 4.38 × 1010) and has
thereafter been on an increasing trend (Fig. 5b). The
production value in 2016 (JPY 9.40 × 1010)
exceeded that in 1993.
Japanese eel has by far the largest aquaculture
production, comprising about 70% of the total
freshwater aquaculture production value. The seeds
for eel aquaculture are currently all wild caught, andthe low availability of the glass eel has been
problematic in recent years. Japanese eel is listed as
an endangered species in the IUCN Red List of
Threatened Species. The unit price for the glass eel
is soaring, marking the highest value (JPY 3.0 ×
106/ kg) in 2018 (Fisheries Agency, 2019), while it
was JPY 1.6 × 105 / kg in 2003. Studies on artificial

123

spawning of Japanese eel were started in the 1960s,
and the National Research Institute of Aquaculture,
Japan Fisheries Research and Education Agency
finally succeeded in the complete aquaculture ofJapanese eel at laboratory scale in 2010. However,technical difficulties remain to be overcome inscaling up the seed production in laboratory tanks tofull industrial scale. Prompt establishment of mass
production technologies for eel seeds is desired.

This is not simply a chicken-and egg-situation.
Japan is facing a labor shortage due to demographic
problems; Japan’s population is shrinking and theaverage age is rising because of a low fertility rate.
The rising education level of workers makes themreluctant to be engaged in menial jobs. It seems thatthe socio-economic reconstruction of the fisheriessociety is needed for future development ofaquaculture and fisheries in Japan.

Declining aquaculture activities in JapanAs above, aquaculture production volume of many
species has been on a decreasing trend in Japan forthe past two decades. The earthquake and tsunamis
had a big impact on the production of some of thespecies in 2011, but the long-term decreasing trend
is not attributed to natural disasters. Althoughproduction values of some species are increasing
due to increasing unit price, it can be said thataquaculture activities are generally dwindling inJapan. This is also true for fisheries activities as awhole in Japan.

REFERENCES
Fisheries Agency. (2019). A handout of the 95th
meeting of the working group on fisheries
resource management, fisheries policy council(in Japanese)
http://www.jfa.maff.go.jp/j/council/seisaku/ka
n ri/attach/pdf/190605-20.pdf
Higurashi, T. 1912. Suisan-yoshoku-gaku(aquaculture study) Shokabou, Tokyo, Pp. 376(in Japanese).
IUCN Red List of Threatened Species (Japaneseeel, Anguilla japonica)
https://www.iucnredlist.org/species/166184/11
17791)
Tanigawa, E., T. Tamura, M. Kanamori, and D.
Arai. 1966. Suisan-gaku-tsuron (complete
fisheries science). Koseishakoseikaku, Tokyo,
Pp. 306 (in Japanese).

One of the causes for the production declines is
considered to be the reduced number of aquaculture
management bodies due to the aging of theoperators and a shortage of successors. The numberof management bodies of yellowtail aquaculture, forinstance, decreased 81.8% from 3,473 in 1978 to
632 in 2013. Soaring feed prices are puttingpressure on the profits of fed aquaculture of teleostfish. Similarly, the number of management bodies
of nori culture decreased by 92.6% from 1963
(51,354) to 2013 (3,819). The number ofaquaculture farmers is decreasing in Japan. As of
2016, the fishing industry employed 166,000
people, which was about half the number in 1993
(325,000 people). The average age of fishingindustry employees is 56.7 years old as of 2016.
Merger is one of the reasons for the decrease in thenumber of the management bodies, but the majorityof them are still small-scale, self-owned businesses,
which are less competitive than enterprises within alarge capitalist economy.

ANNOTATED BIBLIOGRAPHY OF KEY
WORKSDemura, M. 2010. Trend in fishmeal price and its
effects on aquaculture. Norinkinyu
2010.10:45- 49. (in Japanese,
https://www.nochuri.co.jp/report/pdf/n1010jo1.pdf)
Despite the increasing global demand foraquaculture products, aquaculture business
management is not necessarily sustainable in Japan.

124Some aquaculture companies producing pufferfish
and red seabream went bankrupt in 2010 due to low
fish prices and increasing feed prices. The author
analyzed the supply and demand and price change
trends of fishmeal and discussed their effects on

aquaculture business in Japan. The author urged theimportance of counter measures for insufficient
fishmeal supply, such as use of unutilized fish anddevelopment of low fishmeal feed.

125Preliminary Experiments on Using the Seaweed Extract, AMPEP, to Enhance
Thermal Tolerance and Growth of the Sugar Kelp, Saccharina latissima, from
New England
Schery Umanzor1,3*, Sookkyung Shin2, Simona Augyte1, Charles Yarish1, and Jang K. Kim2
1

Department of Ecology & Evolutionary Biology, University of Connecticut, Stamford CT 06901- 2315, USA
Department of Marine Science, Incheon National University, Incheon 22012, Korea
3
Department of Marine Biology, University of Alaska Fairbanks, Juneau AK 99801
2

Corresponding author: Sumanzor@alaska.edu
Keywords: Ascophyllum nodosum, biostimulants, thermal stress, suboptimal temperature
latissima, when exposed to suboptimal
temperatures.

ABSTRACT
Seaweed extracts are widely used as biostimulants
to enhance the performance of land crops. AcadianMarine Plant Extract Powder (AMPEP), an extract
derived from Ascophyllum nodosum, that has also
been reported to enhance growth of Kappaphycus
alvarezii, a red seaweed crop. Here we assessed if
AMPEP can enhance the thermal tolerance of thekelp Saccharina latissima. To assess the effects of
AMPEP, we first exposed early sporophytes todifferent concentrations (0.001, 0.005, 0.05, 1 and 5
mg L-1) of the product and allowed individuals togrow for 20 days at an optimal temperature of 12±1℃. After determining that there were no
significant differences in sporophyte growth related
to the concentration of AMPEP, we transferred thesporophytes to the suboptimal temperature of 18℃
where they remained for 14 days. Throughout theexperiment, the photoperiod was maintained at
12:12 L:D with a photosynthetically active radiation(PAR) of 90 ±10 µmol m-2 s1. We found thatsporophytes previously exposed to AMPEP showeda higher percentage of survival and growth thancontrol sporophytes never exposed to AMPEP.
Sporophytes also showed thicker blades and adarker brown color than control sporophytes. These
preliminary results indicate that AMPEP may
enhance growth of the sugar kelp, Saccharina

INTRODUCTION
The kelp farming industry is undergoing a fast
global expansion, becoming one of the fastest
growing industries in western countries(Cottier-Cook et al. 2016, Kim et al. 2017). In the
United States for example, kelp farming, mainly of
the sugar kelp – Saccharina latissima – is emerging
as a sustainable aquaculture industry that isrevitalizing the working waterfronts of New
England by offering new economic opportunities
for seafood production while at the same time
removing excess nutrients from the coastal marine
environment (Yarish et al. 2017). To keep its
momentum, kelp aquaculture in the region needs toovercome challenges that may result in slowing its
growth. For instance, it is necessary to invest in thedevelopment of strains that can resist diseases,
biofouling, and increasing seawater temperature,
while maintaining rapid growth rates and higher
yields (Kim et al. 2017).
Rising seawater temperatures are already impacting
most temperate marine ecosystems, affecting wildbiomass production as a whole (Wernberg et al.
2016, 2018). Because kelp farming is a key
component of aquaculture production (De Silva and126

Soto 2009, Kim et al. 2017), rapid adaptive
measures should be considered to support
expanding operations. In the United States, there areseveral ongoing selective breeding programs as partof a national effort to domesticate and improve(e.g., enhance thermal tolerance) native kelp
cultivars, including Saccharina spp. (MARINER,
2017).

by fertilizing gametophytes (SL18-UCONN-CC)
isolated and cultivated in laboratory conditions fromparental sporophytes collected from Cape Cod
Canal (lat. 41.773818N; long. 70.499448W).
A stock of AMPEP solution was prepared bydissolving 10 g of AMPEP in 1L of filtered andsterilized seawater. We then prepared five dilutions(0.001, 0.005, 0.1, 0.5, and 1 g L -1) and a control
solution consisting of 100 ml filtered and sterilized
natural seawater (30 ppt). Each of the AMPEP
solutions and the control was then used to dip 36
kelp juvenile sporophytes (500 µm in length, 15days old); 6 kelp sporophytes per treatment plus 6
controls, where they remained for 45 minutes.
Immediately after the 45-minute dipping time, kelp
sporophytes were rinsed with seawater and two
sporophytes (pseudoreplicates) per treatment andcontrol were transferred to 50 ml Erlenmeyer flasks
containing nutrient-enriched seawater (half strength
Provasoli Enriched Seawater (Provasoli 1968) plus
germanium dioxide (GeO2) following Lewis (1966)
to inhibit the development of diatoms. Each pair of
blades (N= 3 per treatment) was allowed to growfor 20 days at 12±1 ℃ with a PAR of 90 ±10 µM
photons m-2 s-1. Blades were photographed every
five days using a PixeLINK® camera mounted onto
an Olympus (SZH) dissecting scope. Photographs
were processed as binary images using Fiji by
Image J (Schindelin et al. 2012) to calculate the area
occupied by each blade as a proxy to measure
growth.
After the 20 days, the sporophytes were transferred
to 125 ml Erlemeyers flasks containing media as
described above. They were allowed to grow for 15
additional days inside an incubator set at the
suboptimal temperature of 18° ±0.5℃. This allowed
us to measure if the exposure to AMPEP promoted
an increase in thermal tolerance by facilitating ahigher percentage of survival and growth.
Sporophytes were photographed at the end of the15- day period and final blade area was calculated.
After photographing, all of the survivors werecross-sectioned using a razor blade andphotographed again to examine what appeared to bepotential differences in the thickness of treated andnon- treated blades.

Experiments conducted principally on theeconomically important red alga, Kappaphycus
alverezii, highlight what could be a faster
alternative to enhance seaweed strains (Loureiro etal. 2014b, Marroig et al. 2016, Souza et al. 2018).
These studies have shown that commercial extracts
obtained from certain seaweeds can be used to
improve the resistance and vigor of othernon-related seaweeds and terrestrial plants. For
example, similar to the results obtained inagriculture (Khan et al. 2009), applying AMPEP(Acadian Seaplants Ltd., Canada), a seaweed
extract from Ascophyllum nodosum, to cultivars of
Kappaphycus alvarezii resulted in growthenhancement (Hurtado et al. 2009; Tibubos et al.
2017; Souza et al. 2018), reduction in epiphyte
loads, and higher thermal tolerance (Loureiro et al.
2014b; Hurtado and Critchley 2018).
Saccharina latissima, as other kelp species, isvulnerable to changes in water temperature. Itshows an optimal growth between 10 and 15°C
(Lee and Brinkhuis 1988, Egan et al. 1989, Yarish
et al. 1990, Augyte et al. 2017), which decreases
with temperatures exceeding 16°C, while 100%
mortality is observed for temperatures exceeding
23°C (Lee and Brinkhuis 1986). Here we report on
the effects of applying AMPEP to S. latissima as apotential method to increase thermal tolerance andenhance growth under optimal and suboptimal
temperature conditions.

METHODS
The experiment consisted of testing the effect of
AMPEP on the thermal tolerance and growth ofjuvenile sporophytes (500 µm in length) of
Saccharina latissima. Sporophytes were produced

127

Data analysis
The specific growth rate (SGR, expressed as percent
increase day-1) of juvenile kelp sporophytes wascalculated as:
𝑆𝐺𝑅 = (𝐿𝑛(𝐴1) − 𝐿𝑛(𝐴2)) ÷ ( 𝑇1 − 𝑇2) 𝑥 100
where A1 and A2 are the area (mm-2) at time T1
and T2, respectively (Kim et al. 2009).
Differences in SGR were analyzed using repeatedmeasures ANOVA as a function of concentration of
AMPEP (0, 0.001, 0.005, 0.1, 0.5, and 1 g L -1) andtime. For all of the analyses, significance was P >0.05 unless otherwise noted.

RESULTS
Early juvenile sporophytes of Saccharina latissima
showed the highest specific growth rates (SGR) at
day ten post-treatment with AMPEP (Fig.1). Results
show no significant difference in the SGR as afunction of the concentration of AMPEP whengrowing at 12°C (P ˃ 0.05). Nonetheless, when
sporophytes were exposed to 18°C, treated samples
did show a higher percentage of survival (Fig. 2),
darker brown color, and increased overall growth(Fig. 3) compared with control sporophytes, whichlooked pale.

Figure 1. Specific growth rates (mean values ±SE, N=3) of
Saccharina latissima as a function of AMPEP concentration andtime. The absence of error bars indicates only one survivor per
treatment.

128

land crops (refer to Craigie 2011 for a review).
Similarly, studies conducted on red seaweeds haverevealed a variety of benefits with an overall
increase in seaweed vigor (Hurtado et al. 2012,
Loureiro et al. 2014a, Marroig et al. 2016, Souza etal. 2018). To date, we are not aware of any study
evaluating the effect of a brown seaweed extract(intertidal, Fucales) on kelp (Laminariales).
Hurtado and colleagues (2012) found that the use of
AMPEP on Kappaphycus alvarezii growing in
optimal conditions triggered higher growth rates
than control thalli never exposed to AMPEP, andthat low concentrations of the product (< 0.1 g L-1)
resulted in higher growth rates than higher
concentrations (i.e. 1.0 g L-1). In our study, we
found that applying AMPEP to Saccharina
latissima juvenile sporophytes growing at ideal
12°C did not result in increased growth rates.

Figure 2. Percentage of survival of juvenile sporophytes of
Saccharina latissima growing at 18°C after exposure to different
AMPEP solutions.

Cross-section measurements also revealed thattreated sporophytes showed thicker blades than nontreated individuals. Blade thickness of treatedsporophytes (avg. 0.8 cm in length) was on average
1.5 ±0.2 mm, while thickness of control
sporophytes was 0.4 ±0.03 mm. We could notconfirm significance for either survival, growth, or
thickness due to a reduced number of survivors
across treatments.

However, similar to our observations related to thedarker color exhibited by treated sporophytes, otherexperiments conducted on K. alvarezzi also reportthat at suboptimal temperatures samples without
any AMPEP treatment showed bleaching of thethallus (Loureiro et al. 2014b). The dark browncoloration exhibited by the surviving kelp
sporophytes exposed to AMPEP could be related tovarying concentrations of fucoxanthin, however
further analyses are required to establish thisassociation.
Furthermore, Loureiro et al. (2014) also found that
applying AMPEP to Kappaphycus alvarezii
increased its tolerance to survive when exposed to
suboptimal colder temperatures. Here we detectedthat by applying AMPEP to sugar kelp, individuals
seem to increase their probability of thriving at thesublethal higher temperature of 18°C. Additionally,
as in land plants, seaweed extracts applied to otherseaweeds stimulate the increase of phytohormones,
which in turn promote cell division and elongation(Khan et al. 2009, 2017, Hurd et al. 2014, Li and
Mattson 2015, Hurtado and Critchley 2018),
possibly related to the increased thickness observedon our treated blades. Nevertheless, although a

Figure 3. Final area of treated and control blades of Saccharina
latissima exposed to 18°C for 15 days.

DISCUSSION
The commercial extract AMPEP derived from
Ascophyllum nodosum has been used widely inagriculture and horticulture for the beneficial effects
of enhancing yields and reducing damage caused byexternal biotic and abiotic stressors in a variety of129higher number of treated sporophytes survived andthese exhibited overall thicker and bigger blades
than non-treated samples, a larger number of
replicates is required to confirm significance. Also,
field experiments are required to test how treatedsamples will perform over time in open ocean
conditions and if there are any inheritable
responses. However, our data suggests that applying
AMPEP to juvenile kelp sporophytes while in thenursery could enhance thermal tolerance of
Saccharina latissima, allowing treated blades togrow larger than non-treated counterparts over thesame period.

seaweed aquaculture industry. United Nations
University (INWEH) and Scottish Association
for Marine Science Policy Brief.
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23:371–93.
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and aquaculture: potential impacts, adaptationand mitigation In: Climate Change
Implications for Fisheries and Aquaculture:
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Gerard, V.A. and Mann, K.H. 1979. Growth andproduction of Laminaria
longicruris(phaeophyta) populations exposed
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Lobban. 2014. Seaweed Ecology andPhysiology. 2nd ed. Cambridge University
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Hurtado, A.Q. and A.T. Critchley. 2018. A review
of multiple biostimulant and bioeffector
benefits of AMPEP, an extract of the brown
alga Ascophyllum nodosum, as applied to theenhanced cultivation and micropropagation of
the commercially important red algal
carrageenophyte Kappaphycus alvarezi. J.
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radical scavenging, and iron chelating
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ACKNOWLEDGEMENTS
This study was supported by Macroalgae Research
Inspiring Novel Energy Resources(MARINER, DE-FOA-0001726) to CY as a
sub-award to Contracts DE-AR0000912, DEAR0000911; and DE-AR000915. This study was
also supported by the Basic Science Research
Program through the National Research Foundationof Korea (NRF) funded by the Ministry of
Education (NRF-2017R1A6A1A06015181). We
would like to thank D. Bailey and S. Lindell(Woods Hole Oceanographic Institution) for theirsupport in collecting the parental sporophytes.

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spindle inhibitors, and plant growth hormones.
J Appl Phycol. 29: 2345-2349.
This is a recent contribution describing the effect of
AMPEP K+ on the formation of vegetative shoots of
Kappaphycus alvarezii. AMPEP K+ is a Canadian
commercial extract of the brown seaweed
Ascophyllum nodosum with enhanced levels of
potassium. The authors tested this extract in
combination with colchicine or oryzalin, in addition
to synthetic plant growth regulators. They foundthat 5 mg L−1 of AMPEP K+ with plant growth
regulators produced the longest direct axis shoots(9.6 ± 0.33 mm), followed by 0.1 mg L−1 of
AMPEP K+ combined with 1.0 mg L−1 oryzalin andplant growth regulators (8.7 ±0.00 mm). Theirresults suggest that using AMPEP K+ could
improve shoot formation of K. alvarezii plantlets inland-sea-based nursery cultivation.
Hurtado, A.Q. and A.T. Critchley. 2018. A review
of multiple biostimulant and bioeffector
benefits of AMPEP, an extract of the brown
alga Ascophyllum nodosum, as applied to theenhanced cultivation and micropropagation of
the commercially important red algal
carrageenophyte Kappaphycus alvarezii andits selected cultivars. Journal of Applied
Phycology https://doi.org/10.1007/s10811018-1407-4

ANNOTATED BIBLIOGRAPHY OF KEY
WORKS
Craigie, J.S. 2011. Seaweed extract stimuli in plantscience and agriculture. J Appl Phycol.
23:371–393.
This contribution derives from the author’s
presentation at the “XX International Seaweed
Symposium”. Craigie provides an overview of theutilization of seaweeds by humans, and more
relevant, a summary of seaweeds used inagriculture. Furthermore, this paper details with
examples how the application of seaweed as biostimulants enhances land crops in different ways.
The author includes brand names, countries oforigin, and nutritional content of various
commercially available products.

The majority of publications related to using
seaweed extracts on the aquaculture of otherseaweeds have assessed the effect of Ascophyllum
nodosum extracts on Kappaphycus alvarezii. This
review mainly summarizes studies made on K.
alvarezii exposed to different treatments of A.
nodosum extracts. A. nodosum extracts are utilized
to mitigate biotic and abiotic stressors of K.
alvarezii subject to farming conditions. The authors
provide examples related to micropropagation, field
cultivation, endophyte mitigation, and impacts on
the resulting carrageenan qualities of different K.
alvarezii cultivars. In addition, the authors alsomention alternative seaweed extracts that have been

Tibubos, K.R., A.Q. Hurtado, and A.T. Critchley.
2017. Direct formation of axes in newplantlets of Kappaphycus alvarezii (Doty)
Doty, as influenced by the use of AMPEP K+,

132

Ascophyllum extracts as biostimulants for
Kappaphycus aquaculture.

applied to boost the growth of other cultivatedseaweeds. Altogether, this review paper provides
evidence of the favorable results of using133

Challenges to Establishment of Tetraploid Breeding Stocks for Shellfish
Aquaculture
Huiping Yang
Fisheries and Aquatic Sciences Program, Institute of Food and Agricultural Sciences, University of Florida,
Gainesville, FL 32653
Corresponding author: huipingyang@ufl.edu
Keywords: tetraploid, shellfish, aquaculture

ABSTRACTINTRODUCTION

In the Gulf of Mexico region, oyster farming has
been rapidly increasing in recent years, in part, tohelp meet the seafood market demand that has
resulted from a decline in fishery landings since2012. For the oyster farming industry, triploids arerecognized as the preferred products because of
their fast-growth, better meat quality, andyear-round harvest. With the rapidly growing oyster
farming industry, there has been a significant
increase in demand for oyster triploid seed. Seedshortages have occurred in the past several years
and are becoming a major constraint to the industry.
To address the demand for triploid oyster seed,establishment of tetraploid breeding stocks areneeded because tetraploids are key for commercial
all-triploid seed production. With support from theGulf States Marine Fisheries Commission incooperation with the National Oceanic andAtmospheric Administration’s National Marine
Fisheries Service, tetraploid induction research was
initiated. It is expected that establishment of
tetraploid breeding stocks will be useful for triploidseed production with diversified genetic resources
to support the oyster farming industry. In thispublication, challenges for tetraploid production
were discussed to provide insights and better
understanding of triploid-tetraploid technology forshellfish aquaculture.

Molluscan shellfish aquaculture has traditionallybeen a major component of aquaculture production
worldwide and occurs mostly in marine water
environments (98.3% of the total production). In
2016, the total molluscan aquaculture production
worldwide was 17.1 million metric tonnes ($29.2
billion) and the cultured molluscan species included
oysters, clams, scallops, and mussels (FAO, 2018).
Sustainability of this large-scale aquacultureindustry requires considerable efforts to increase
product quantity and quality, improve aquaculture
environments, and enhance the well-being of thecommunity. Use of genetic breeding is one of theimportant strategies because genetically selectedbroodstock can produce well-adapted offspring or
seed. For shellfish aquaculture, triploid-tetraploid
technology has been widely recognized by theindustry and utilized in many farming species,
especially oyster species (Guo, 2004). Triploids
grow fast, have better meat quality in summers, andprobably resist diseases in aquaculture bivalve
species (Guo et al., 2009, Yang et al., 2018). So far,
farming of triploids has become an important part of
the global oyster industry. For Pacific oysters,
Crassostrea gigas, triploids account for about 50%of the production on the west coast of the UnitedStates, 100% of the hatchery seed production in
France (Degremont et al., 2016), and over 2.3

134billion triploid seed in China (Yang et al., 2019).
For eastern oysters, Crassostrea virginica, triploids
account for nearly 100% of the seed production in
the Chesapeake Bay (Peachey and Allen, 2016) anda majority of oyster seed in the Gulf of Mexico(Wadsworth et al., 2019). For Sydney rock oysters,
Saccostrea glomerata, triploids account for about
15% of the production in Australia (Peachey &Allen 2016).

2012), and Catarina scallops Argopecten
ventricosus (Maldonado et al., 2003). Tetraploidstocks have been mostly established in oyster
species by using this method and applying it tocommercial triploid seed production.
Challenges for Tetraploid Production
The process to produce tetraploids using thisinnovative method includes the following steps: 1)
produce triploids from normal diploids and culture
to adult stage; 2) identify triploid individuals andfind fecund triploid females; 3) collect oocytes from
triploid females; 4) induce tetraploids by fertilizing
oocytes from triploids with sperm from diploids andfollowing polar body inhibition; 5) culture putative
tetraploid larvae to beyond metamorphosis andadult stage, and 6) confirm tetraploid individuals by
non- lethal ploidy determination as founders forbreeding stocks. Each of these steps is connected to
one another and problems which could occur at any
step would result in failure of the tetraploid
production.

Triploids can be produced by directly inhibiting thefirst or second polar body or crossing of diploids
and tetraploids (Yang et al., 2018). Commercially,
production of triploids is usually achieved bycrossing (mating) oocytes from diploids with spermfrom tetraploids in most hatcheries worldwide (Guoet al., 1996) because this method can produce 100%
triploids and ease to operation by commercial
hatchery staff (Yang et al., 2018). Therefore,
establishment of tetraploid breeding stocks is
becoming a critical factor for the application of
triploid-tetraploid technology in shellfish
aquaculture.

During this process, the big challenges for
successful tetraploid production are the infrequent
occurrence of fecund triploid females, limited
availability of oocytes from triploid females, andpoor survival of putative tetraploid larvae to beyond
metamorphosis (Yang et al., 2019).

Establishment of Tetraploid Breeding
Stocks
Tetraploid induction in shellfish was initiated in the
1980s and has been a challenge due to the poor
survival of tetraploid larvae to beyond themetamorphosis stage (Guo et al., 2009, Yang et al.,2019). In 1994, a unique methodology for tetraploid
induction was reported in Pacific oysters and theinduced tetraploid larvae survived to the adult stage(Guo and Allen, 1994c) and were used for
all-triploid seed production by crossing with normal
diploids (Guo et al., 1996). This innovative method
used oocytes from triploid females to fertilize with
sperm from diploid males and was followed byinhibition of the first polar body (PB 1) (Guo andAllen, 1994c). So far, this method has been applied
to several other shellfish species and produced
viable tetraploids in Pearl oysters Pinctada
margaritifera (He et al., 2000), eastern oysters
Crassostrea virginica (Guo et al., 2002), Sumino
oysters Crassostrea ariakensis (Allen et al., 2005),
bay scallops Argopecten irradians (Surier et al.,1) Occurrence of fecund triploids.
Triploids possess three sets of chromosomes andcould have abnormal chromosome synapsis andsegregations during gametogenesis. Therefore,
triploids generally exhibit abnormal gonad
development. To obtain fecund triploid females fortetraploid induction, a large number of triploids
need to be produced and screened. For oyster
species, occurrence of fecund triploids differeddepending on species. For example, about one
female out of 1,600 triploid eastern oysters (i.e.,
0.06%) was observed (Supan, 2000), but for Pacificoysters, higher proportions (58%) of fecund females
were observed in triploids (Guo and Allen, 1994a).
For Crassostrea hongkongensis, most of thetriploids exhibited polymorphic sterility with135

atrophic gonads and abnormal gametogenesis(Zhang et al., 2017). For Soccostrea commercialis,
triploids were found highly retarded (Cox et al.,1996). In other shellfish groups, the occurrence of
fecund triploid females also varied. For soft-shellclams, Mya arenaria, 77% of triploids were females(Allen et al., 1986), for Dwarf surfclams, Mulina
lateralis, 59% of triploids were females (Guo andAllen, 1994b), and for Noble scallops, Chlamys
nobilis, 25% of triploids were females (Komaru andWada, 1989). Ongoing research on eastern oysters
in my laboratory showed the female occurrences in
one-year old triploids were 0.6% (5 out of 840),
3.5% (23 out of 630), and 1.2% (14 out of 1142) inthree populations originating from different
geographic locations.

mature gametes at sexual maturity) withaccumulation of abnormal gonia (Jouaux et al.,2010).
2) Limited availability of oocytes from
triploid females.
Oocytes from triploid organisms usually haveabnormal meiosis due to the three sets ofchromosomes. Thus, the number of oocytes from
triploid females is usually low (Gong et al., 2004).
Efforts have been made to improve gonad
development in triploids, such as applying estradiol
hormones to eastern oyster triploids (Quintana,2005). The availability of oocytes varied from 100
to 1.6 million per female in one-year-old triploids (n= 41, Yang, unpublished data), and could be related
to culture locations and seasons sampled.

Additionally, for the same species (eastern oysters),
different cohorts of triploids may have significantly
different occurrences of fecund triploids (Dr. John
Supan, personal communication). Even for the samecohort of triploids (eastern oysters), the occurrencesof fecund triploids varied greatly in different culture
locations (Yang, personal observation in 2019). A
comparison of diploid and triploid gametogenesis in
Pacific oysters indicated that high levels (42 to50%) of gametogenesis occurred in triploids in all
nine triploid batches in different culture locations,and could be the reason for the summer heavymortality of these triploids (Houssin et al., 2019).

3) The poor survival of induced tetraploidlarvae.
This is a major challenge for tetraploid production.
In Pacific oysters, the survival of putative tetraploidlarvae to beyond metamorphosis was 0% in two
replicates and 0.0739% in one replicate (Guo andAllen, 1994c). Thus, to obtain viable tetraploidjuveniles (after metamorphosis), it is necessary tohave enough oocytes (at least 10,000) from triploid
females. Additionally, tetraploid mussel larvae were
found smaller than diploids and triploids in thesame cohort (Scarpa et al., 1993) and the same
situation was observed in Pacific oysters(Benabdelmouna and Ledu, 2015). Therefore,
smaller larvae may be disposed of during larval
culture. Extensive care is needed for culture ofputative tetraploid larvae by saving slow growers
and maintaining good culture conditions to ensure
they can survive beyond metamorphosis.

For the mechanism of triploid female
gametogenesis, a specific gene (Nanos-like gene)
was reported to be perhaps responsible because thisgene was expressed in the gonads of diploid females
matching with gonad seasonal development, andwas expressed significantly lower in the gonads ofsterile triploids (Xu et al., 2018). Another two genes
were also found to be correlated with triploid
gametogenesis (Jiang et al., 2017). Observation of
gametogenesis in triploids of Pacific oysters
identified two types of gametogenesis: α-pattern,which corresponded to animals displaying
numerous proliferating gonia and resulted inabundant gametes, and p-pattern, which was
associated with locked gametogenesis (only few

SUMMARY
Triploid-tetraploid technology has been applied tocommercial oyster aquaculture in many countries
for years. Establishment of tetraploid breeding
stocks is the key for the application of thistechnology, but many challenges exist. For
successful tetraploid production, great efforts are136

needed to address the key challenges, especiallyfurther investigations on triploid gametogenesis,
which can increase the occurrence of triploid
females for oocyte collection for tetraploid
production.

Guo. 2004. Chromosome inheritance intriploid Pacific oyster Crassostrea gigas
Thunberg. Heredity, 93: 408-415.
Guo, X. 2004. Oyster breeding and the use of
biotechnology. Bulletin of the Aquaculture
Association of Canada, 104: 26-33.
Guo, X. and S.K. Allen. 1994a. Reproductive
potential and genetics of triploid Pacificoysters, Crassostrea gigas (Thunberg).
Biological Bulletin, 187: 309-318.
Guo, X. and S.K. Allen. 1994b. Sex determinationand polyploid gigantism in the Dwarf
surfclam Mulinia lateralis Say. Genetics, 138:
1199-1206. Guo, X. & Allen, S. K. 1994c.
Viable tetraploids in the Pacific oyster
Crassostrea gigas Thunberg produced byinhibiting polar body I in eggs from triploids.
Molecular Marine Biology and Biotechnology,
3: 42-50.
Guo, X., G.A. DeBrosse, and S.K. Allen. 1996. Alltriploid Pacific oysters Crassostrea gigas(Thunberg) produced by mating tetraploids
and diploids. Aquaculture, 142: 149-161.
Guo, X., J. Wang, B.J. Landau, L. Li, G.A.
DeBrosse, and K.D. Krista. 2002. The
successful production of tetraploid easternoyster Crassostrea virginica Gmelin. Journalof Shellfish Research 21: 380-381.
Guo, X., Y. Wang, Z. Xu, and H. Yang. 2009.
Chromosome Set Manipulation in Shellfish.
Pages 165-194 in G. Burnell & G. Allen
editors. New Technologies in Aquaculture:
Improving Production Efficiency, Quality andEnvironmental Management. Woodhead
Publishing Limited, Abington, Cambridge.p.
165-194.
He, M., Y.G. Lin, Q. Shen, J.X. Hu, and W.G. Jiang.
2000. Production of tetraploid pearl oyster
Pinctada martensii Dunker by inhibiting thefirst polar body in eggs from triploids. Journalof Shellfish Research, 19: 147-151.
Houssin, M., S. Trancart, L. Denechere, E. Oden, B.
Adeline, M. Lepoitevin, and P. Pitel. 2019.
Abnormal mortality of triploid adult Pacificoysters: Is there a correlation with highgametogenesis in Normandy, France?

ACKNOWLEDGEMENTS
This project received funding under ACQ‐210‐039‐
2017‐UFL and UFL2 from the Gulf States Marine
Fisheries Commission in cooperation with NOAA
Fisheries. In addition, this project was supported by
the National Institute of Food and Agriculture,
United States Department of Agriculture Hatch
Fund FLA-FOR-005935.

REFERENCES
Allen, S. K., A.J. Erskine, E.J. Walker, and G.A.
DeBrosse. 2005. Production of tetraploidsuminoe oysters, Crassostrea ariakensis.
Aquaculture, 247: 3.
Allen, S. K., H. Hidu, and J.G. Stanley. 1986.
Abnormal gametogenesis and sex-ratio in
triploid soft-shell clams Mya arenaria.
Biological Bulletin, 170: 198-210.
Benabdelmouna, A. andC. Ledu. 2015.
Autotetraploid Pacific oysters Crassostrea
gigas obtained using normal diploid eggs:
Induction and impact on cytogenetic stability.
Genome, 58: 333-348.
Cox, E. S., M.S.R. Smith, J.A. Nell, and G.B.
Maguire. 1996. Studies on triploid oysters inAustralia 6. Gonad development in diploidand triploid Sydney rock oysters Saccostrea
commercialis (Iredale and Roughley). Journalof Experimental Marine Biology and Ecology
197: 101-120.
Degremont, L., C. Ledu, E. Maurouard, M.
Nourry,and A. Benabdelmouna. 2016. Effectof ploidy on the mortality of Crassostrea
gigas spat caused by OsHV-1 in France using
unselected and selected OsHV-1 resistantoysters. Aquaculture Research, 47: 777-786.
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Aquaculture, 505: 63-71.
Jiang, Q., Q. Li, H. Yu, and L.F. Kong. 2017.
Expression and DNA methylation pattern ofreproduction-related genes in partially fertile
triploid Pacific oysters Crassostrea gigas.
Genes & Genomics, 39: 997-1006.
Jouaux, A., C. Heude-Berthelin, P. Sourdaine, M.
Mathieu, and K. Kellner. 2010. Gametogenic
stages in triploid oysters Crassostrea gigas:
Irregular locking of gonial proliferation andsubsequent reproductive effort. Journal of
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395: 162-170.
Komaru, A. and K.T. Wada. 1989. Gametogenesis
and growth of induced triploid scallops
Chlamys nobilis. Nippon Suisan Gakkaishi,
55: 447-452. Maldonado, R., Ibarra, A. M. &Ramirez, J. L. 2003. Induction to tetraploidy
in catarina scallop, Argopecten ventricosus(Sowerby II, 1842). Ciencias Marinas, 29:229-238.
Peachey, B. L. and S.K. Allen. 2016. Evaluation ofcytochalasin B and 6-dimethylaminopurine fortetraploidy induction in the Eastern oyster
Crassostrea virginica. Aquaculture, 450:
199-205.
Quintana, R. 2005. Effect of estradiol-17β on the
gonadal development of diploid and triploid
female eastern oysters. Pages 139. Louisiana
State University, Baton Rouge, LA.
Scarpa, J., K.T. Wada, and A. Komaru. 1993.
Induction of tetraploidy in mussels by
suppression of polar body formation. Nippon
Suisan Gakkaishi, 59: 2017-2023.
Supan, J. E. 2000. Tetraploid eastern oysters: anarduous effort. Journal of Shellfish Research
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Surier, A., R. Karney, X. Guo, and Y. Wang. 2012.
Challenges to creating a tetraploid broodstock
for the Bay scallop Argopecten irradians.
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Walton. 2019. Elevated mortalities of triploideastern oysters cultured off-bottom in northern
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Xu, R., Q. Li, H. Yu, and L.F. Kong. 2018. Oocyte

maturation and origin of the germline asrevealed by the expression of Nanos-like in
the Pacific oyster Crassostrea gigas. Gene,
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Yang, H., X. Guo, and J. Scarpa. 2019. Inductionand establishment of tetraploid oyser breeding
stocks for triploid oyster production.
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https://edis.ifas.ufl.edu/fa215.
Yang, H., N. Simon, and L. Sturmer. 2018.
Production and performance of triploid oysters
for aquaculture. Pages 9. Electronic Data
Information Source - UF/IFAS Extension.
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and Z.N. Yu. 2017. A comparative study ofthe survival, growth and gonad developmentof the diploid and triploid Hong Kong oyster,
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ANNOTATED BIBLIOGRAPHY OF KEY
WORKSGuo, X. and S. Allen. 1994. Viable tetraploids in the
Pacific oyster (Crassostrea gigas Thunberg)
produced by inhibiting polar body I in eggs
from triploids. Molecular Marine Biology andBiotechnology, 3: 42-50.
Guo, X., G.A. DeBrosse, and S.K. Allen. 1996. Alltriploid Pacific oysters Crassostrea gigas(Thunberg) produced by mating tetraploids
and diploids. Aquaculture, 142: 149-161.
Tetraploid production for all-triploid seedproduction
Tetraploid production has been a challenge inshellfish due to their poor survival to spat and adultstages despite strong efforts on this research topic
since the 1980s. In 1994, Guo and Allen reported aunique methodology for tetraploid induction in the
Pacific oyster. The tetraploids survived to an adultstage and were used for all-triploid seed production
by crossing with normal diploids (Guo et al., 1996).
This innovative method for viable tetraploid spat138

and adult production uses oocytes from triploid
females to fertilize with haploid sperm, followed byinhibition of the first polar body (PB 1) (Guo andAllen 1994). Later, this method was applied to otheroyster species and led to the production of viable
tetraploids in the Pearl oyster Pinctada
margaritifera, eastern oyster Crassostrea virginica,
and Somonie oysters Crassostrea ariakensis (He etal. 2000, Guo et al. 2002, Allen et al. 2005). This
unique methodology has been patented by Rutgers
University (US5824841A) and adopted by a privatecompany. So far, tetraploid breeding stocks havebeen successfully established mostly in oyster
species (including Pacific oysters, eastern oysters,
Sydney rock oysters, and Suminoe oysters) by using
this method and applied for commercial triploidseed production.

Pages 165-194. in G. Burnell & G. Allen
editors. New Technologies in Aquaculture:
Improving Production Efficiency, Quality andEnvironmental Management. Woodhead
Publishing Limited, Abington, Cambridge.
Reviews and summaries
This book chapter systematically reviewed shellfish
polyploid breeding, including triploid and tetraploid
production, concepts, mechanisms, history, updates,
and future application for the aquaculture industry.
In this publication, two comprehensive tables areincluded to summarize triploid production, growth,and performance, and tetraploid induction andbreeding. In addition, there are several figures
included in this review to describe the chromosome
segregation mechanism for triploid, tetraploid andaneuploid production.

McCombie, H., C. Ledu, P. Phelipot, S. Lapegue, P.
Boudry, and A. Gerard. 2005. A
complementary method for production oftetraploid Crassostrea gigas using crosses
between diploids and tetraploids withcytochalasin B treatments. Marine
Biotechnology, 7: 318-330.

Yang H., N. Simon, L. Sturmer. 2018 Production
and performance of triploid oysters foraquaculture. Electronic Data Information
Source - UF/IFAS Extension,
http://edis.ifas.ufl.edu/fa208.
Yang H, X. Guo, and J. Scarpa. 2019. Induction andestablishment of tetraploid oyster breeding
stocks for triploid oyster production.
Electronic Data Information Source UF/IFAS Extension.
https://edis.ifas.ufl.edu/fa215.

An alternative method for tetraploid
production by using tetraploid founders
Complementarily, another novel approach has beenreported in the Pacific oyster using tetraploid
founders (McCombie et al. 2005) to produce viable
tetraploids by fertilizing oocytes from diploids with
sperm from tetraploids followed by inhibition of thepolar body 2 (PB2). This method offers analternative way for producing more tetraploids after
production of initial founders and avoids the use ofoocytes from triploid females, which is always a big
challenge. This is an approach to diversify, change,and enrich the genetic background of tetraploid
populations (50% from mother in the 1st generation,and probably increased to 100% from mother in thesecond generation, depending on the chromosome
segregation in sperm formation).

Updated reviews in extension-style
publications for oyster triploid-tetraploid
technology
These two publications summarized the history,
mechanisms, procedures, and updates about oyster
triploid-tetraploid technology. Different from otherjournal publications, these two publications were
written in extension style with glossaries asappendixes that are understandable for generalpublic and industry communities. Download ofthese two publications is free through the website
links stated above from the Electronic Data
Information Source, Institute of Food andAgricultural Sciences Extension, University of
Florida.

Guo, X., Y. Wang, Z. Xu, and H. Yang. 2009.
Chromosome Set Manipulation in Shellfish.
139

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