Preface
The UJNR (The U.S.-Japan Cooperative Program in Natural Resources) Aquaculture Panel was established in
1968, and the business meeting and symposium have been held every year since 1971. Through the long history
of UJNR, Aquaculture Panel has contributed to the development of aquaculture researches of both countries bymeans of various cooperative activities, such as the exchange of scientists and literatures, and the promotionof joint research projects. The Aquaculture Panel is highly appraised as one of the most active UJNR panels inboth countries.
The 47th business meeting of the UJNR Aquaculture Panel was conducted at Okinawa Industry Support
Center, Okinawa prefecture, Japan on November 12, 2019, and the scientific symposium was held at the same
venue from November 12 to 13. The symposium theme was “Marine aquaculture in a changing environment”
which was the 10th three-year plan commenced in 2017. As the final year of the three-year plan, we discussed
about application of aquaculture technology to provide sustainable seafood and reduce impacts of environmental
change. Seventeen oral presentations were made on topics such as new techniques to assess and mitigate
impacts of environmental changes on aquaculture, impact assessment of environmental changes andconservation of fisheries environment in coral reef areas, production management under environmental changes
in bivalve aquaculture, and aquaculture technologies to respond to environmental changes in seaweed breeding
and feed development. The proceedings of the 47th UJNR Aquaculture Panel scientific symposium “Marine
aquaculture in a changing environment” is published as the special issue of the Bulletin of Japan Fisheries
Research and Education Agency. With great pleasure, this UJNR proceedings containing high quality papers
authored by selected American and Japanese aquaculture scientists will hopefully help improve the aquaculture
environment programs, which is expected to contribute to the development of the aquaculture industry in boththe United States and Japan.
Finally, I would like to express my sincere gratitude to the colleagues involved in the UJNR Aquaculture
Panel for their efforts in the preparation and organization of the symposium. I would also like to deeply thankthe editorial board members for publishing the proceedings.

Hideaki Aono, Ph.D.
Chair of UJNR Aquaculture Panel
Executive Director
Japan Fisheries Research and Education Agency

Participants in 47th UJNR Aquaculture Panel Symposium, held in Okinawa Industry Support Center,
Okinawa, Japan, November 12-13, 2019

CONTENTS
Preface … ……………………………………………………………………………………………………………

1

Group Photo …………………………………………………………………………………………………………

2

Contents ………………………………………………………………………………………………………………

3

th

Program for The 47 Scientific Symposium of UJNR Aquaculture Panel… ………………………

5

Contributed Papers and Abstracts
1.

Bacterial community composition of the sediment in the coastal regions of Kashima-Nada andKujukurihama, Japan (Tomoko Sakami, and Toru Udagawa) … ………………………………………

2.

9

Applications of environmental DNA data in support of aquaculture (Thomas Noji, Daniel Wieczorek,
Beth Phelan, Yuan Liu, Lisa Milke, Renee Mercaldo-Allen, Julie Rose, Lauren Sassoubre, and BruceNash)… …………………………………………………………………………………………………………

3.

A stable isotopic approach to investigate nitrogen pathways in a coastal aquaculture area (Satoshi
Watanabe)… ……………………………………………………………………………………………………

4.

25

Immunological assays of hemocytes in molluscan bivalves as biomarkers to evaluate stresses foraquaculture (Huiping Yang) … ………………………………………………………………………………

5.

19

31

Preliminary feasibility assessment of purple sea urchin, Strongylocentrotus purpuratus, roeenhancement (Luke Gardner, Helaina Lindsey, Katherine Neylan, Walan Chang, Katrina Herrmann,
Max Rintoul, and Katherine Roy)… …………………………………………………………………………

6.

Potential impacts and management of ocean acidification on Japanese marine fisheries andaquaculture (Haruko Kurihara)… ……………………………………………………………………………

7.

55

Sustain seafood resources in the U.S. affiliated Pacific islands- status and strategies (Cheng-Sheng
Lee)… ……………………………………………………………………………………………………………

8.

47

67

Towards effective coral community restoration for sustainable fishery of a coral reef grouper
Epinepherus ongus: implications of ecosystem-based management (Atsushi Nanami) ……………… 75

9.

Scaling up coral restoration to meet the demands of a collapsing ecosystem (Tali Vardi) ………… 83

10.

Sustainable large-scale coral restoration by establishing “artificial spawning hotspots” (Go Suzuki) ……

11.

The influence of climate and environment on the growth and survival of Pacific oyster seed in US

91

West Coast estuaries (Brett Dumbauld, Evan Durland, Konstantin Divilov, Kelly Muething, Anna
Bolm, Ylva Durland, and Brooke Mcintyre)………………………………………………………………… 101
12.

Comparative study of the impact of environmental changes on oyster culture between USA andJapan, as collaborative research under UJNR (Natsuki Hasegawa, Brett R. Dumbauld, Masakazu
Hori, Satoshi Watanabe, Michael Rust, and Zachary Forster)…………………………………………… 115

13.

Oyster aquaculture using seagrass beds as a climate change countermeasure (Masakazu Hori,
Masami Hamaguchi, Masaaki Sato, Réjean Tremblay, Alana Correia-Martins, Valerie Derolez,
Marion Richard, and Franck Lagarde)……………………………………………………………………… 123

14.

Kelp, Saccharina spp, population genetics in New England, US, for guiding a breeding programof thermally resilient strains (Simona Augyte, Jean-Luc Jannink, Xiaowei Mao, Mao Huang, Kelly
Robbins, Matt Hare, Schery Umanzor, Michael Marty-Rivera, Yaoguang Li, Scott Lindell, DavidBailey, and Charles Yarish)…………………………………………………………………………………… 135
15.

Cell selection technique for establishment of low salinity tolerance strain in Pyropia tenuipedalis(Mahiko Abe, Tomomi Ohashira, Noboru Murase, and Masanobu Kishioka) ………………………… 141

16.

New diets with potential for enhancement of juvenile bivalve seed production and culture
techniques (Yasuhiro Yamasaki) … ………………………………………………………………………… 149

17.

Development of a sustainable diet for Japanese white trevally Pseudocaranx dentex juveniles (Jonas
Miller, Shuhei Tanaka, Hiroki Kihara, Shinichi Yamada, Fumiaki Takakuwa, Keitaro Kato, Amal
Biswas, and Hideki Tanaka)… ……………………………………………………………………………… 159

Program
The 47th Scientific Symposium of UJNR Aquaculture Panel
Marine Aquaculture in a Changing Environment
Date:
November 12

９：15 - 12：00

November 13

９：15 - 17：00

Venue:
Okinawa Industry Support Center，1831－１ Oroku，Naha，Okinawa

Aim of the Symposium
The UJNR Aquaculture Panel is a cooperative research exchange between the U.S. and Japan
jointly addressing environmental and technical issues that affect the aquaculture industries of bothnations.
The 47th UJNR Aquaculture Symposium is the final symposium of a 3-year cycle with the theme
Marine Aquaculture in a Changing Environment. Environmental changes impact aquaculture
in many ways. Nutrient pollution is driving eutrophication and dead zones; ocean acidification ischanging water chemistry, and climate change is already influencing our food supply, freshwater
availability, weather and way of life. Aquaculture will be impacted by, and can also impact, theseenvironmental changes over various scales. Aquaculture of finfish, shellfish and seaweed havedifferent threats, benefits and opportunities related to environmental change.
Over the last two years, we discussed the potential of aquaculture to mitigate impacts ofenvironmental change (e.g. carbon sequestration, bioextraction of nutrients and CO2, antacidity, andoxygen production), impacts of environmental change on aquaculture production (e.g. effects of ocean
acidification on shellfish aquaculture), and science to mitigate these impacts (countermeasures). Thisis the final year of the three year-plan, and the symposium theme is the Application of Aquaculture
Technology to Provide Sustainable Seafood and Reduce Impacts of Environmental Change. This
theme includes development of technology to increase marine aquaculture production to offset
seafood deficit due to loss of capture fisheries impacted by environmental change, and to augment
food deficits due to impacts of environmental change on inland agriculture.

Tuesday, November 12, 2019
Registration: ９：00 - ９：15
Opening Session

Welcome and Aim of the Symposium
Hideaki Aono (Japan Panel Chair, Japan Fisheries Research and Education Agency)………… ９：15 - ９：25

Session I. New techniques to assess and mitigate impacts of environmental changes onaquaculture(Moderators: Simona Augyte and Natsuki Hasegawa)1. Bacterial communities in marine sediments; a biological parameter to evaluate coastal environment
Tomoko Sakami (National Research Institute of Aquaculture, FRA)… ………………… ９：25 - ９：55
2. Applications of environmental DNA data in support of aquaculture
Tom Noji (Northeast Fisheries Science Center, NOAA Fisheries)… …………………… ９：55 - 10：25
3. Stable isotopic approach to investigate nitrogen pathway in coastal aquaculture area
Satoshi Watanabe (National Research Institute of Aquaculture, FRA)… ……………… 10：25 - 10：55
4.	Establishment of immunological assays and baseline profile of hemocytes in the hard clam Mercenaria
mercenaria as evaluation biomarkers for environmental stresses
Huiping Yang (School of Forest Resources and Conservation, Institute of Food and Agricultural
Sciences, University of Florida)… …………………………………………………………… 10：55 - 11：25
5.	Purple urchin barrens: an opportunity for aquaculture and fisheries to work together to solve anenvironmental issue
Luke Gardner (California Sea Grant)… ……………………………………………………… 11：25 - 11：55

Wednesday, November 13, 2019
Session II. Impact assessment of environmental changes and conservation of fisheries
environment in coral reef areas(Moderators: Luke Gardner and Masakazu Hori)
6. Impacts of ocean acidification on Japan coastal water and marine fisheries
Haruko Kurihara (Invited speaker)(University of the Ryukyus)… ……………………… ９：15 - ９：45
7. Sustain seafood resources in the U.S. affiliated Pacific islands- status and strategies
Cheng-Sheng Lee (Center for Tropical and Subtropical Aquaculture, United States Department ofAgriculture)……………………………………………………………………………………… ９：45 - 10：15
8.	T owards effective coral community restoration for sustainable fishery of a coral reef grouper
Epinepherus ongus: implication of ecosystem-based management
Atsushi Nanami (Seikai National Fisheries Research Institute, FRA)…………………… 10：15 - 10：45
9. Scaling up coral restoration to meet the demands of a collapsing ecosystem
Tali Vardi (ECS for NOAA Fisheries Office of Science ＆ Technology)………………… 10：45 - 11：15
10. Sustainable large-scale coral restoration by establishing “artificial spawning hotspot”
Go Suzuki (Seikai National Fisheries Research Institute, FRA)…………………………… 11：15 - 11：45
Lunch Break…………………………………………………………………………………………… 11：45 - 13：00

Session III. Production management under environmental changes in bivalve aquaculture(Moderators: Cheng-Sheng Lee and Atsushi Nanami)
11.	The influence of climate and environment on the growth and survival of Pacific oyster seed in US
West Coast estuaries
Brett Dumbauld (Agricultural Research Service, United States Department of Agriculture)…………………………………………………………………………………………… 13：00 - 13：30
12.	Comparative study of the impact of environmental changes on oyster culture between USA andJapan, as collaborative research under UJNR
Natsuki Hasegawa (Hokkaido National Fisheries Research Institute, FRA)… ………… 13：30 - 14：00
13. Oyster aquaculture using seagrass beds as a climate change countermeasure
Masakazu Hori (National Research Institute of Fisheries and Environment of Inland Sea, FRA)…………………………………………………………………………………………… 14：00 - 14：30
Coffee Break…………………………………………………………………………………………… 14：30 - 14：45

Session IV. Aquaculture technologies to respond to environmental changes; Seaweed
breeding and feed development(Moderators: Brett Dumbauld and Satoshi Watanabe)
14.	Kelp, Saccharina spp, population genetics in the Northwest Atlantic for guiding a breeding program ofthermally resilient strains
Simona Augyte (Dept. of Ecology and Evolutionary Biology, University of Connecticut)… 14：45 - 15：15
15. Cell selection technique for establishment of low salinity tolerance strain in Pyropia tenuipedalis
Mahiko Abe (Applied Aquabiology, National Fisheries University, FRA)……………… 15：15 - 15：45
16.	Improvement of dietary effect on juvenile Ruditapes philippinarum using the dietary-supplements andnew diet microalga
Yasuhiro Yamasaki (Applied Aquabiology, National Fisheries University, FRA)……… 15：45 - 16：15
17.	Exploration of alternative protein sources in the development of a sustainable Japanese white trevally
Pseudocaranx dentex juvenile diet
Jonas Miller (Uragami Station, Aquaculture Research Institute, Kindai University)…

16：15 - 16：45

Science Symposium Closing
Michael Rust (US Panel Chair, NOAA Fisheries Office of Aquaculture)………………………… 16：45 - 17：00

水 研 機 構 研 報， 第 50 号，9 － 17， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，9－17，2021

9

Bacterial community composition of the sediment in the coastal regionsof Kashima-Nada and Kujukurihama, Japan

Tomoko SAKAMI＊1 and Toru UDAGAWA＊2
Abstract: Environmental monitoring of marine coastal areas is becoming increasingly importantbecause of accelerated global climate change and rapid industrial development. The composition of
the bacterial community in bottom sediments is strongly related to the surrounding environment.
Thus, surface sediments were collected at the coastal regions of Kashima-Nada and Kujukurihama
to elucidate how bacterial communities vary where the confluence of the cold Oyashio and warm
Kuroshio Currents occur. The bacterial communities collected at water depths of 10 m weredifferent between the Kashima-Nada and Kujukurihama areas, but were not different at water
depths of 30 m. The chlorophyll a content in the sediment and bottom water temperature wereindicated as the major environmental parameters influencing the composition of the bacterial
communities. At 10 m depth in the Kujukurihama area, operational taxonomic units (OTUs) ofBacteroides, which are known as high molecular weight organic matter degraders, were abundant,
whereas OTUs of Nitrospinae and Nitrosospira, which are related to the nitrification process, wereless abundant. The taxonomic groups characteristic of the sediment seemed to be related to theproperties of organic matter quality in the sediment.
Key words: bacterial community composition, sediment quality, Kashima-Nada, Kujukurihama

Introduction

monitoring physical and chemical parameters is
not enough to evaluate integrated environmental

Terrestrial materials such as soil, inorganic

conditions for living organisms. As a biologicalnutrients, and organic matter are discharged into

parameter, macrobenthos fauna are often examined

the sea through river water flow or by land erosion,in order to monitor coastal environmental conditions.

with considerable consequences for the coastal

This requires time and labor, and the amount ofenvironment (Bergamaschi et al., 2012). In addition

data is less than what is obtained by physical andto industrial development on land, global climate

chemical monitoring. In contrast, approximatelychange, which is predicted to bring about increases

one hundred million bacterial cells can be observedin torrential rain and storms (Yagi et al., 2015), willin a single gram of marine sediment (Schmidt etchange material inputs from land to coastal areasal., 1998). Data collection for bacterial community(Philippart et al., 2011; Viitasalo et al., 2015). To deal

composition analysis is a mechanical processwith prospective environmental changes, we need tothat augments physical and chemical parametermonitor certain environmental parameters precisely.

measurement. Typically, millions of bacterial

Using physical and chemical monitoring techniques,sequences can be retrieved from one sample, and

it is now possible to obtain highly accurate data

they can be clustered into hundreds or thousands

owing to advances in measuring equipment andof species. Therefore, it may be possible to detect

information technology (Bean et al., 2017). However,certain integrated environmental changes which
2020年12月11日受理（Accepted on December 11, 2020）＊1
National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 422-1 Nakatsuhamaura, Minami-ise, Mie, 5160193, Japan＊2
National Research Institute of Fisheries Engineering, Japan Fisheries Research and Education Agency, 7620-7 Hasaki, Kamisu, Ibaraki, 3140408, Japan
E-mail: sakami “at” affrc.go.jp

10

Tomoko SAKAMI and Toru UDAGAWA

cannot be detected by physical and chemical

and the exact dimensions of this band depend also

parameters alone.

on the year of the survey (Shimizu, 2001). Bacterial

Bacterial community composition of coastal bottom

communities in sediments may change in different

sediments is more diverse than that of seawater

ways from the larger organisms and may help

(Zinger et al., 2011). Community compositions are

elucidate bottom environmental conditions in more

often related to environmental conditions such asdetail.

the particle size of the sediment, and the dissolved

We examined bacterial community composition

oxygen concentration in the bottom water (Zheng

in the sediments collected at Kashima-Nada andet al., 2014), or the organic matter content in the

Kujukurihama by using 16S ribosomal RNA (rRNA)

sediment (Qiao et al., 2018). Salinity change in thegene sequences. By analyzing relationships between

brackish water area (Vidal-Durà et al., 2018) andthe community composition and sediment quality

anthropogenic contamination, such as metal andparameters, such as organic matter content andpolycyclic aromatic hydrocarbon contaminants

mud content, we aimed to clarify how the bacterial

from land (Sun et al., 2013), can also affect sedimentcommunity varies in this area, and how the sedimentbacterial communities. In land soil, bacterial

qualities influence or reflect these variations.

communities are more affected by the biochemical
Materials and Methods

conditions of the soil, such as organic matter content,
than by the origin or geographic location of thesoil (Lin et al., 2019). In marine bottom sediments,

Sample collection

however, it is not clear what difference the sea area

Sediment samples were collected at three sites

makes. Factors such as the geographical distanceadjacent to the shorelines at a water depth of 10 mfrom land, or overlying seawater characteristics

and 30 m, at the Kashima-Nada and Kujukurihama(the current type), may affect bacterial communities

beaches in July, August, and September 2017 (Fig.

together with the bottom environmental parameters1). The annual water temperatures ranged from 25℃

such as particle size or organic matter content.

(highest; range: 23-27℃) to 12.5℃ (lowest; range:

Kashima-Nada and Kujukurihama are open, large,

9-14℃) at Kashima-Nada, and from 26℃ (highest;

sandy beaches, which are located at the northernrange: 25-27℃) to 15℃ (lowest; range: 12-16℃) at

and southern parts of Cape Inubo, respectively, on

Kujukurihama area between 2014 and 2018 (Tokyo

the Pacific coast of Japan. In these areas, primaryDistrict Meteorological Observatory). A Smith-production is high, and there are high fisherycatches of bivalves and whitebait (Okunishi et al.,2000). The cold Oyashio Current and the warm
Kuroshio Current run along the Pacific coast of

140°E

141°E

142°E

Japan, and they meet at a confluence point, off
Pacific
Ocean

Cape Inubo. It is well known that the biologicalcommunity of the Pacific coast of Japan changes,
with Cape Inubo marking the border of the northernand southern fauna and flora (Shimizu, 2001). In

36°N

addition, the Tone river mouth, which is the largest

Cape Inubo

river in Japan, is north of Cape Inubo. Together

Kujukurihama

with the large amount of river water discharge andthe high wind in this area, a strong tidal stream

Kashima-Nada

35°N

occurs around Cape Inubo, and complex waterexchange and sediment transportation occurs (Yagiet al., 2002; Arai et al., 2006; Uzaki et al., 2016). The
distribution patterns of macrobenthos fauna vary
within a certain band north and south of Cape Inubo,

Fig. 1. Sample collection points at the coastal regionsof Kashima-Nada and Kujukurihama on the PacificFig. 1.Japan.
SampleOpencollection
pointsat the coastal
regionsandofcoast,
symbols
indicate
10 m depth,
Kashima-Nada
andKujyukurihama
onthePacific
coast,
solid symbols indicate 30 m depth.
Japan. Open symbols indicate 10 m depth, and solid
symbols indicate 30 m depth.

Bacterial community composition in coastal sediments

11

McIntyre grab sediment sampler (1/20 m2, RIGO,

taxonomic annotation using the Mothur Miseq

Osaka, Japan) was used to collect sediment samples.

standard operating procedure (SOP) (Kozich et al.,A surface sediment sample (top 2 cm) was collected2013). Inverse Simpson diversity index was alsousing a plastic corer of 23 mm diameter, and theestimated using this procedure.

sample was frozen immediately in a sterilized plastic

The analyses below were done using the statisticalbag. The sediment samples were kept at -60℃

package R version 3.5.3 (R Core Team, 2018).

until further processing. Temperature and salinity

Detrended correspondence analysis was performed

were measured using a multi-parameter handy

and produced an axis length of less than four,

conductivity temperature depth profiler (RINKO

indicating a linear relationship between community

Profiler, ASTD152, JFE Advantech, Nishinomiya,composition and the examined environmental

Japan).

parameters: temperature, salinity, median particle
diameter, mud content, ignition loss, and chlorophyll

Sediment quality analysis

a content. Therefore, relationships of the bacterial

The following variables were analyzed: (i) particle

community composition across the sampling sites

size was determined using a laser diffraction particle

were examined using non-metric multi-dimensional

size analyzer (SALD-3100; Shimadzu, Kyoto, Japan),scaling (NMDS) analysis, and relationships of the(ii) ignition loss was evaluated by igniting thebacterial community composition and environmental

sediment samples at 550℃ for 12 h, (iii) chlorophyllparameters were examined using redundancy

a content was determined by fluorescence using aanalysis (RDA). Relative abundance of subphyla

spectrophotometer (10AU, Turner Designs, San Jose,of the top three phyla, namely Proteobacteria,

USA).

Bacteroidetes, and Firmicutes, and phyla whose
relative abundance was more than 0.001 wereDNA extraction and sequencing

compared among four sampling areas: Kashima-

DNA was extracted from a 0.4 to 0.6 g sedimentNada-10 m, Kashima-Nada-30 m, Kujukurihama-10

sample using the Fast DNA SPIN Kit for soil (MPm and Kujukurihama-30 m. A heatmap figure was

Biomedicals LLC., Ohio, USA) according to themade using the 16 taxonomic groups that were

manufacturer’s instructions. Barcodes were attachedsignificantly different among the four areas (ANOVA,

to DNA fragments in a 2-step PCR amplificationp < 0.05).

that targeted the variable V1/V2 region of the 16S
rRNA gene based on the protocol of Sakami et al.

Results(2016). First PCR was conducted using 27Fm and338R primers (Kim et al., 2013), then the sequences

Environmental parameters

were modified by adding 34 and 36 bp snippets,

Median particle diameter (d50) was larger in therespectively, in order to later add molecular

Kashima-Nada-30 m, and mud content was higher

identifier (MID) tags. Pooled amplicons were paired-in Kujukurihama-30 m. Ignition loss was higher

end sequenced (2×300 bp) on the Miseq device

at Kujukurihama at a depth of 10 m and lower at(Illumina, Tokyo, Japan).

Kashima-Nada at a depth of 30 m. Chlorophyll acontent was higher at a depth of 30 m in both areas.

Bacterial community analysis
The sequences obtained by extraction wereBacterial community composition

quality filtered, and chimeras were removed using

The number of sequences obtained from sediment

the UCHIME algorithm (Edger et al., 2011) withprofiles ranged from 60,000 to 138,000 per sample.

alignment to the SILVA database (SSU_Ref_NR

The community coverage was estimated to bever. 119) reference alignment. Sequences that

more than 0.999 for all samples. The sequences

passed through the filtering process were divided

were clustered into 803 operational taxonomic units

into operational taxonomic units (OTUs) using a(OTUs). Community diversity (Inverse Simpson

3% similarity threshold and were identified withindex) ranged from 5.8 to 11.4, and it tended to be12

Table 1. Average values of environmental parameters andbacterial community
diversity at the sampling areas
Tomoko SAKAMI and Toru UDAGAWA

Table 1. Average values of environmental parameters and bacterial community diversity at the sampling areas

Kashima-Nada-10m
Kashima-Nada-30m
Kujukurihama-10m
Kujukurihama-30m

Central particle
diameter(um)

MudContent(%)

Ignition
Loss(%)

Chlorophyl acontent(ug/cm2)

Invsimpson
index

33.7b

187 b

0.18 b

1.4 bc56.3 b

6.8b

17.0b

33.8 b239 a0.05 b

1.4 c

203 a

7.4ab

21.6a

34.0 ab166 b0.36 b2.1 a88.8 b8.4ab

19.3ab34.3 a160 b0.99 a

1.8 ab

167 a

8.9a

Temp

Salinity

(°C)

(psu)

20.7ab* Numbers with different superscripts are different at the significance level of p < 0.05.
100%
90%

* Numbers with different superscripts are different Acidobacteria
at theActinobacteria
significance level of p < 0.05..
Bacteroidetes

80%
70%
60%
50%
40%
30%
20%

Kashima
10m

Kujukuri30m

JK1030SK1030
JK0830
AK0830SK0830SK0330

Kujukuri
10m

JS0930SS0930JS0630AS0630SS0630JS0330SS0330
JInu30SInu30JK1010SK1010
JK0810AK0810SK0810
JK0310AK0310SK03100%JS0910AS0910SS0910SS0610JS0610AS0610SS0310JS0310

10%

系列21
系列20
系列19
Chloroflexi
系列18
Cyanobacteria/Chloroplast
系列17
Firmicutes
系列16
Fusobacteria
系列15
Ignavibacteriae
系列14Latescibacteria
系列13
Nitrospinae
系列12Planctomycetes
系列11
Alphaproteobacteria
系列10
Betaproteobacteria
系列9Deltaproteobacteria
系列8
Epsilonproteobacteria
系列7Gammaproteobacteria
系列6Proteobacteria_unclassified
系列5
Spirochaetes
系列4Verrucomicrobia
系列3
etc
系列2
Bacteria_unclassified
系列1

Kashima
30m

Fig. 2. Bacterial community composition in the surface sediment collected at 10 and 30 m depths off the coastal
regions of Kashima-Nada and Kujukurihama in July, August and September, 2017.
0.3 the surface sedimenthigh at Kujukurihama(Table 1). Of
the OTUs, composition
33%
Fig. 2. Bacterial
communityin

to 53% belonged
to the phylum,collected
at 10 orProteobacteria,
30 m depths andoff the coastal
regions of Kashima0.2

the major subphyla
wereGammaproteobacteria
Nada andKujyukurihama
in July, August, and September of 2017.
(21%–30%) and Deltaproteobacteria (5%–21%).
Actinobacteria (3%–9%) and Acidobacteria (2%–6%)(Fig. 2).
The community composition of each sample wasplotted in two dimensions following NMDS (nonmetric multi-dimensional scaling) analysis (Fig. 3).
Bacterial communities at a depth of 30 m at both
Kashima-Nada and Kujukurihama were plotted
together in the top area of the graph. Bacterial
communities at a depth of 10 m at Kashima-Nada
were plotted below the 30 m communities, and those
off Kujukurihama were plotted in the bottom area of
the graph.

NMDS 2Other major phyla were Bacteroidetes (7%–33%),

0.1
0

-0.1
-0.2
-0.3

-0.7

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

NMDS 1Non-metric multi-dimensional
scaling plotof sedimentNon-metricmulti-dimensional
scaling
plot ofFig.Fig.
3. 3.
bacterial bacterial
communitiescommunities
with environmental
parameters.
Black
sediment
withenvironmental
symbols indicate the Kujyukurihama area, and gray symbols
parameters.
Black symbols
indicate
Kujukurihama
indicate the Kashima-Nadaarea. Opensymbols
indicate a depth of
10 andm andgraysolid symbols
indicate
30 m depth.
area,
symbols
indicate
Kashima-Nada area.
Open symbols indicate a depth of 10 m and solid
symbols indicate 30 m depth.

Bacterial community composition in coastal sediments

13

The relationships between the composition

the 10 m depth at Kashima-Nada, and the 10 mof the bacterial community and environmental

depth at Kujukurihama from the right. The Y-axis

parameters were examined using redundancy

represented 4% of the community variation. Bacterial

analysis (RDA) (Fig. 4). The X-axis represented 54%

communities at a depth of 10 m at Kujukurihamaof the community variation. Bacterial communities

were placed on this axis. Large directional arrows of

were placed in order of the areas of 30 m depth,

chlorophyll a content and water temperature were

RDA2-Percent variations explained 4.0％

along the X-axis, implying that these parameters
were strongly related to variation in bacterial

150

community composition. Small directional arrows

100

for mud content, salinity, and ignition loss were on

50

Temp

d50

the lower right. No obvious directional arrows were

Chla

Mud

0

observed along the Y-axis.

Sal

-50

The relative abundance of the major taxonomicILgroups, whose abundance was different among the

-100
-150

four sea areas, was shown in a heat map (Fig. 5).

-200

The taxonomic groups were clustered into two-250groups (cluster I and II), with the first cluster
-120

-100

-80

-60

-40

-20

0

20

40

60

80

100

RDA1-Percent variations explained 54％

divided further into three subgroups (cluster

Ia, Ib, Ic). Cluster Ia included Acidobacteria andFig.
4.
Redundancy
analysis(RDA)ofsediment
Proteobacteria, except for beta and epsilon types,
Fig. 4. Redundancy analysis (RDA) of sediment bacterial communities withenvironmental
symbolsb a c tparameters.
e r i a l c oBlackmmu
n i t i eindicates w i t theh Kujyukurihama
e n v i r o n m earea,n t aandl gray and abundance tended to be low at a depth ofsymbols indicate the Kashima-Nada area. Open symbols indicate 10 m depth, and solidparameters. Black symbols indicate Kujukurihama
symbols indicate 30 m depth. IL, Chl a, d50, and mud represent ignition loss,
10 m off the Kujukurihama coast. Cluster Ib
area,and mediangray particle
symbols
indicate
Kashima-Nada
chlorophyll
a content,
size, andmud content,
respectively.
included Chloroflexi and Ignavibacteriae, and thearea. Open symbols indicate 10 m depth, and solid
abundances tended to be low at a depth of 30 m
symbols indicate 30 m depth. IL, Chl a, d50, and mud
at Kashima-Nada and at Kujukurihama. Cluster
represent ignition loss, chlorophyll a content, median
Ic included Betaproteobacteria, Planctomycetes,

particle size, and mud content, respectively.

KashimaNada
10m

Kujukurihama
30m

KashimaNada
30m

JS0910AS0910SS0910SS0610JS0610AS0610SS0310JS0310JK1010SK1010
JK0810AK0810SK0810
JK0310AK0310SK0310JS0930SS0930JS0630AS0630SS0630JS0330SS0330
JInu30SInu30
JK1030SK1030
JK0830
AK0830SK0830SK0330

Kujukurihama
10m

Alphaproteobacteria
Proteobacteria unclassified
Gammaproteobacteria
Cluster Ia
Deltaproteobacteria
Acidobacteria
Chloroflexi
Cluster Ib
Ignavibacteriae
Betaproteobacteria
Planctomycetes
Clostridia
Cluster Ic
Spirochaetes
Nitrospinae
Sphingobacteriia
Verrucomicrobia
Cluster II
Flavobacteriia
other Bacteroidetes

Fig. 5. Hierarchically clustered heatmap of bacterial communities from the four areas at the phylum orsubphylum level. Rows represent the relative abundance of each bacterial group, and columns indicate different
sediment samples. The relative abundance of each bacterial taxon is depicted by color intensity with the legend
indicated at the top of the figure.

Fig. 5. Hierarchically clustered heatmap of bacterial communities
from the four areas at the phylum or subphylum level. Rows
represent the relative abundance of each bacterial group, andcolumns indicate different sediment samples. The relative
abundance of each bacterial taxon is depicted by color intensity

Tomoko SAKAMI and Toru UDAGAWA

Relative abundance

14

0.016

Nitrosospira OTU

bacterial community composition was different
between Kashima-Nada and Kujukurihama at depthsof 10 m, but no obvious difference was observed

0.012

at 30 m. The water depth, chlorophyll a content,
and mud content was related to the variation in

0.008

bacterial community composition in addition to thewater temperature, indicating that the bacterial

0.004

communities present in the examined areas0

environment rather than with the differences invaried primarily with the conditions of the bottom

KJ- KS- KJ- KS10m 10m 30m 30m

current. On land soil, bacterial community structureand diversity were shown to be affected moreby parent material differences or biogeographicalFig. 6. Relative abundance of a Nitrosospira OTU in
separation of the soil than the soil properties, such as
the four areas. KJ indicates Kujukurihama and KS
Fig. 6. Relative
abundance of a Nitrosospira OTU in theorganic
four matter content (Lin et al., 2019). In marine
indicates Kashima-Nada.
ecosystems, there is evidence that the benthic

areas. KJ indicates the Kujyukurihama and KS indicatesbacterial
the community composition is affected more
Kashima-Nada.
Clostridia, Spirochetes, and Nitrospinae, and theby the geographic location than the ecosystem typeabundance tended to be low at 10 m depths in theand time or upper water productivity (Zinger et al.,Kujukurihama area. Relative abundance of an OTU2011). Since the bacterial community compositions

affiliated to genus Nitrosospira accounted for 73% ofwere affected by sediment qualities rather thanbetaproteobacterial OTUs. It was low at a depth of

the geographical distance, we suggest that there

10 m at Kujukurihama (Fig. 6). Cluster II included

are considerable exchanges of sediment materials

Flavobacteriia, Sphingobacteriia, other Bacteroides,

between the northern and the southern areas ofand Verrucomicrobia, and the abundance tended toCape Inubo.

be high at 10 m depth in the Kujukurihama area.

It has been shown that bacterial community
composition in marine bottom sediments changes

Discussion

according to the primary production in the sea(Zinger et al., 2011). The Tone River water

It is well known that the fauna and flora change

discharge to the north side of Cape Inubo, increases

at the border of the northern and southern junction

primary productivity in both the Kashima-Nada andat Cape Inubo, on the Pacific coast of Japan. For

Kujukurihama areas, and the influence fluctuates

example, the distribution of marine bivalves andwidely with precipitation and wind conditions

snails changes from tropical to cold-water varieties(Arai et al., 2006). The primary productivity in theat the border of the Boso Peninsula (Shimizu, 2001).

Kashima-Nada area was estimated to be 0.4 g C/

The warm Kuroshio Current which runs along them /day, and 76% of the production was estimated towestern Japanese Pacific coast changes direction

depend on the regeneration of the nutrient due toto the east at Cape Inubo, leaving the main islandorganic matter degradation within the area (Okunishi

of Japan. Therefore, the Kujukurihama area is

et al., 2000). Although primary production and a

influenced by the warm Kuroshio Current. In

contribution from regeneration production were notcontrast, a confluence of the warm Kuroshio and theindicated at the Kujukurihama area, the taxonomic

cold Oyashio Currents occurs at the northern end offeatures observed in the bacterial community

Cape Inubo, the Kashima-Nada area. Moreover, thecomposition may indicate different biologicalfour examined areas, viz. the 10 m and 30 m depths

production processes between the two areas.

2

at the Kashima-Nada and Kujukurihama sites, had

Concerning the abundance of bacterial

different bottom environments as indicated by theirtaxonomic groups in the four sea areas, cluster IIparticle size and organic matter content data. The(which was abundant at 10 m at Kujukurihama)Bacterial community composition in coastal sediments

15

Acknowledgements

included Flavobacteriia, Sphingobacteriia andVerrucomicrobia, which belong to Bacteroidetes.
Bacteroidetes are generally considered to be

The authors greatly thank Dr. Hiroshi Yagi of thespecialists in the degradation of high molecular

National Defense Academy for fruitful discussions

weight organic matter (Thomas et al., 2011). The

about coastal environment management and thetotal organic matter (ignition loss) was high, anddevelopment of new monitoring techniques. The

the chlorophyll a content was low at the depth

authors also thank the crew of the R.V. Taka-Maruof 10 m at Kujukurihama, suggesting that thefor their support during sample collection. This work

sedimentary organic matter was relatively poor in

was supported by JSPS KAKENHI Grant Number

fresh material. Flavobacteriia are often observed

JP17H03317.

abundantly at the end of phytoplankton blooms
References(Teeling et al., 2012; Sakami et al., 2016). They may
degrade not only fresh phytoplankton materials,
but also labile, high molecular weight organic

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matter, which is deposited in the sediment. AmongT., and Yagi H., 2006: Effect of Tone and Naka

Bacteroidetes, Cytophagaceae members oftenRiver on Primary Production in the Coastal

digest macromolecules such as polysaccharides

Region of Kasima-Nada and Kujukurihama. Ann.

and proteins (McBride et al., 2014), but their

J. Coast. Engineer., JSCE, 53, 1101-1105. (inabundance did not differ among the four areas

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Bean T. P., Greenwood N., Beckett R., Biermann

Verrucomicrobia are also reported to be candidates

L., Bignell J. P., Brant J. L., Copp G. H., Devlin

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M. J., Dye S., Feist S. W., Fernand L., Foden

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less abundant in the shallow area off Kujukurihama,

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Combining Historic and Contemporary Methods

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水 研 機 構 研 報， 第 50 号，19 － 24， 令 和 ３ 年
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19

Applications of environmental DNA data in support of aquaculture
Thomas NOJI＊1, Daniel WIECZOREK＊1, Beth PHELAN＊1, Yuan LIU＊2, Lisa MILKE＊2,
Renee MERCALDO-ALLEN＊2, Julie ROSE＊2, Lauren SASSOUBRE＊3, and Bruce NASH＊4


Abstract: With advances in analytical and computational technologies the data for environmental
DNA or eDNA are becoming rapidly and increasingly available. eDNA data have been applied
successfully to assess presence of fish species, impacts of human activities on benthic biota andmore recently to a limited extent to assess biomass. Because of the relative ease with which eDNA
data can be collected, the number of proposed applications is increasing rapidly; this includes
applications to support aquaculture. Some of the applications of particular interest for aquaculture
operations include measurement of eDNA as a surveillance tool for pathogens, protected
species and escapees from e.g. net pens; as an indicator of benthic impacts and efficacy of stockenhancement operations; and health of cultured species.
Key words: environmental DNA, genetics, aquaculture, seafood, production
Introduction

leading global importer of fish and fishery products;
90% of USA seafood is imported and half of this is

Environmental DNA or eDNA is collected fromfrom aquaculture.

the environment and not directly from organisms

Not surprisingly there is now a strong expectation

larger than about half a micrometer in size, asthat marine aquaculture will be a significant factor

determined by pore size of filters used. The

in increasing the USA’s seafood productivity,

collection, extraction and analysis of eDNA havethereby reducing the USA’s reliance upon import ofbecome a popular method in recent years. This

seafood and helping to assure food security for thewas largely made possible by the development of

nation. Toward this end, multiple applications exist

technologies to analyze the genetic sequences offor eDNA data to support aquaculture to increase

DNA and to the high-powered computing systems

seafood production in an environmentally friendly

easing the analysis of the large data sets acquired.

and sustainable manner. This short paper is a

Aquaculture in Japan is a well-established andsummary of a presentation at the US-Japan Bilateralthriving industry with an annual production in

Meeting in Okinawa, Japan, in 2019 and gives a brief

2017 of about 700 thousand tons of freshwater andoverview of some of those applications.

marine animal products; most of this is seafood. In
Japan another approximately 500 thousand tons of

Why environmental DNA?

aquatic plants are also produced annually (http://
www.fao.org/fishery/countrysector/naso_japan/

There are several benefits, which make eDNAen). In contrast in the USA freshwater and marine

a promising research tool. Firstly, the sampling isaquaculture produced only 270 and 41 thousand

non-invasive or only minimally invasive. If you cantons, respectively, in 2017 and ranked 17th in global

collect water, you can collect eDNA, e.g. from rivers,

aquaculture production. Ironically the USA is thelakes, bays, surface and deep-sea water, and pore
2020年12月11日受理（Accepted on December 11, 2020）＊1
Northeast Fisheries Science Center, NOAA Fisheries, Sandy Hook, NJ 07732, USA.
＊2
Northeast Fisheries Science Center, NOAA Fisheries, Milford, CT 06460, USA.
＊3
School of Engineering and Applied Sciences, University of Buffalo, Buffalo NY, 14260, USA＊4
DNA Learning Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
E-mail: thomas.noji “at” noaa.gov

20

Thomas NOJI, Daniel WIECZOREK, Beth PHELAN, Yuan LIU, Lisa MILKE, Renee MERCALDO-ALLEN,
Julie ROSE, Lauren SASSOUBRE, Bruce NASH

water in sediments. Further the set of eDNA datacomprises a comprehensive overview of all livingorganisms which contributed DNA into the sampled
volume of water in the recent past (generally up to afew days). Moreover, the methodologies for filtering
the water, extracting the DNA, amplifying it, andsequencing the genetic material have advanced tothe point that eDNA measurements are routine andtaught even in high school courses. That said, there
is continual (a) technological development to improve
the sequencing and amplification of genetic material,(b) increasingly high powered computing systems
to analyze millions of data points into Operational
Taxonomic Units or OTUs, and (c) expanding
Fig. 1.

Fig. 1. Conceptual model of eDNA distribution in
the water column. From Sassoubre et al. (2016).

Quantitative polymerase chain reaction or qPCR

requirements. Whether a decay rate of hake DNA

can perform species-specific analyses on eDNA

is 2 days or 4 days is probably not significant for

to provide quantitative findings of the target

assessing distribution in the middle of the Pacificspecies. In contrast, next generation barcoding

Ocean, but that same difference can be extremelyor metabarcoding provides an estimate of theimportant for estimating distribution of Atlantic

relative abundance of the suite of organismal DNA

salmon in a river in Maine or distribution ofcollected on your filter. One of the major questions

aquaculture escapees in an embayment.

reference libraries to correlate OTUs with species.

for researchers today is whether metabarcoding

Conceptually, to fully understand eDNA dataof eDNA can be used for quantitative species

from the field surveys, one would need to accountassessments. Another question is whether eDNA canfor all the processes affecting eDNA distribution as

be differentiated between different life-history stages

shown Fig. 1, including shedding, enzymatic andof the same species. Answering these questions

other types of degradation, and physical processes

would significantly add to the value of eDNA as asuch as advection, dispersion and export forfield survey tool. Still even with limitations to using

example as feces. Realistically, we are likely to makeeDNA for biomass and aging estimates in the field,

faster progress in using eDNA as a survey tool

there are multiple ways in which eDNA can and isby comparing field eDNA with trawl and acoustic

used to support fisheries and aquaculture.

survey data, and using the laboratory experiments
to help in interpretation.

Challenges of Interpreting eDNA Data
Ecosystem Services
As illustrated in Fig. 1, the quantity of eDNA inseawater is affected by species properties including

Environmental DNA is often used to identify

size, age, behavior such as spawning; as well as

presence or absence of species. This can be e.g.

environmental parameters such as temperature,

an indicator of ecosystems services such as preysalinity, and UV light. Understanding the processes

species’ refugia or the impact of aquaculture on

affecting your sample is critical to interpreting yourfinfish diversity. In Milford, CT, USA, a team of

data. Further, being aware of the biases inherent

researchers from the NOAA Milford laboratory

in the amplification process, i.e. some genetic

has been measuring eDNA along with use of

sequences amplify more efficiently than others, isunderwater video to evaluate the effect of caged

important (Kelly et al., 2019). The extent to which

oyster on fish diversity (Liu et al., 2019). In thethese uncertainties are significant for your research

first year of investigation they identified 23 species

depends much upon temporal and spatial scalingfrom a total of 49 samples or 20 million reads from

Environmental DNA and aquaculture

21

Fig. 2.
Fig. 2. Environmental DNA as a tool to assess spatiotemporal finfish distribution in relation to shellfish
aquaculture operations. From Liu et al. (2019).
the sequencing data (Fig. 2). Notably, the species

benthic community composition. What is necessary

identified by eDNA overlapped but were not

to make eDNA a more useful quantitative survey

identical to the species identified with video, thereby

tool?

leading researchers to conclude that employing

As illustrated in Fig. 1, the quantity of eDNA

both methods concurrently would give a morein seawater is affected by species-specific rates ofcomprehensive result.

metabolism and physiology as well as environmental
parameters. In collaboration with the Cold Spring

When is Quantity Important?

Harbor Laboratory, University of Buffalo, Stanfordand Monmouth Universities, NOAA scientists

Of course scientists often want to know more

conducted laboratory experiments in a closed

than presence – absence of species in the field, andrecirculating system at Sandy Hook, NJ, USA to

being able to estimate quantity even if in relative

determine species-specific shedding and decay rates

terms can be important. This is true, for example,of eDNA under different environmental conditions.

for detection of pathogens in relation to critical

To date we have run trials with adult black sea

thresholds for human consumption or permissible

bass, juvenile winter flounder, and currently adulttransport, estimating the frequency and magnitude

summer flounder. Some results from the black sea

of encounters of protected species with aquaculture

bass run in Fig. 3 show eDNA in equilibrium andgear, estimating the effect of aquaculture on the

eDNA degradation. Most eDNA degraded withinfood web, or estimating the effect of aquaculture on

about two days.

22

Thomas NOJI, Daniel WIECZOREK, Beth PHELAN, Yuan LIU, Lisa MILKE, Renee MERCALDO-ALLEN,
Julie ROSE, Lauren SASSOUBRE, Bruce NASH

107

Black Sea Bass System 1〇 Below Limit of Quantification
× Non-detect106

copies per liter

105

103
102

Used tocalculate theshedding rate

Environmental DNA can be an important tool forbenthic surveillance to assess habitat preferences
cultured fish on sediments and associated biota.
There are clear correlations reported for changes
in OTUs and species as measured using eDNA

101

in relation to input of organic waste, e.g. from100

aquaculture, to sediments (Keeley et al., 2019). This10-1

has also been explicitly demonstrated for penned
0Fig. 3.

from small vessels.

(Takahara et al., 2019) as well as impacts of caged

Used to calculate thedecay rate

104

construct a midwater eDNA sampler for deployment

50

100

Time (hours)

150

Fig. 3. Quantification of Environmental DNA
shedding and decay rates for black sea bass. From
Kirtane et al. (in preparation).

salmon (Dowle et al., 2015). Notably even if species
cannot be identified from the OTUs due to lack ofappropriate reference libraries, the OTU response toa disturbance reflects a biological impact and can beanalyzed without species identification. Thus eDNA
is a promising tool to quickly measure biological

To apply such shedding and degradation rates

impacts in sediments due to aquaculture and can be

to field investigations, Sandy Hook staff conducted

an important method to help identify Aquaculture

sampling on cruises with the Marine Academy of

Opportunity Areas and to monitor operations

Science & Technology (MAST) in NJ in early May,

impacts.

2019, as well as on the Northeast Fisheries Science

It is also important to note that disease is one ofCenter fall 2019 bottom trawl survey. On both

the greatest impediments to successful aquaculture

cruises, water was filtered for later analysis; results

operations. Tracking and modeling the spread ofare pending and should be available as the US

disease will be key to managing the epidemiology of

response to the current pandemic permits.

viruses, bacteria and parasites on cultured shellfish

One of the important next steps to making thisand finfish. eDNA is a tool which can be used fora quantitative survey tool is to develop eDNA

pathogen surveillance in the field. In conjunction

particle transport models, as has been done off

with environmental and hydrographic information,

Southern California. These models are intended

this will assist modelers to predict rates and areas

to indicate the geographical origin of collectedof spread of specific diseases. Notably, this also allow

eDNA (Andruszkiewicz et al., 2017). In addition toforecasts in relation to changing climate. Further,

supporting stock assessments, this approach can be

the potential is large for application of eDNA for

useful to tracking the distribution and abundance

seafood inspection, measuring not only fish andof e.g. escapees from penned cultured salmon andshellfish pathogens but also human pathogens suchother species.

as Vibrio.

Notably, a promising application of environmental
DNA in field surveys will be to pair this approach

Aquaculture Operations Conditions and Fish Healthwith acoustic surveys. This is particularly attractive
because it would be relatively easy to develop aeDNA is potentially a valuable tool for studying

strategy for rapid sampling and rapid analysis,

health-related effects of aquaculture operations

using eDNA species data to ground truth acoustic

including use of pharmaceuticals and otherdata. The development of towed midwater eDNA

chemicals on cultured fish and shellfish. Microbial

sampling equipment will significantly facilitate thisdiversity of gut microflora has been studied inmethodology; several efforts are currently underway

various fish species collected from Japan’s coastal

to develop this equipment. For example NOAA staff

waters using next-generation sequencing. In one

in Sandy Hook, NJ have partnered with MAST to

study metabolites and bacterial eDNA in feces wereEnvironmental DNA and aquaculture

analyzed as indicators for fish health. The potential

23

journal. pone.0176343)

of this approach as a non-invasive inspection

Asakura T., Sakata K., Yoshida S., Date Y., andtechnique in aquaculture was suggested by Asakura

Kikuchi J., 2014: Noninvasive analysis of

et al. (2014).

metabolic changes following nutrient input
into diverse fish species, as investigated byProtected Species

metabolic and microbial profiling approaches.
PeerJ, 2, e550. (doi.org/10.7717/peerj.550)

In the USA, one of the greatest concerns impeding

Dowle E., Pochon X., Keeley N., and Wood S. A.,

the permitting of offshore aquaculture is the risk of

2015: Assessing the effects of salmon farming

entanglement of protected species such as the rightseabed enrichment using bacterial community

whale in the NE USA. To date, predicting species

diversity and high-throughput sequencing.

distributions and migrations have largely relied

FEMS Miclobiol. Ecol., 91(8), fiv089.

on models of food supply availability and habitat(doi:10.1093/femsec/fiv089)

suitability of the species of interest such as right

Keeley N., Valdemarsen T., Strohmeiera T., Pochon

whales. Use of environmental DNA as a surveillanceX., Dahlgren T., and Bannister R., 2019: Mixed-

tool for protected species may enable us to go fromhabitat assimilation of organic waste in coastal

modeling their distribution to actual observation

environments –It’s all about synergy! Sci.

for assessing encounter rates of aquaculture gear

Total Environ., 699, 134281. (doi.org/10.1016/with target species. With the use of in situ deployedj.scitotenv.2019.134281)

automated samplers and sequencers (e.g. from

Kelly R. P., Shelton A. O., and Gallego R., 2019:

Monterrey Bay Aquarium and Research Institute)

Understanding PCR Processes to Draw

eDNA is a promising tool to actually record the

Meaningful Conclusions from Environmental

presence of protected species or their prey in the

DNA Studies. Sci. Rep., 9, 12133. (doi.org/

direct vicinity of aquaculture sites. Getting more

10.1038/s41598-019-48546-x)

field observation data will be pivotal to informing

Kirtane A., Sassoubre L., Wieczorek D., Phelan

and verifying the reliability of entanglement models.

B., Nash B., and Noji T., in Preparation.
Quantification of Environmental DNA

Summary(eDNA) Shedding and Decay Rates for Three
Marine Fish in Support of Quantitative Field

Sampling and processing of environmental DNA

Investigations.

samples are relatively rapid and inexpensive. eDNA

Liu Y., Wikfors G. H., Rose J. M., McBride R. S., Milke

data can support multiple applications to support

L. M., and Mercaldo-Allen R., 2019: Applicationaquaculture. The applications can be qualitativeof environmental DNA metabarcoding toand quantitative. Applications are useful to describe

spatiotemporal finfish community assessment

species diversity; to monitor for finfish escapees,

in a temperate embayment. Front. Mar. Sci., 6,

pathogens and protected species; to assess impact

674. (doi.org/10.3389/fmars.2019.00674)

of aquaculture operations on habitats; to evaluate

Sassoubre L. M., Yamahara K. M., Gardner L.

suitability of habitats for cultured shellfish; to assessD., Block B. A., and Boehm A. B., 2016:the health of cultured species; and other applications.

Quantification of Environmental DNA (eDNA)
Shedding and Decay Rates for Three Marine

References

Fish. Environ. Sci. Technol., 50(19), 1045610464.

Andruszkiewicz E. A., Starks H. A., Chavez F. P.,

Takahara T., Ikebuchi T., Doi H., and Minamoto T.,

Sassoubre L. M., Block B. A., and Boehm A.

2019: Using environmental DNA to estimate theB., 2017: Biomonitoring of marine vertebrates

seasonal distribution and habitat preferences ofin Monterey Bay using eDNA metabarcoding.

a Japanese basket clam in Lake Shinji, Japan.

PLoS ONE, 12(4), e0176343. (doi.org/10.1371/

Estuar. Coast. Shelf. Sci., 221, 15-20.

24

Thomas NOJI, Daniel WIECZOREK, Beth PHELAN, Yuan LIU, Lisa MILKE, Renee MERCALDO-ALLEN,
Julie ROSE, Lauren SASSOUBRE, Bruce NASH
Annotated Bibliography of Key Works

PCR for the successful application of eDNA data toestimate biomass. The investigation is a modeling(1) Sassoubre L. M., Yamahara K. M., Gardner L. D.,approach and describes how the proportional indices

Block B. A., and Boehm A. B., 2016: Quantification ofof amplicon reads capture trends in taxon biomass

Environmental DNA (eDNA) Shedding and Decay

with high accuracy.

Rates for Three Marine Fish. Environ. Sci. Technol.,50(19), 10456-10464.

(4) Liu Y., Wikfors G. H., Rose J. M., McBride

A key publication on experiments for shedding

R. S., Milke L. M., and Mercaldo-Allen R., 2019:and decay rates of eDNA from marine finfish.

Application of environmental DNA metabarcoding

Also includes a much cited conceptual model forto spatiotemporal finfish community assessment in aprocesses in the field affecting concentrations oftemperate embayment. Front. Mar. Sci., 6, 674. (doi.

eDNA.

org/10.3389/fmars.2019.00674)
Describes the field investigations addressing(2) Andruszkiewicz E. A., Starks H. A., Chavez F. P.,

the beneficial effect of caged oyster on finfish

Sassoubre L. M., Block B. A., and Boehm A. B., 2017:

biodiversity in Long Island Sound, USA.

Biomonitoring of marine vertebrates in Monterey
Bay using eDNA metabarcoding. PLoS ONE, 12(4),(5) Kirtane A., Sassoubre L., Wieczorek D., Phelan B.,

e0176343. (doi.org/10.1371/journal. pone.0176343)

Nash B., and Noji T., in Preparation. Quantification

This paper is the first to my knowledge to applyof Environmental DNA (eDNA) Shedding andnumerical modeling to predict the origin of eDNA

Decay Rates for Three Marine Fish in Support ofcollected in the field. The outputs include levels of

Quantitative Field Investigations.

uncertainty for the calculations.

Quantification of Environmental DNA (eDNA)
Shedding and Decay Rates for Three Marine Fish

(3) Kelly R. P., Shelton A. O., and Gallego R., 2019:

in Support of Quantitative Field Investigations.

Understanding PCR Processes to Draw Meaningful

Describes the results from laboratory experiments

Conclusions from Environmental DNA Studies. Sci.

on one pelagic fish species and two flatfish. Also

Rep., 9, 12133. (doi.org/10.1038/s41598-019-48546-x)

relates these data to field surveys for these species

This paper presents guidelines for the use ofwith comparisons to trawl data.

水 研 機 構 研 報， 第 50 号，25 － 30， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，25－30，2021

25

A stable isotopic approach to investigate nitrogen pathways
in a coastal aquaculture area
Satoshi WATANABE＊



Abstract: Coastal eutrophication and consequent environmental deterioration have been
problematic in many parts of the world. However, oligotrophication and resulting reduction incoastal fisheries and aquaculture productivity have also been a recognized problem in someparts of Japan. Some studies argue that declining fisheries production of coastal resources andunfed aquaculture production is partially attributable to the reduced nutrient concentrationsin the coastal waters in recent years. Eutrophication mitigation efforts have reduced theterrestrial nutrient load to the coastal environment over the past forty years in Japan. Excessive
oligotrophication has allegedly reduced primary productivity and hence carrying capacity ofcoastal ecosystems supporting coastal fisheries and aquaculture. Fisheries production of the Manila
clam, Ruditapes philippinarum, has decreased by 95% over the past three decades. Insufficient
food supply is one amongst many speculated factors causing the reduction. However, complex
nutrient flow in coastal environments has not necessarily been elucidated. Our stable isotopic(δ13C and δ15N) studies have suggested that there are large and small scale variations in the
nutrient pathways from inorganic nutrients to the clam. Clams inhabiting the same tidal flat only
10 m apart from each other can have a different food environment as indicated by δ13C. Pervasive
effects of terrestrial nutrient load on the food availability of the clam may be ascertained by δ15N.
Stable isotopic methods may also be used to determine nutrient flow within an integrated multitrophic aquaculture (IMTA) system, which has potential to enhance unfed aquaculture production
in an oligotrophic environment.
Key words: eutrophication, oligotrophication, stable isotope ratio, nitrogen, carbon, IMTA
Introduction

as unfed aquaculture production of inorganic andorganic extractive species is partially attributable to

Coastal eutrophication due to increasing

the reduced nutrient levels in the coastal waters inanthropogenic activities and consequent

recent years (Tanda and Harada, 2012; Yamamoto,environmental deterioration, such as anoxia,

2003).

harmful algal blooms and hydrogen sulfide

Eutrophication mitigation efforts have reduced theemission have been problematic in many parts ofload of nitrogen and phosphorus of terrestrial andthe world, sporadically causing mass mortality of

aquaculture origins to the coastal environment overaquatic organisms. While this holds true for Japan,

the past forty years in Japan. Allegedly excessive

oligotrophication and resulting reduction in coastal

oligotrophication has reduced primary productivity

fisheries and unfed-aquaculture productivity haveand hence carrying capacity or coastal ecosystems

also been a recognized problem in some parts ofsupporting coastal fisheries and aquaculture.

Japan. Some studies argue that ever-dwindling

However, complex nutrient flows within coastal

fisheries production of coastal resources, as wellecosystems are not necessarily well understood.


2020年12月11日受理 （Accepted on December 11, 2020）＊
National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency, 422-1 Nakatsuhamaura, Minami-ise, Mie, 516-0193
Japan
E-mail: swat “at” affrc.go.jp

Satoshi WATANABE

200

1.7x105 t (1983)

150

Although the mechanism of the clam production
decline is not necessarily elucidated, insufficient
food supply associated with recent coastal
oligotrophication is one amongst many speculated

100

7.7x103 t (2019)

factors causing the reduction. The relationship50

between the annual mean dissolved inorganic0

Manila clam fisheries production in Ise Bay from

nitrogen (DIN) level (Mie Prefecture) and the

1964
1969
1974
1979
1984
1989
1994
1999
2004
2009
2014
2019

Production (103 t/yr)

26

Fig. 1. Manila clam fishery production in Japan.

1985 to 2015 (e-Stat) showed a significant negative2

correlation (Fig. 2) (r =0.49, p < 0.001, N=30). Like
many other bivalves, the Manila clam is thought

Clam production (t/yr)

to filter-feed microalgae. Reduced nutrient levelin coastal waters is thought to diminish primary

16000

12000
Fig.1. Manila clam fishery production in Japan
8000
4000
0

productivity, thereby reducing the carrying capacityof the area. However, this hypothesis has not been
supported yet because of the complexity of nutrient
cycles in coastal areas.
Small Scale Variation in Food Items of the Clam

0

5
10
15
20
Dissolved Inorganic N (M)

Fig. 2. Relationship between the annual meandissolved inorganic nitrogen level and Manila clam
fisheries production in Ise, Bay, Mie Prefecture from1985 to 2015.

Food availability to the clam is often represented
by chlorophyll a levels in a water column. In manystudies, however, researchers take water samples
from water columns far above the bottom water thatthe clams inhale and filter-feed. Collection of bottom
water within a few centimeters from the bottom islaborious, and water parameters measured for other

This paper describes a stable isotopic approach togeneral purposes are often used to ascertain theFig.2. Relationship between the annual mean
investigate nutrient
pathwaysin coastal
food availability. Also, information on the availability
dissolved
inorganic
nitrogenlevelaquacultureand Manila clam
areas, withproduction
Manila clam,inRuditapes
fisheries
Ise, Bay,philippinarum,
Mie Prefecture and nutritional quality of different microalgal species
and Integrated
Multi-Trophic Aquaculture (IMTA)
is limited. These issues make accurate determinationfrom1985 to 2015.
as examples.

of food availability and nutrient flow within the foodchain difficult.

Manila Clam Fisheries Production in Japan

13

Stable isotope ratios of carbon (δ C) and nitrogen(δ15N) are widely used in the studies of interactions

Manila clam is one of the iconic coastal species

of food webs in aquatic ecosystems. Our stablefacing serious stock depletion in Japan. National

isotopic study (Watanabe et al., 2009a) has suggested

fisheries production, inclusive of bottom culture,that the food source of the clam may differ within aof the Manila clam has decreased by 95% over thesmall area depending on the bottom topography ofpast three decades (Fig. 1) (e-Stat). The production

the habitat.

reached the peak at 169,621 t in 1983, and it

Many filter feeding bivalves are generally held

gradually and continuously decreased to 7,736 t in

to be phytoplanktivorous; however, they also feed

2018. Japan used to be the largest producer of theon benthic microalgae. Provisional ratio of theclam in the world, but it imported 42,482 t of theplanktonic (i.e. pelagic) to benthic microalgae seems

clam, amounting to JPY 8.6 billion from countries

to be affected by the extent of resuspension ofsuch as China and South Korea in 2017 (Ministry of

the latter from the sediment to the bottom water

Finance, Trade statistics of Japan).

to make them available to the clam. In our study

Nutrient cycling in coastal aquaculture areas

the clam. Resuspension of benthic microalgae may

-10
-20

12
11

-30
0

(B)

20 40 60 80 100 120
Distance from shoreline (m)

-15

-40

13C (‰)

-10
-20
-30
-17

Clam C/N ratio(C)

0

20 40 60 80 100 120
Distance from shoreline (m)

-40

0

4

-10
-20

3.6
3.4

-30

3.2

-40

0
15

20 40 60 80 100 120
Distance from shoreline (m)both planktonic and benthic microalgae near thedune had higher δ13C, and they seemed to havebetter nutritional condition as indicated by higher
C/N.
Benthic microalgae are important food source forfood availability to the clam.
Large Scale Variation in Nutrient Pathway to the Clam
Anthropogenic nitrogen is known to have ahigher nitrogen stable isotopic (δ15N) signature thannitrogen from atmospheric deposition ( 2‰ - 8‰)
and nitrogen fixed by cyanobacteria (-2‰ - 0‰)(McClelland et al., 1997; Oowada et al., 2003). Treated

4.2
3.8

benthic microalgae to the clam. The clams utilizing

the bottom water should be analyzed to determine

0-16

of higher wave action, increasing the availability of

the Manila clam, and chlorophyll a concentration in

Relative ground height(cm)

15N (‰)

13

be more active near a dune presumably because

Relative ground height
(cm)

0

14

water of sewage, for example, contains DIN withRelative ground height
(cm)

(A)

27

13

Fig. 3. δ N (A), δ C (B) and C/N ratio (C) of wholesoft tissue of Manila clam at eight stations on an- offshoretransect in tidal flat in Yokohama
Fig.3.inshore
d15N (A),d13C (B)and C/N ratio (C) of whole soft
Marine Park (after Watanabe et al., 2009a).

tissue of Manila clam at eight stations on an inshore offshore transect in tidal flat in Yokohama Marine
Park (after
et al., 2009a)
13 Watanabe
15of δ C and δ N of the clams collected along ahigher δ15N elevated by denitrification during thetreatments (Macko and Ostrom, 1994). Agricultural
fertilizer also enhances soil denitrification andincreases δ15N in groundwater (Ogawa et al., 2001).
Thus, δ15N in DIN acts as an indicator of the levelof anthropogenic nitrogen loads to coastal waters.
The δ15N in the soft tissues of the Manila clam
was found to be positively correlated to the total
DIN (i.e. sum of nitrate, nitrite and ammonium
nitrogen) concentration in the bottom water intidal flats in Kanagawa, Shizuoka and Fukuoka
Prefectures with different eutrophication levels(Watanabe et al., 2009b). This indicates that the clam
δ15N can be a proxy for the anthropogenic nitrogenload to the coastal environment.

inshore-offshore transect in an artificial tidal flat in

The concentration and δ15N of the total DIN were

Yokohama Marine Park, clams inhabiting the same

positively correlated with each other in the studied

tidal flat only 10 m apart from each other had aareas (Fig. 4), indicating that input of terrestrial

different δ13C and δ15N signature, as well as carbon

nitrogen with higher δ15N elevates the DIN levelto nitrogen ratio (C/N) depending on the positionin coastal waters. The δ15N of the clam soft tissue,

on the sandwave (Fig. 3). This indicates that theparticulate organic matter (POM) in seawaterclam assimilates different food items depending on

and sediment organic matter (SOM) in tidal flat

the position on the sandwave. In general, benthic

surface were higher in areas with higher total DIN

13

microalgae tend to have higher δ C than doconcentration of bottom water (Fig. 5). The higher

phytoplankton, and higher C/N indicates higher

δ15N in the clam was considered to be attributable

glycogen (i.e. energy reserve for bivalves) content of

to higher δ15N in the food particles (POM and SOM),

Satoshi WATANABE

28

15Ntotal DIN (‰)15(A) 16

R² = 0.91

15Nclam (‰)

10
5
0
1

10
[total DIN] (log M)

Fig. 4. Relationship between the mean concentrationand mean δ15 N in total DIN in bottom watercollected from different tidal flats in Japan (after
Watanabe
et al., 2009b). between the mean
Fig.4.. Relationshipconcentration and mean d15N in total DIN in
bottom waterfrom different tidal flats
especiallythose incollected
SOM.
inThisJapan(afterWatanabeet elevatedal., 2009b)
study demonstrates that
δ15N in(C)

fisheries productivity.
Nutrient Flow within Integrated Multi-Trophic
Aquaculture
Integrated multi-trophic aquaculture (IMTA) is
a technique to use unfed aquaculture of inorganicand organic extractive species (e.g. algae andmacrobenthos) to consume effluent from fed
aquaculture of finfish (Chopin, 2006). Although IMTA
is usually proposed as a mitigation measure for15NSOM (‰)

by the clam δ N. This can be a powerful tool
oligotrophication of coastal waters to the clam

1

10
[total DIN] (log M)

100

10
8
6

15

to understand the effects of eutrophication and

8

R² = 0.95

loads is reflected in the δ15N in the clam. Thus,
food availability to the clam may be ascertained

10(B) 12

DIN in coastal waters due to anthropogenic nitrogen
pervasive effects of terrestrial nutrient load to the

12

6

100

15NPOM (‰)-5

R² = 0.91

14

1

10
[total DIN] (log M)100

12R² = 0.97

10
8
6
4

1

10
[total DIN] (log M)

100

Fig. 5. Relationship between the mean concentration
Fig.5. Relationship between the mean concentration(±SE) of the total DIN and δ15N in
Manila clam (A),(±SE) of the total DIN and d15N in Manila clam (A),POM in the bottom water (B) and SOM in the tidal
POM in the bottom water (B) and SOM in the tidal
flat surface (C) (after Watanabe et al., 2009b).

flat surface (C) (after Watanabe et al., 2009b)

eutrophication by intensive fed aquaculture, it has aboth of which can be a nutrient source for the

potential to alternatively enhance unfed aquacultureorganic extractive species.

production in an oligotrophic environment. In either

We compared the δ13 C and δ15 N of fecescase, nutrient tracing is important to design ancollected from five different finfish species (Asianefficient IMTA system.

seabass, mangrove red snapper, milkfish, snubnose

In order to trace nutrient flow within an IMTA

pompano and orange-spotted spinefoot) commonlysystem using stable isotopic method, not only is

produced in aquaculture in Southeast Asia (Watanabe

it necessary to determine isotopic fractionation

unpubl. data). We fed the same formulated feed to

between organic extractive species (e.g. sea

these fish to determine difference in δ15N, δ13C,

cucumber, Watanabe et al., 2013) and food, but toand contents of nitrogen and carbon between thehave information on how stable isotopic signatures

feed and feces. We found that while the δ15N ofdiffer between the aquaculture feed and fish feces,

feces showed no significant difference from that of

Nutrient cycling in coastal aquaculture areas29

the feed among all the species, δ13C and C/N ratioof nitrogen isotope ratio of gobiid fish (Isaza)

were different among the species. Therefore, δ15N

specimens and sediments in Lake Biwa, Japan,

can be used to determine the contribution of theduring the 20th century. Limnol. Oceanogr.,

nitrogen from fed aquaculture to growth of organic

46(5), 1228–1236.

extractive species, but it cannot determine whether

Oowada S., Kouzu Y., Fukuda Y., and Yamatake S.,

the nitrogen was used by the organic extractive

2003: Characterization of nitrogen stable isotope

species as leftover feed or fish feces.

ratios at different kinds of nitrate nitrogen

In order to using δ13C to trace carbon flow within
13

sources. Ann. Rep. Ehime Pref. Inst. of Public

an IMTA system, the relationship of δ C between

Health, 6, 46–49. (in Japanese with English

feed and fish feces must be determined beforehand

abstract)

for each fish species and feed. The difference we
13

Tanda M., and Harada K., 2012: Setonaikai toubu

observed in δ C and C/N ratio may be attributable(harimanada) no eiyouen kankyou to gyogyou

to different digestibility of each fish species.

(nutrient environment and fishery in Eastern

Formulated feed usually contains plant ingredients

Seto Inland Sea). Aquabiology, 199, 132-141. (insuch as wheat flour and rice bran as a binder.

Japanese)

Herbivorous species (orange-spotted spinefoot)Watanabe S., Katayama S., Kodama M., Cho N.,

may be able to digest and assimilate more plant

Nakata K., and Fukuda M., 2009a: Small-scaleingredients (i.e. rich in carbon) than do carnivorous

variation in feeding environments for the Manila

species (Asian seabass).

clam Ruditapes philippinarum in a tidal flat inTokyo Bay. Fish. Sci., 75, 937–945.

References

Watanabe S., Kodama M., and Fukuda M., 2009b:
Nitrogen stable isotope ratio in the manila clam,

Chopin T., 2006: Integrated multi-trophic

Ruditapes philippinarum, reflects eutrophicationaquaculture: what it is, and why you should carelevels in tidal flats. Mar. Pollut. Bull., 58, 1447-

and don’t confuse it with polyculture. Northern

1453.

Aquaculture. July/August, p.4.
e-Stat, Statistics of Japan, https://www.e-stat.go.jp/
en, (Cited in November 2019)
Macko S. A., and Ostrom N. E., 1994: Pollution

Watanabe S., Kodama M., Sumbing J. G., andLebata-Ramos M. J. H., 2013: Diet-tissue stable
isotopic fractionation of tropical sea cucumber,
Holothuria scabra. JARQ, 47, 127-134.

studies using stable isotopes, in “ Stable Isotopes

Yamamoto T., 2003: The Seto Inland Sea –in Ecology and Environmental Science” (ed.

eutrophication or oligotrophication? Mar. Pollut.

by Lajtha K., and Michener R. H.), Blackwell

Bull., 47, 37-42.

Scientific Publications, Oxford, pp. 45–62.
McClelland J. W., Valiela I., Michener R. H., 1997:

Annotated Bibliography of Key Works

Nitrogen-stable isotope signatures in estuarine
food webs: A record of increasing urbanization(1) Watanabe S., Kodama M., Sumbing J. G., andin coastal watersheds. Limnol. Oceanog., 42(5),Lebata-Ramos M. J. H., 2013: Diet-tissue stable

930–937.

isotopic fractionation of tropical sea cucumber,

Mie Prefecture: Senkai Teisen Chosa (shallow sea

Holothuria scabra. JARQ, 47, 127-134.

fixed line survey), http://www.pref.mie.lg.jp/

In order to provide a basis for stable carbon andsuigi/hp/79877017487.htm, (Cited in November

nitrogen isotope ratio (δ C /δ N) analysis that will

2019)

allow determination of assimilated organic matter

13

15

Ministry of Finance: Trade statistics of Japan,

in the sea cucumber, Holothuria scabra, diet-tissue

http://www.customs.go.jp/toukei/srch/index.

fractionations were experimentally determined byhtm, (Cited in November 2019)

mono-feeding rearing with diatoms. While δ15N

Ogawa N. O., Koitabashi T., Oda H., Nakamura T.,

fractionation of the whole body wall ( 2.4‰) was

Ohkouchi N., and Wada E., 2001: Fluctuations

similar to the commonly accepted value ( 2.6–4‰),

Satoshi WATANABE

30

δ13C fractionation of the body wall ( 4.2‰) showed

proportion of shrimp aquaculture in South-Eastconsiderable discrepancy with the commonly

Asian countries has switched from black tiger

accepted value ( 0–1‰) due to the high content

shrimp (Penaeus monodon) to P. vannamei, an exotic( 35% dry wt/wt) of calcareous spicules (CaCO3) in

species originally imported from Latin America. One

the body wall, which had significantly higher δ13Cof the causes of disease outbreaks is thought to be(-8.6‰) than the organic fractions. Computational

poor water and sediment conditions in the shrimp

elimination of spicules based upon spicule content

ponds, which may aggravate disease symptoms. To

and spicule δ13C reduced the δ13C fractionation

obtain basic information for co-culture methods ofof the body wall to 1.5‰, close to the common

black tiger shrimp and sandfish (Holothuria scabra)

value. δ13 C fractionation after spicule removal

for possible mitigation of shrimp-pond eutrophicationby acid decarbonation and subsequent rinsing

and prevention of disease outbreaks, basic laboratory(3.2‰) did not agree with the common value, andexperiments were conducted. A feeding trial ofδ 15N fractionation was significantly elevated byjuvenile sandfish showed that they do not grow well

15

13

decarbonation. δ N and δ C fractionations of thewith fresh shrimp feed on hard substrate. Anotherintestine ( 1.5 and 2.2‰, respectively) did not agree

trial indicated that sand substrate enhances thewith the common values. Since δ13C and δ15N ofgrowth of juvenile sandfish fed with shrimp feed.

the feces did not differ significantly from those of

A feeding trial using shrimp tank detritus, shrimp

the diet, feces may be used to determine ingested

feces and Navicula ramosissima (a benthic diatom) as

organic matter in the wild.

food sources showed that sandfish grew fastest withthe feces, followed by detritus and N. ramosissima.

(2) Watanabe S., Kodama M., Zarate J. M., Lebata-Dissolved oxygen consumption and acid-volatile

Ramos M. J. H., and Nievales M. F. J., 2012: Ability ofsulfur levels in the shrimp tank detritus were

sandfish (Holothuria scabra) to utilise organic matter

reduced by sandfish feeding. This suggests thatin black tiger shrimp ponds. ACIAR Proceedings,

sandfish are capable of growing with organic matter

136, 113-120.

in shrimp ponds and can bioremediate shrimp-pond

Due to frequent viral disease outbreaks, a large

sediment.

水 研 機 構 研 報， 第 50 号，31 － 45， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，31－45，2021

31

Immunological assays of hemocytes in molluscan bivalves asbiomarkers to evaluate stresses for aquaculture
Huiping YANG＊



Abstract: As an important aquaculture section, molluscan aquaculture has traditionally accounted
for about 60% of total marine aquaculture production worldwide. The molluscan aquaculture
species are majorly bivalves, including oysters, clams, scallops, and mussels. Challenges inmolluscan aquaculture include diseases, environmental stresses, coastal pollutions, and seed
quality and quantity. This mini review summarized current research updates on immunologicalassays of hemocytes of molluscan bivalves against the biotic (e.g. bacteria, viruses, or protozoan
parasites) and abiotic stresses (e.g. temperature shock, fluctuated salinity, or environmental toxins).
As the frontline of immune system, hemocytes play a significant role against these stresses. The
immunological assays of hemocytes could be used as effective biomarkers to evaluate the effects ofbiotic and abiotic stresses in aquaculture operation and breeding programs.
Key words: molluscan bivalves, immunological assays, hemocyte, stress, biomarker
Introduction1966). Hemocytes in bivalves have been studied
extensively since the 1970s and summarized in two

Molluscan aquaculture is a US$23.9 billion industry

review publications (Hine, 1999; Anisimova, 2013). In

worldwide for seafood production (FAO, 2018).

recent years, the molecular mechanisms and signal

Sustainability of molluscan aquaculture industry

pathways of bivalve immune system have been

faces many challenges including seed quality

becoming research focus to understand the immune

and quantity, environmental stresses and climate

protective strategies from various pathogens andchanges, diseases, natural and genetic resources,

environmental stresses (Song et al., 2010).

best practices of operations, and regulatory scrutiny

This mini review summarized the immunological(Dumbauld et al., 2009). Accordingly, efforts haveassays of hemocytes from aquaculture molluscan

been made on overcome these challenges throughbivalves in response to biotic and abiotic factors, andgenetic breeding for stock enhancement (Hulata,

it is expected that these hemocyte assays could be2001), disease diagnosis and control by use ofused as effective biomarkers for disease diagnosis,

probiotics (Hoseinifar et al., 2018), natural resources

evaluation of environmental stresses, and breeding

conservation management (Beck et al., 2011),

tools.

improvement of water quality through land use
management, employment of best practices, andImmune System in Molluscan Bivalves

extensive education programs.
Immune system in molluscan bivalves was firstly

Immune system in vertebrates includes a series ofreported in Mytilus californianus about hemolymph

collectively effective defenses against diseases andagglutinins (Tyler, 1946), which were observed later

pathogen invasions. The first defense is the physicalin butter clams, Saxidomus giganteus (Johnson, 1964),

barriers, such as the skin, which can preventand eastern oysters, Crassostrea virginica (Tripp,from colonization with other organisms and move
2020年12月11日受理（Accepted on December 11, 2020）＊
School of Forest Resources and Conservation, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32653.
E-mail: huipingyang “at” ufl.edu

32

Huiping YANG

inhaled materials using the mucociliary apparatus

Humoral immunity can be achieved through(ciliated epithelial cells and mucus-secreting cells).

antimicrobial peptides, which is an evolutionarily

The second defense is innate immunity which isconserved component of innate immunity in alla primitive nonspecific immunity to against any

classes of life and represent the main form of

pathogens that enter the body rather than targeted

invertebrate systemic immunity. In bivalves, thespecific invaders. The third defense is a complex,

identified humoral factors together with their

specific, and long-lasting adaptive immunity which

immune functions included atrial natriuretic peptides

relies on the accumulated memory cells after(ANPs) in hemolymph and heart of the eastern

exposure to pathogens.

oyster, Crassostrea virginica (Vesely et al., 1993),

Molluscan bivalves possess an open circulatory

catecholamines in the giant scallop, Placopecten

system. Bivalves pump hemolymph into the open

magellanicus (Pani and Croll, 2000), lectins in the

body cavity (hemocoel), circulates in hemolymph

pearl oyster, Pinctada maxima (Flower et al., 1985),

vessels and sinuses as well as throughout softand the giant clam, Hippopus hippopus (Puanglarp ettissues, thus the hemolymph can bath the internalal., 1995), and hemagglutinins in the eastern oysterorgans and deliver nutrients and gases. It is believed(Li and Flemming, 1967) and the northern quahogthat bivalves use their hemocytes and humoral(Tripp, 1992). The profile of total protein, ions,proteins in circulatory system to provide internaland sugars composition in hemolymph have been

defensive functions against various pathogen anddocumented in Mya arenaria and connected withenvironmental stresses (Bayne, 1983).

their immune functions (Sunila and Dungan, 1992;
Rees et al., 1993).

1. Physical barriers

Hemocyte immunity is the fundamental immune

The external shells in molluscan bivalves are thefeature and usually achieved by recognition ofmost important physical barrier to protect theirforeign substances and subsequent ingestion (Pilasoft tissues and organs and prevent from predators,

et al., 2016). The immunological assays of hemocyte

parasites, harmful substances, and environmental

in molluscan bivalves were stated as follows in nextchanges. For example, the northern quahog (also

Section.

called hard clam), Mercenaria mercenaria, can keep
their shells closed without any movement and3. A d a p t i v e i m m u n i t y ( a l s o c a l l e d a c q u i r e dejection for days at -1.0 to 1.9℃ seawater (Loosanoff,

immunity)1939); the Pacific oyster, Crassostrea gigas, can close

Adaptive immunity is a more sophisticated system

their shells tightly at 4℃ air dry for 47.8 days (50%

to recognize and destroy specific invaders basedlethal time) (Kawabe et al., 2010); scallops can swim

on cellular memory. The process of this defensive

away by flapping their shells to escape predators orreaction normally uses specific antigens which areenvironmental stresses. Additionally, bivalves canactivated by exposure to pathogens. Therefore, it

use their gills and labial palps to select food particles

is antigen-specific functions through cell-mediatedand wrap un-selected particles with mucus for

system. Adaptive immunity system uses anejection (Shumway et al., 1985; Ward and Shumway,

immunologic memory to learn about the pathogen

2004).

and enhance the immune response, accordingly. Thissystem is more effectively and specifically to the

2. Innate immunity
The innate immune in molluscan bivalves

pathogens, but usually much slower to respond to
threats and infections than the innate immunity.

is believed to achieve through humoral innate

Generally, adaptive immunity is consideredimmunity, which involves in molecules (e.g. proteins)

to exist only in vertebrates. However, in recent

in the body humors to stop the growth of pathogens

years, adaptive immunity has been identified

or clump them together, and cellular immunity of

in invertebrates and even bacteria, such as thehemocytes, which involves in phagocytes to ingest

CRISPR/cas9 system which can recognize andand degrade pathogens (Bayne, 1983).

destroy the invaded virus RNA sequence (Zhang etImmunological assays of hemocytes in molluscan bivalves

33

al., 2012). For bivalves, the first evidence of antiviralmorphological observation include light microscopy,

immune priming was just reported in the Pacifictransmission electron microscopy, flow cytometry,oyster (Lafont et al., 2020) against the herpes-likeand monoclonal antibody (Noël et al., 1994).

virus Ostreid herpesvirus 1, a major viral disease(1) Granulocytes

triggers the Pacific oyster mortality syndrome

Granulocytes were found to be the major(Segarra et al., 2010). The injection of various nucleic

hemocyte type in bivalves. Granulocytes wereacids showed the capability to trigger oysters tousually characterized with cytoplasmic granules

protect them against a subsequent viral infection.

and have a low nucleus: cytoplasm ratio. Depending

Additionally, specific genes in adaptive immunity

on the granular features granulocytes can be sub-pathway in abalones were found to be up/down

categorized as eosinophilic granulocytes which

regulated when exposure to thermal shock and/or

contain cytoplasmic large eosinophilic granules andhypoxia (He et al., 2017; Zhang et al., 2019) and in

stain with acid stains (such as eosin with pink color),

the Pacific oyster when exposure to environmental

basophilic granulocytes which contain small granules

stresses (Guo et al., 2015).

and stain with alkaline stains (such as methylene
blue with blue color). In several studies, granulocytes

Immunological Assays of Hemocytes inwere also divided and named as small and large

Molluscan Bivalves

granulocytes. The nuclei of granulocytes are usually
uninucleate or binucleate with eccentric, spherical,

Hemocytes in bivalves have been reported toor occasionally ovoid morphology and stains as darkparticipate a variety of physiological and immune

blue with DNA staining by Giemsa.

functions, such as wound repair, shell formation and(2) Agranulocytes (Hyalinocytes)

healing, nutrient transport and digestion, excretion,

Agranulocytes were also named as hyalinocytes inand internal defense (Anderson and Good, 1976;

many publications based on microscopic observations.

Song et al., 2010; Pila et al., 2016). When exposure to

As the name reflects, these cells are characterizedinvasive pathogens, hemocytes can encapsulate themwith few or without visible cytoplasmic granules inand subsequently destroy them via enzyme activity

cytoplasm and have relative larger nucleus. Basedand oxygen metabolite release defense the invadingon the cell size, agranulocytes can be classified

microbes (Song et al., 2010). A comprehensive

into large hyalinocytes (agranulocytes) and small

review has summarized the morphology andhyalinocytes (agranulocytes, also called blast-likefunctionality of hemocytes in bivalves for further

cells in several publications) with a central ovoid

reading (Anisimova, 2013).

or spherical nucleus surrounded by a rim of scant

In this publication, a literature search was
performed about bivalve hemocyte functions under
different stresses (Table 1). Briefly, the hemocyte
assays in bivalves are as follows.

cytoplasm lacking organelles (Bachère et al., 1988).
(3) Other types
Besides the granulocyte and agranulocyte
types, other hemocyte types were observed inbivalve species. For example, cells had the general

1. Hemocyte morphology and cell types

appearance of granulocytes, low nucleus: cytoplasmic

Based on the morphological characteristics

ratio and round nucleus, but had few or no granules.

such as cell sizes and cytoplasmic inclusions, theThese cells were regarded as fibrocytes in thehemocytes in bivalves are classified into two types

eastern oyster and the northern quahog (Foley- granulocyte and hyalinocyte (agranulocyte).

and Cheng, 1972) and later were considered as

For some species, a third type of hemocytes

degranulated granulocytes following phagocytosis

with different characteristics was reported with

(Mohandas and Cheng, 1985). Depending on celldifferent names. So far, two reviews have madecharacterizations, different names have been used tocomprehensive summaries on bivalve hemocyte

describe these hemocytes by different authors withcells types for further reading (Hine, 1999;

no systematic rules (Hine, 1999).

Anisimova, 2013). The methodologies for hemocyte

SPECIES
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea madrasensis
Crassostrea rhizophorae
Crassostrea rhizophorae
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Crassostrea virginica
Ostrea edulis
Ostrea edulis
Ostrea edulis; C. gigas
Ostrea edulis
Pinctada fucata martensii
Pinctada fucata martensii

STRESS*
Spatial and temporal
T and infection
Seasonal changes
Herpesvirus
Hypoxia
Bacteria
Air dry, hypoxia
Virus infection
Disturbances, salinity, and T
T, TBT, and trace metals
Repeated bleeding
Vibrio anguillarum
Virus – T3-coliphage
Vibrio alginolyticus
Seasonal change
Virus infection
Cadmium
Heat shock to 28℃
Perkinsus marinus
Fungicide
SalinityT, salinity, food, Dermo
TBT
Repeated bleeding
Haplosporidium nelsoni
Starvation, salinity, T
Cercariae
Bacteria
Bacillus megaterium
Minchinia nelsoni (MSX)Micrococcus lysodeikticus
Bonamia ostreae
Salinity, T
Bonamia ostreae
Bonamia ostreae
Post-operative care
Diseased oyster fluids

IMMUNOLOGICAL ASSAYS OF HEMOCYTES**
Hemolymph microbiota status
Hemolymph microbiota status
Persistence, seasonal dynamics, and pathogenic potential of Vibrio
Antiviral activity of hemolymph protein against herpesviruses
Regulation of an isoform of AMP-activated protein kinase
Antimicrobial peptides analysis in hemolymph
Changes of pO2, pCO2, pH, and osmolality in hemolymph
Antiviral activity in hemolymph
Catecholamine changes in the hemolymph
The alteration of spontaneous hemocyte aggregation
THC, phagocytosis, hemolymph pH, and pO2
Agglutination activity after challenge to Vibrio
Hemocyte neutralizing activity
THC, serum protein, lysozyme activity
Carbonic anhydrase to reflect environmental contaminations
Evaluation of antiviral activity in hemolymph
Apoptosis
Changes of hemocyte cell types and viability
Function of hemocytes to kill Perkinsus marinus
Phagocytosis, reduced pyridine nucleotides, and ROS
Atrial natriuretic peptides (ANPs) in the circulation hemolymph
Hemolymph lysozyme activity and protein profile
Kinetic regulation of chloride-ion and osmotic-pressure in hemolymph
Hemolymph protein and parasitism infection of Haplosporidium nelsoni
Hemolymph proteins from resistant and susceptible oysters
Protein and carbohydrate levels in the hemolymph
Encapsulation of cercariae by hemocytes
Clearance of enteric bacteria from the hemolymph
Aminopeptidase activity in hemocytes and hemolymph
Hemolymph enzyme activities during oyster-MSX interaction
Hemocyte lysozyme capability
Apoptosis of hemocytes
Expression of HSP/C70 or metallothionein genes in hemocytes
Enzymatic activities – a commercial kit for detection of 19 enzymes
Hemolymph function against Bonamia ostreae
Hemolymph hemagglutination activity
Hemocyte viability and survival of infected pearl oysters

Table 1. The immunological assays of hemocytes in molluscan bivalves under challenges of environmental and biotic stresses
REFERENCE(Lokmer et al., 2016)(Lokmer and Wegner, 2015)(Wendling et al., 2014)(Green et al., 2014)(Guevelou et al., 2013)(Defer et al., 2013)(Kawabe et al., 2010)(Olicard et al., 2005)(Lacoste et al., 2001)(Auffret and Oubella, 1997)(Jones et al., 1993)(Olafsen et al., 1992)(Bachere et al., 1990)(Ittoop et al., 2010)(dos Santos et al., 2017)(Carriel-Gomes et al., 2006)(Sokolova et al., 2004)(Hegaret et al., 2003)(Volety and Fisher, 2000)(Baier-Anderson and Anderson, 2000)(Palmer et al., 1994)(Chu and Lapeyre, 1989)(Bokman and Laughlin, 1989)(Ford, 1986b)(Ford, 1986a)(Fisher and Newell, 1986)(Font, 1980)(Hartland and Timoney, 1979)(Yoshino and Cheng, 1976)(Douglass and Haskin, 1976)(Mcdade and Tripp, 1967)(Gervais et al., 2018)(Corporeau and Auffret, 2003)(Xue and Renault, 2000)(Cochennec et al., 1992)(Sano et al., 2017)(Morizane et al., 2002)

34
Huiping YANG

Disease individuals
Vibrio sp.
Vibrio alginolyticus
StarvationT, salinity, air dry
Pesticide
Diatom (Domoic Acid)
Tissue implantation
TiO2
Carbon nanofibers
Heavy metals
Air dry, hypoxia
Air dry
Freshwater
Crude oil and dispersant
Zinc pyrithione
Hypoxia
Bacteria challenge
Estrogens
BaP, 4NQO
TiO2, hypoxia
Acute salinity
Bacteria
Fluctuating Salinity
Heat (10℃ for 48 hr)
Salinity
QPX disease, T
CdCl2, BaP
Heavy metals
Spatial and temporal change
Bacillus megaterium
Dinoflagellate algae
Heat and cold shock
VibrioVibrioSpatial and temporal change
TBT
Repeated sampling

Hemocyte morphological changes
Hemocyte morphological and functional changes
Antibacterial activity in the hemolymph
THC, ROS, acid phosphatase in hemolymph
Catecholamines in hemolymph
Cholinesterase activities of hemocytes
Changes in hemolymph pH, pCO2, and pO2
Hemocyte cell type, phagocytosis, and implant rejection
THC, viability, phagocytosis, lysosome, MN; MMP, ROS
Gene expression of hemocytes
Transportation of heavy metals in the circulatory system
Hemolymph acid-base status - pCO2, ammonia, pO2, and pH
Changes of pH and pO2 in hemolymph
Changes of the electrolyte contents
ROS, viability, phagocytosis, MXR, ACI
THC
Cell type, ROS, viability
Bacterial adherence and association with hemocytes
Vitellogenin levels in hemolymph
DNA damage of hemocytes
Cell type and number, THC, viability
Hemocyte osmolality change
Identification of a potent serine protease inhibitor
Osmotic pressure and ionic concentrations of hemolymph
Expression of HSP70 in thermally stressed hemocytes
Viability, ROS, lysosome activity, phagocytosis
ROS, phagocytosis, lysozyme activity
Identification of a small HSP gene and expression
Phagocytosis, cell number, and viability
Prevalence of leukemia in hemolymph
Lipase activity in hemolymph and hemocytes
Genotoxicity in gills and hemolymph
Identification of stress-immune response genes in hemocytes
Cell count, viability, lysozyme activity, assay of bacterial infection
Total protein and leucine aminopeptidase in hemocytes
Hematocrit, phagocytosis, lysozyme activity
Superoxide dismutase, lysozyme activity
Total protein, ions composition, glucose, glycerol(Maeno et al., 2001)(Vieira et al., 2017)(Luna-Gonzalez et al., 2007)(Xu et al., 2008)(Chen et al., 2008)(Owen et al., 2002)(Jones et al., 1995)(Bayne et al., 1979)(Wang et al., 2019)(Barrick et al., 2019)(Devoid et al., 2007)(Booth et al., 1984)(Jokumsen and Fyhn, 1982)(Khlebovich et al., 1981)(Katsumiti et al., 2019)(Katalay et al., 2019)(Andreyeva et al., 2019)(Zampini et al., 2003)(Riffeser and Hock, 2002)(Bihari et al., 1990)(Wang et al., 2014)(McFarland et al., 2013)(Khan et al., 2008)(Djangmah et al., 1979)(Park et al., 2007)(Jauzein et al., 2013)(Perrigault et al., 2011)(Li et al., 2013)(Brousseau et al., 1999)(Craig et al., 1989)(Cheng and Yoshino, 1976)(Florez-Barros et al., 2011)(Menike et al., 2014)(Allam et al., 2000)(Oubella et al., 1994)(Matozzo et al., 2003)(Matozzo et al., 2002)(Rees et al., 1993)* T: temperature; TBT: tributyltin; BaP: benzo[a]pyrene; 4NQO: 4-nitroquinoline-N-oxide; TiO2: titanium dioxide.
**THC: total hemocyte count; ROS: reactive oxygen species in cells; MN: mitochondrial number; MMP: mitochondrial membrane potential; MXR: multi-xenobiotic resistance; ACI: actin cytoskeleton disruption.

Pinctada fucata martensii
Pteria hirundo
Argopecten ventricosus
Chlamys farreri
Chlamys farreri
Euvola (Pecten) ziczac
Mytilus californianus
Mytilus californianus
Mytilus coruscus
Mytilus edulis
Mytilus edulis
Mytilus edulis
Mytilus edulis
Mytilus edulis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus galloprovincialis
Perna viridis
Perna viridis
Perna viridis
Anadara senilis
Laternula elliptica
Macrocallista nimbosa
Mercenaria mercenaria
Meretrix meretrix
Mya arenaria
Mya arenaria
Mya arenaria
Ruditapes decussatus
Ruditapes philippinarum
Ruditapes philippinarum
Ruditapes philippinarum
Tapes philippinarum
Tapes philippinarum
Tridacna gigas

Immunological assays of hemocytes in molluscan bivalves

35

36

Huiping YANG

2. T otal hemocyte number and proportions ofas important assays for hemocytes in bivalves, suchdifferent types

as in the eastern oyster against cadmium exposure

The total hemocyte number and relative(Sokolova et al., 2004) and in the flat oyster Ostrea

proportions of different types were the most direct

edulis against parasite Bonamia ostreae (Gervais etmeasurement of hemocyte immune responses.

al., 2018). Apoptosis and cell cycle assays could be

Although variations in hemocyte number may exist

performed by flow cytometry or genomic sequences.

among individuals, any significant changes would
be more likely linked to the metabolic condition

5. Phagocytosischanges. Counting of different types of hemocyte

Phagocytosis is the most fundamental role fornumbers could be accomplished by microscopic

hemocytes in bivalves to defense invasive pathogens,observation, but this method is time consuming withsuch as bacteria (Canesi et al., 2002), and involves

the process of making slides, staining, and counting.

in collaboration of humoral defense factors such as

Flow cytometer is a fast and accurate method toagglutinins. Hemocytes can recognize, bind, andcount single-cell suspension such as sperm (Yangphagocytize the microbes, and the encapsulated

et al., 2016) and can distinguish different types of

microbes would be eventually degraded by cellular

hemocytes based on side scatter (SSC, measurement

enzymes and oxidization to decrease the number ofof cell granularity) and forward scatter (FSC,

microbes.

measurement of cell size) (Ashton-Alcox and Ford,

Phagocytosis of hemocytes on foreign substances

1998). Therefore, flow cytometry is becoming thewas firstly observed in the Pacific oyster (Feng,

mostly used approach for measuring the changes of1965), and has been reported to accomplish majorly

hemocyte cell types.

by granulocyte hemocytes (Pipe, 1990), especiallythe eosinophil granulocytes (Hine, 1999; Anisimova,

3. Hemocyte viability

2013; Pila et al., 2016). The commonly used method

Hemocyte viability is a measure of the proportion

to evaluate phagocytosis is to incubate fluorescenceof alive cells to evaluate the overall hemocyte health.

labelled beads (e.g. The Bangs Laboratories, Inc.,

Viability assays can be assessed based on cellular

https://www.bangslabs.com/) or actual microbes atmetabolism, enzyme activity, or cell membranecertain temperature for a period of time, and theintegrity. The widely used approach was double

quantification of hemocytes with phagocytic beads

fluorescence staining with membrane permeable

can be performed by use of direct microscopic

nuclear dyes, such as SYBR, and the membrane

examination, fluorometric evaluation, or flow

impermeable dyes, such as propidium iodide (PI),cytometry.

and detected by use of fluorescence microscopy or

For bivalves, phagocytosis has been studied inflow cytometry (Allam et al., 2002). Alternatively,

many species (Table 1). The phagocytosis process

because cell membrane damage can cause releaseinvolves in humoral defense factors such asof cytosolic contents into the extracellular space

agglutinins and lysosomal enzymes, and the surface-

including the enzyme lactate dehydrogenase (LDH),

bound factors play a significant role in the bacteria-

measurement of the extracellular LDH has also been

hemocyte interactions leading to the phagocytosis.

used as an effective assay for hemocyte viability (ChuPhagocytosis in bivalves can be affected by the

et al., 2002).

environmental temperatures and other seasonal
factors, but underlying factors influencing

4. Hemocyte apoptosis and cell cycle
Hemocyte apoptosis is a fundamental biologicalphagocytosis are still not completely understood (seea comprehensive review in Canesi et al., 2002).

process in immune system for defensive functions(Sokolova, 2009). Hemocyte proliferation in cell

6. Reactive oxygen species (ROS) production

number due to cell division in a sample can be used

ROS are natural byproducts of the normal

as an indicator to evaluate the cell health status.

metabolism of oxygen in cell signaling

Therefore, apoptosis and cell cycle have been usedand homeostasis. When exposure to environmental

Immunological assays of hemocytes in molluscan bivalves37

stress, ROS production within cells would increasein mammalian cells (Ishii et al., 2019), and may bedramatically because of the damage to cellapplied for bivalve hemocytes.

structures. After phagocytosis, the encapsulated
microbes could be degraded by the oxidization
process and cause ROS changes.

8. Molecular pathways for hemocyte immunity
In recent years, molecular signal pathways for

ROS production can be measured by use of ahemocyte immunity have been investigated innonfluorescent analogue 2’,7’-dichlorofluoresceinaquaculture bivalves. The molecular mechanisms fordiacetate (DCFH-DA) (Eruslanov and Kusmartsev,

hemocyte immune recognition, signal transduction,2010). After diffusing into the cells, DCFH-DA is

and effector synthesis have been reviewed in twohydrolyzed into 2’,7’-dichlorofluorescein (DCFH)

recent publications together with humoral immunity

which would be trapped within the cells. The(Song et al., 2010; Zhang et al., 2019).

intracellular DCFH can be oxidized to highly
fluorescent 2’,7’-dichlorofluorescein (DCF) by ROS,

Application of Hemocyte Immunological Assays

and measurement of DCF fluorescence at 530 nm

for Aquaculture

can be used to quantify the ROS production byuse of a flow cytometer (Lambert et al., 2003) orEnvironmental stresses, such as temperature,proteomic approach (Sheehan and McDonagh, 2008).

salinity, dissolved oxygen, pollutions, and red-In molluscan bivalves, the production of ROS hastide algal toxins, are the challenges for molluscan

been reported in hemocytes of many bivalve species,

aquaculture. To overcome these challenges,

including oysters, mussels, scallops, and clams

molluscan bivalves would close their shells asagainst environmental and biotic stresses (Donaghy

immediate responses and use their hemocyte

et al., 2012).

immune system to respond (Table 1). However,with prolonged exposure to environmental

7. Lysosome enzyme activity

stresses, molluscan bivalves could be subsequently

Lysosomes are membrane-bound vesicles

susceptible to pathogens, increase disease outbreaks,

containing digestive enzymes, such as glycosidases,

and eventually suffer heavy mortality. Therefore,

proteases, and sulfatases, which can digest engulfed

immunological responses of hemocytes in molluscan

foreign microbes. In addition, lysosomes can destroy

bivalves could show different levels and link to theirtargeted organelles through autolysis, and beconsiderable resilience to adverse environmental

responsible for digesting protein from cell surface

conditions. This suggests that, similar to the bloodpresented via endocytosis. Therefore, lysosome

tests as diagnostic tool for health evaluation in

enzyme activity is a parameter to evaluate thehuman and livestock, hemocyte immunological assays

status of hemocytes after phagocytosis.

in bivalves could be used as effective parameters to

In molluscan bivalves, the role of lysosomes

evaluate the impact of the environmental stresses,

following phagocytosis has been studied widely

serve as measuring tools for genetic breeding, and(Cheng, 1983). Lysosome enzyme activities wasprovide diagnosis tools to guidance the operation

demonstrated in hemocytes of the north quahog

management.

after exposure and phagocytosis of single-cell algae

With the fast development of DNA sequencing(Moore and Gelder, 1985) and other stresses (Table

technology, genomic tools such as immunological1). The measurement of lysosomal enzyme activities

related genes, molecular pathways, and specific up-

was usually performed by incubating with specificor down-regulation genes, have been investigated in

substrates and quantification of enzymatic products

responses to different environmental stresses. It isthrough comparing with negative controls without

expected that combination of organism level, cellular,

substrate (Moore and Gelder, 1985). Alternatively,

and molecular immunological assays could provide aprobes linked to the factor controlling lysosomal

full spectrum of immunological assays and serve as

homeostasis was identified and used as an effective

tools for improvement of molluscan aquaculture.

and efficient tool for measuring lysosomal activity

Huiping YANG

38
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45

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Flow cytometric characterization of hemocytes

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parameters (oxidative activity, lysosomal content,

Flow cytometric analysis of haemocytes fromphagocytosis capacity) in sunray venus clams, aeastern oysters, Crassostrea virginica, subjected

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249-265.

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After

This paper studied the effects of temperature

valve closure, and filtration activity. It wasincrease on hemocyte functions of eastern oysters,interestingly found that hemocytes of sunray venus

including aggregation, viability, phagocytosis,

clam appeared as a unique population, both in terms

and ROS production. This is one of a series ofof morphology and intracellular parameters. Clams

publications on oyster hemocyte functions andafter transferring to 18 and 21 ppt resulted in valve

detailed protocols were documented for readers

closure, mortality, and decreased filtration activity.

to understand the analysis procedure. The results

Low salinities highly impacted hemocyte functions

indicated that temperature increase (from 20 to 28

as follows: increased cell and lysosomal compartment

degree) caused no significant change in hemocyte

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increased oxidative stress and mortality.

hemocyte types, induced significant hemocyte

水 研 機 構 研 報， 第 50 号，47 － 53， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，47－53，2021

47

Preliminary feasibility assessment of purple sea urchin,
Strongylocentrotus purpuratus, roe enhancement.

Luke GARDNER*1, 2, Helaina LINDSEY*2, Katherine NEYLAN*2, Walan CHANG*3,
Katrina HERRMANN*3, Max RINTOUL*2, and Katherine ROY*3
Abstract: Sea urchin barrens and can stretch over 1000s of kilometers and last decades at a time.
They are characterized by a predominance of urchins and coralline algae where kelp forests once
existed. In contrast to barrens kelp forests provide habitat supporting thousands of vertebrates,
invertebrates and plant species. Because kelp forests are keystone hosts their presence is vitalto sustaining commercial and recreational industries including fishing and tourism. However,these kelp forests can collapse and shift to alternate stable states whereby urchin barrens persist.
Over the last 4 decades, transitions between kelp beds and sea urchin barrens have been widelyreported along temperate coastlines globally. During a kelp forest phase, urchin predation is theprimary mechanism keeping sea urchin populations in check. However, due to various factors
including climate change, predator densities can be reduced leading to shifts toward urchinbarrens. Development of urchin fisheries has been implicated several times in recent history as adriver to return urchin barrens to kelp forests. However, this driver most recently has not worked
in California and Oregon, USA where a large barren is persisting and expanding. Both states
already had established urchin fisheries but it has been uneconomical for the fisheries to operate
given urchins in the barrens had little gonad development or undesirable human consumption
traits necessary for commercialization. Aquaculture in the US has potential to restore kelp
forests by collaborating with fisheries to harvest wild urchins from barrens and fatten them in
an aquaculture setting prior to sale. Although sea urchin roe enhancement is not a novel concept
there are still technical limitations to the activity, primarily being the availability of macroalgae
diets given seasonality and the propensity of urchin barrens to deplete kelp forests. Developmentof sustainable alternative diets for urchins is necessary for future commercial urchin aquaculture.
In this student lead study, a preliminary replicated diet trial was performed for enhancing roe
from purple sea urchin (Strongylocentrotus purpuratus) collected from California barrens using 4
diet treatments including giant kelp (Macrocystis pyrifera), ogo (Gracilaria pacifica), formulatedcommercial diet (Urchinomics), and an unfed control. During the 10 week study duration, gonadal
somatic index (GSI) was measured in a subset of urchins (5 individuals) from each replicate tank
every 2 weeks. Baseline GSI at the beginning of the trial was <0.5%. A GSI of 10% was reachedmost rapidly in the formulated diet treatment at 6 weeks, followed by ogo and kelp at 9 and 13
weeks respectively. This study was a preliminary examination of the feasibility of urchin ranchingin California, showing biological potential for alternative diets to develop urchin gonads with aview to restore kelp forests and develop a nascent aquaculture industry in California.
Key words: urchin, barren, aquaculture, kelp, restoration
2020年12月11日受理（Accepted on December 11, 2020）*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
California State University Monterey Bay, 100 Campus Center, Seaside, CA 93955
E-mail: lgardner “at” ucsd.edu

48

Luke GARDNER, Helaina LINDSEY, Katherine NEYLAN, Walan CHANG, Katrina HERRMANN,
Max RINTOUL, and Katherine ROY
Introduction

population before the phase switch to the barren
state (Ling et al., 2015). It is characteristics such as

A s ea ur c hin b arren is a b en t h ic h a bit a t

those described above that make urchin barrens

dominated by urchins, coralline algae, rocky

persistent and long lived resulting in marked

substrate and largely devoid of macroalgae (Filbee-

reduction in ecosystem services and negative

Dexter and Scheibling, 2014). Such barrens haveeconomic effects on associated commercial andbeen recorded throughout the world in coastal

recreational industries (Rocha et al., 2015).

temperate marine waters and can range in size
2

2

Many sea urchin species around the worldfrom 10s of m to 1000s of km . Temporarily, urchin

ordinarily represent valuable fisheries based on

barrens are considered an alternative stable state

harvesting their roe for eventual consumptionof a marine ecosystem persisting in timescales fromin gourmet restaurants. The roe is considered a

one year to several decades. The alternate stabledelicacy in many cultures commanding market

state of the urchin barren is typically a kelp forest.

prices up to $248/kg in Japan (Stefánsson et al.,There are marked differences in the biodiversity2017). Economic analyses suggest that there is an

and general productivity of the urchin barrens andunmet demand for good quality sea urchin roe.

kelp forests. While urchin barrens are characterized

Considering the favorable economics of sea urchin

by a predominance of urchins and coralline algae,

fisheries, the occurrence of an overpopulation of sea

kelp forests provide habitat supporting thousands ofurchins as seen in urchin barrens should represent

vertebrates, invertebrates and plant species. It is thisan opportunity to increase fisheries landings andkeystone host role of kelp that make the presence

profits. However, as previously stated, once urchinof kelp forests the desired stable state for marine

barrens are established in an area, the macroalage

resource management as it supports commercial andthat would ordinarily sustain their roe development

recreational industries including fishing and tourism.

is largely overgrazed resulting in the sea urchins

Over the last 4 decades, transitions between kelp

entering a metabolic dormancy during which little if

beds and sea urchin barrens have been widely

any roe is produced. This characteristic coupled with

reported along temperate coastlines globally.

sea urchinsʼ capacity to exist in low food conditions

During a kelp forest phase, urchin predation is thefor extended periods make the conventional fishingprimary mechanism keeping sea urchin populationsof the sea urchins commercially unfeasible as ain check (Filbee-Dexter and Scheibling, 2014).

solution to remove and restore urchin barrens to

However, due to various factors including both

kelp forests.

natural and anthropogenic, predator densities canAquaculture offers a potential economically viable

be reduced leading to shifts toward urchin barrens.

solution to aid in the removal of sea urchins from

Unlike conventional animal population theory, once

barrens. While full life-cycle aquaculture exists

sea urchin grazing consumes the surrounding

for some sea urchin species itʼs development as

macroalgal biomass, the urchin population does not

an industry has been hindered by relatively slow

collapse or relocate, but rather urchin growth rate

growth (5 years to reach market size) impacting its

decreases and they enter a reproductive dormancy

profitability (James et al., 2015; Unuma et al., 2015;as a result of changing their feeding activity to lessWilliamson, 2015). In contrast, gonad enhancement of

nutritious encrusting algae or microalgal biofilms

wild caught sea urchins can be achieved in relatively(Lawrence, 1982). Hence, once formed, urchinshort time periods, 6 - 12 weeks (Heflin et al., 2016).

barrens are a stable state and dominant feature

Sea Urchin gonad enhancement involves the captureof rocky reefs, surviving decades with individualof urchins with inferior roe qualities and subsequenturchins living up to 50 years on barrens (Ling andculture of the animals to actively improve their roe

Johnson, 2009). Additionally, modelling research

qualities for market. Following capture of the wildsuggests that in order to return an urchin barren to

sea urchins, gonad enhancement typically involves

a kelp forest state, urchin numbers must be reduced

housing the animals in an aquaculture system andto abundances significantly lower than the urchin

fed a diet designed to rapidly increase their gonad

Preliminary feasibility assessment of purple sea urchin, Strongylocentrotus purpuratus, roe enhancement.

size and improve their taste, texture and color

49

gonadosomatic index (GSI) (James et al., 2017).

commensurate with market expectations. With therecent increased prevalence and concern over sea

GSI = (gonad wet weight/total weight)×100

urchin barrens, research efforts have increased
to determine nutrition requirements and optimize

The urchins were fed to satiation with newculture systems for many species of sea urchinsfeed added every 3 days providing ample feed

(Brown and Eddy, 2015).

within the tank to reduce searching behavior or

Currently, a recently formed large sea urchin

periods where the urchins could not readily feed.

barren is affecting coastal kelp beds off California

Remaining uneaten feed after the 3 day period wasand Oregon, USA. It is estimated that purple sea

removed from the tank and replaced with freshurchin (Strongylocentrotus purpuratus) abundance infeed. Mortality was negligible throughout the study

both states have increased 10,000% above baseline

and tanks were cleaned weekly to remove feces

populations and have consumed over 90% of kelp

and biofouling on tank surfaces. Every 14 days, fivecoverage in northern California alone (Rogers-

urchins were randomly selected from each tank,

Bennett and Catton, 2019). This barren has attracted

dissected and GSI measured and recorded.

the attention of marine resource managers andChanges in test diameter and GSI across time

agencies in both states looking to address its affects

were calculated using a linear regression. In orderand restore kelp beds. The research detailed hereinto account for any tank effects, the average GSI

is a preliminary investigation into purple urchin (S.

from each replicate tank was used in the regression

purpuratus) gonad enhancement using alternativefor each treatment. To predict the time it would

feed stuffs as a potential tool to aid in the restoration

take to raise an urchin to marketable roe content

of kelp beds in an economically viable manner.

(GSI = 10%), the regression line equation wasused. In order to find changes in GSI between the

Materials and Methods

treatments over time while accounting for ourhierarchical experimental design, a nested ANOVAThe experiment was conducted at Moss Landing

using a linear mixed effects model was used. All

Marine Laboratories, California USA for a period ofassumptions about normality and homoscedasticity

10 weeks beginning in March, 2019 and concludingin the data and residuals were met. All statisticsin May, 2019. 12 circular (1000 L) flow-throughwere conducted using R.

tanks (8 L/min) were used for the experiment witheach one randomly assigned a diet treatment. The

Results and Discussion

four diet treatments included an unfed control, acommercially formulated pellet diet (Urchinomics),

Expectantly, there were no changes in test

giant kelp (Macrocystis pyrifera) and ogo (Gracilaria

diameter in any of the treatments across the course

pacifica). There were three tank replicates ofof the experiment (Table 1) due their reported

each treatment. The incoming seawater reflected

relative slow growth. However, all of the treatments

nearshore conditions; with the temperature ranging

showed an increase in GSI across the course offrom 10 - 17℃ and the salinity at 32 - 34 ppt. Tanks

the experiment (Table 2, Fig. 1). When accounting

were aerated with air stones and dissolved oxygen

for the ranked experimental design, there weremaintained between 6 - 8 mg/L for the duration ofdifferences in GSI between the treatments

the experiment. Each tank was initially stocked withthemselves and differences across time. The effect40 purple sea urchins, with a minimum test diameterof diet treatment on GSI also changed throughoutof 39 mm. The urchins were collected from a nearby

the course of the experiment as a function of timeurchin barren in Monterey Bay by scuba divers,(Nested ANOVA, Treatment: F = 16.2, p = 0.0009;

about a half a mile from shore. Before stocking

Day: F = 273.8, p < 0.0001, Treatment: Day: F = 15.9,

the tanks, 30 urchins were randomly sampled top < 0.0001). The urchins fed the formulated pellets

determine baseline values for test diameter andshowed the highest increases in GSI, followedLuke GARDNER, Helaina LINDSEY, Katherine NEYLAN, Walan CHANG, Katrina HERRMANN,
Max RINTOUL, and Katherine ROY

50

Table 1. Change in test diameter. The change in test diameter over time for each treatment was analyzed using
linear regression
TreatmentLine Equation

df

F statistic

R2

p-value

Controly = -0.02x + 48.25

1, 14

0.296

0.005

0.589

Formulated

y = -0.04x + 48.36

1, 14

0.9

0.014

0.346

Kelp

y = 0.01x + 47.97

1, 14

0.08

0.001

0.781

Ogo

y = -0.04x + 48.74

1, 14

1.14

0.017

0.289

Table 2. Change in GSI. The change in GSI over time for each treatment was analyzed using linear regression
TreatmentLine Equation

df

F statistic

R2

p-value

Controly = 0.76x - 1.08

1, 14

261

0.952

5.51E-10

Formulated

y = 1.97x - 2.65

1, 14

272.63

0.954

4.20E-10

Kelp

y = 0.83x - 0.92

1, 14

46.5

0.781

1.23E-05

Ogo

y = 1.23x - 1.22

1, 14

158.770.924

1.16E-08

Fig. 1.

Fig. 1. Scatter plot of GSI values over time in weeks. Each data point represents
a replicate tank average per treatment. Data points are distinguished totreatment level by a different color shapes/signs indicated in the figure key.
Solid lines are linear regressions fitted to the respective treatment data points.
by those that were fed ogo. Interestingly, theweeks) to rear the urchins on formulated pellets tourchins that were fed kelp showed similar growtha marketable GSI of 10%. Similarly to reach 10%

to the control treatment wherein urchins were

GSI it would take urchins 63 days (~9 weeks) fed

not provided any feed. Based on the GSI growth

an ogo diet, 91 days (~13 weeks) fed a kelp diet,curves in each treatment, it would take 45 days (~6and 101 days (~14 weeks) for urchins that are notPreliminary feasibility assessment of purple sea urchin, Strongylocentrotus purpuratus, roe enhancement.

51

fed but consuming tank biofilm. Note that the final

diet (Cyrus et al., 2013) but this was not assessed

two predictions are outside the range of the linear

for the study. While the ogo treatment yielded

regression for this study and thus likely unreliable.

relatively slower roe production rates compared

Aquaculture roe enhancement of purple urchinsto the pellet diet, the finding is still encouraging

is a potential tool for resource managers toas the duration is within a time period reported

restore kelp forests while also creating economic

as acceptable for other roe enhancement research.

opportunities for both fisheries and aquaculture

Furthermore, the feeding of a live seaweed capable

sectors. This activity also avoids wanton waste ofof vegetative/fragmentation growth like ogo, offersa marine resource as is typical of urchin cullingother benefits including excellent feed stability andactivities and the antithesis of resource management.

longevity, ability to maintain continuous culture

Feed type is considered the most critical aspect ofonsite and potentially extract dissolved nutrientssea urchin roe enhancement as it heavily influencesfrom the water column as a result of sea urchinboth the rate of roe production, texture, taste andculture. These characteristics and general simplicity

color (Heflin et al., 2016). All these characteristics areof the diet could complement the artisanal natureimportant to the economic feasibility of the activity.

typical of commercial urchin fisheries, affording

This preliminary study was conducted to examine

fishers an opportunity and low entry cost to enhance

gonad production rate for the biological feasibility of

their wild caught sea urchins. In contrast the kelp

urchin roe enhancement in purple urchins collected

positive control treatment and negative control

from barrens off the California and Oregon coasts.

treatment curiously yielded much slower but similar

While roe growth rate is only one aspect of roe

roe production rates. A potential explanation forenhancement the speed at which it develops will

the unfed control treatment displaying roe growthmarkedly affect profitability. The different feed

rates is a verification of sea urchins hardiness. The

types assessed for the study were selected to gauge

negative control tanks experienced observable

relative growth rates on likely feed stuffs that could

biofouling between the weekly cleanings and thus

conceivably be used should the activity become

it is theorized that the biofilm on the tank surfaces

established. They included an easily cultured

provided enough nutrition to support modest

seaweed (ogo) and a commercially formulated pellet

roe production. As all tanks in the experiment

diet (Urchinomics). The natural diet of kelp was

experienced the same biofouling activity it isalso used as a type of positive control to compare

unlikely to have confounded the results of thetheir growth rates, although the authors note thatexperiment overall.

due to the local presence of sea urchin barrens,

In conclusion this preliminary research has

kelp is not a viable feed stuff for roe enhancement.

indicated that rapid roe enhancement of purple

Similarly a negative control was also used, whereinurchin derived from urchin barrens in California,

no exogenous feed was supplied to the sea urchins.

USA is feasible using both a formulated pellet feed

Expectantly the pelleted diet treatment resulted inand a live red seaweed species. While it was not

the most rapid roe production reaching a marketablein the scope of this preliminary experiment, future

GSI of 10% in just over 6 weeks. This is particularly

research for roe enhancement of purple urchinsnoteworthy when considering the starting GSI of theshould include a more thorough assessment of

sea urchins was >0.5%. Such a roe production rate

nutrition requirements including proximate analysis

is on par with some of the fastest roe enhancementof feed types, measurements of digestibility andrate reported elsewhere for other species (James,

intake rate as well as quantitatively assessing the2006). Also encouraging is that the ogo seaweed

resulting roe for important market characteristics

diet yielded relatively rapid roe production rates as

including color, texture and taste. Based on thewell taking approximately 9 weeks to reach market

outcome of these and other biological considerations

size. The disparity between these two treatments isof purple urchin (S. purpuratus) roe enhancement, an

likely due to caloric density and digestibility being

economic feasibility is also required to determine thegreater in the pellet diet compared to the seaweedcommercial potenital of this aquaculture activity for

52

Luke GARDNER, Helaina LINDSEY, Katherine NEYLAN, Walan CHANG, Katrina HERRMANN,
Max RINTOUL, and Katherine ROY

transforming urchin barrens back to kelp forests.

Mar. Ecol. Prog. Ser., 374, 113–125.
Ling S. D., Scheibling R. E., Rassweiler A., Johnson

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assessment. Nofima Report, 7/2017, 1-22.

Williamson J. E., 2015: Sea urchin aquaculture in

James P., Siikavuopio S. I., and Mortensen A.,

Australia, in “Echinoderm Aquaculture” (ed.

2015: Sea urchin aquaculture in Norway, in

by Brown N. P., and Eddy S. D.), John Wiley &"Echinoderm Aquaculture" (ed. by Brown N.

Sons, Inc., Hoboken, New Jersey, pp.225-243.

P., and Eddy S. D.), John Wiley & Sons, Inc.,

Wiley Online Library (doi:10.1002/9781119005810.

Hoboken, New Jersey, pp. 147-173. Wiley Online

ch10)

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James P. J., 2006: A comparison of roe

Annotated Bibliographyenhancement of the sea urchin Evechinus
chloroticus in sea-based and land-based cages.

(1) Filbee-Dexter K., and Scheibling R. E., 2014:

Aquaculture, 253, 290–300. (doi.org/10.1016/

Sea urchin barrens as alternative stable states ofj.aquaculture.2005.03.050)

collapsed kelp ecosystems. Mar. Ecol. Prog. Ser.,

Lawrence J. M. (ed.), 1982: Echinoderms: Proceedingsof the international conference, Tampa Bay.
CRC Press. Boca Raton, Florida, 572pp.

495, 1–25. (doi.org/10.3354/meps10573)
This paper provides an excellent description of
sea urchin barrens as to what they are comprised of,Ling S. D., and Johnson C. R., 2009: Populationand their extent both temporally and spatially acrossdynamics of an ecologically important range-

the world. The authors examine and list the drivers

extender: kelp beds versus sea urchin barrens.

of phase shifts between barrens and kelp forests.

Preliminary feasibility assessment of purple sea urchin, Strongylocentrotus purpuratus, roe enhancement.

53

They describe different thresholds for forward(3) Unuma T., Sakai Y., Agatsuma Y., and Kayaba(to barrens) and reverse (to kelp beds) shifts, inT., 2015: Sea Urchin Aquaculture in Japan, in

accordance with alternative stable-state dynamics.

“Echinoderm Aquaculture” (ed. by Brown N. P.,

They surmise that accelerating climate change andand Eddy S. D.), John Wiley & Sons, Inc, Hoboken,

increasing anthropogenic impacts play important

New Jersey, pp.75-126. Wiley Online Library (doi.

roles in altering alternative stable-state dynamics

org/10.1002/9781119005810.ch5)

and triggering phase shifts.

This is the most recent review of urchinaquaculture in Japan. Japan is the foremost(2) Heflin L. E., Makowsky R., Taylor J. C., Williams M.

consumer of urchins and sigificnfact producers of

B., Lawrence A. L., and Watts S. A., 2016: Production

urchins both from ranching and closed life cycleand economic optimization of dietary protein andaquaculture. The chapter details the history ofcarbohydrate in the culture of Juvenile Sea Urchinurchin fisheries in Japan and the rise of urchin

Lytechinus variegatus. Aquaculture 463, 51–60. (doi.

barrens and urchin aquaculture. The review

org/10.1016/j.aquaculture.2016.05.023)

discusses the diver mediated destruction of urchinsThis manuscript is a through collection of urchinto bring back kelp beds as well as reseeding efforts

feeding experiments to understand the nutrient

to restore overfished urchin grounds. The review

requirements of urchins for aquaculture. The paper

also discusses the development of full life-cycle

creates predictive models of growth, production andaquaculture to meet both reseeding and commercial

efficiency outcomes and generates economic analysis

production requirements. Also the movement ofmodels in relation to these dietary outcomes forurchins from barrens to other kelp grounds andurchins held in culture. The models compare dietary

aquaculture facilities for commercial fattening are

requirements and growth outcomes in relation

detailed.

to economic costs and provide insight for futurecommercialization of sea urchin aquaculture水 研 機 構 研 報， 第 50 号，55 － 66， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，55－66，2021

55

Potential impacts and management of ocean acidification on
Japanese marine fisheries and aquaculture.
Haruko KURIHARA＊

Abstract: Marine fisheries have great importance economically and for food production,
particularly in Asian countries including Japan. Although the total value of fisheries production
has decreased in Japan since 1982, it still has enormous commercial value accounting for about
US$157 billion (JPY15,700 billion). Additionally, the world demand for seafood is expected tofurther increase with the growth of human population and income in developing countries. Ocean
acidification (OA), which is caused by the increase of atmospheric CO2, is now an increasing
cause for concern as a major threat to marine fisheries together with global warming. Because
OA causes a decrease in calcium carbonate saturation, most marine calcifiers, which include anumber of commercially important shellfish such as mollusks and crustaceans, are expected tobe particularly affected by OA. In this study, an overview is provided of the scientific knowledge
of OA with regard to commercially important organisms and the potential impacts on marine
fisheries and aquaculture in Japan. Potential management and adaptive strategies to mitigate
impacts of OA on marine fisheries in Japan are also discussed.
Key words: ocean acidification, fisheries, Japan, aquaculture, calcifiers
Introduction

temperature due to global warming is now causingchanges in species distribution and productivity ofMarine fisheries have great importancethe ocean (Cheung et al., 2013). In addition, “ocean

economically and with food production, particularly

acidification (OA)” is being highlighted as a threat toin Asian countries including Japan. Seafoodmarine fisheries and aquaculture (Doney et al., 2009).

consumption has steadily risen since the 1960s (FAO,

In this paper, an overview is provided regarding the2009) and reached around 80 million tonnes recently

potential impacts of ocean acidification on marine(Watson and Pauly, 2001). The demand for seafood

fisheries and aquaculture in Japan, which is one ofis expected to further increase with the growth of

the world’s largest fishery industries. Additionally,

the human population and income in developing

potential management and mitigation solutions arecountries. Continuously increasing pressure on

discussed.

supply of marine food sources is a principal threat
to future fisheries. In addition to fishing activities,

Background of Ocean Acidification

impacts of climate change are emerging as anotherserious challenge for the marine fisheries industry

Since the Industrial Revolution, atmospheric(Brander, 2007). A number of studies have showncarbon dioxide (CO 2) concentration has steadily

that global environmental change can severely affectincreased from an average of 280 µatm to thethe entire marine ecosystem and directly affectpresent 400 µatm value (IPCC, 2014). Increased

economically important fish and shellfish species

atmospheric CO 2 is quickly absorbed into the(Allison et al., 2009, Cheung et al., 2010). Risingseawater and dissolved into bicarbonate ion (HCO3-)


2020年12月11日受理（Accepted on December 11, 2020）＊
University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan
E-mail: harukoku “at” sci.u-ryukyu.ac.jp

Haruko KURIHARA

56

Fig. 1or carbonate ion (CO 32-) and hydrogen ions (H +).

350

decreases. Because seawater calcium carbonate
saturation (Ω) is defined by the amount of calcium
ion concentration ([Ca2+]) and [CO32-],

Ω will also

decrease with OA.
Ω = [Ca2+] [CO32-] / Ksp*


(Ksp* = calcium carbonate solubility product)

2017

2014

2011

2008

2005

2002

1996

1993

1990

1987

1984

0
1981

pH decreases, CO 3 2- reacts with H + forming

Marine fisheries

1978

50

1960

to seawater carbonate chemistry, as the seawater
HCO3- and hence the CO32- concentration ([CO32-])

Marineaquaculture

100

1975

the end of this century (IPCC, 2014). According

150

1972

the pH to decrease by another 0.1-0.4 units by

1969

Revolution, and IPCC scenarios (RCP2.6-8.5) expect

Island fisheries

200

1966

already decreased by 0.1 unit since the Industrial

Island aquaculture

250

1963

2003). The present seawater average pH (8.1) has

300
Catch value (Billion USD)

increase in atmospheric CO2 (Caldeira and Wickett,

1999

Therefore, seawater becomes more acidic with an

Year

Fig. 1. The transition of marine fisheries, marine
aquaculture, inland fisheries and inland aquaculture
total catch values in Japan since 1960. The fisheries
catch value steeply increased since 1960 and peakedin 1982. Since then the catch value continuously
decreased to the present value of about US$157
billion (JPY15,700 billion).

The decline of Ω is suspected to affect marine
calcifiers including mollusks, echinoderms andt) around 1982, however, the fisheries catch amountcrustaceans. For the formation of calcium carbonate

has since decreased to present levels of around(CaCO 3 ) shells and skeletons, marine calcifiers

4 million t (MAFF, 2019) (Fig. 1). Nevertheless,actively excrete H+ by ATPase pumps to increase

the present value of Japanese fisheries is still ofthe Ω at the compartment they extract CaCO 3

major commercial importance with a total value of(Allemand et al., 2011). Therefore, OA is expected

about US$157 billion (JPY15,700 billion as of 2018).

to affect the CaCO3 production of marine calcifiers

Japanese marine fisheries are mainly classified into 3

because they will need more energy to compensate

categories: marine fisheries, marine aquaculture andfor acidosis at those compartments (Al-Horani etisland fisheries. Marine fisheries are further dividedal., 2003; Cohen and Holocomb, 2009). In addition

into 3 categories according to the location they are

to affecting calcification processes, many otherconducted: distant marine fisheries operated at highbiological processes, such as metabolism andseas and foreign countries EEZ, offshore marine

protein synthesis, are also suspected to be affectedfisheries operated within Japan’s and neighboring

by OA because acidosis can cause the decline ofcountries EEZ, and coastal fisheries operated in

intracellular pH unless the organism has the ability

Japanese coastal waters. The clear trends in Japan’s

to compensate their internal acid-base balance

fisheries are that the catch amount in the distant(Pörtner and Farell, 2008). Therefore, organisms thatand offshore marine fisheries have decreased fromhave less compensation capacity, such as marine

2.3 and 6.7 million t in 1986 to 0.3 and 2.0 million t ininvertebrates and those in early developmental

2018, respectively (Fig. 2). During this same period,

stages, are expected to be particularly affected by

the amount of aquaculture has remained stable andOA.

the relative percentage value of aquaculture to thetotal fisheries value has steadily increased since 1960
Potential Impacts of Ocean Acidification on

from 10% to about 38% with a total value of about

Marine Fisheries in Japan

US$60 billion (MAFF, 2019) (Fig. 2). Therefore,
the Japanese fisheries is relying more and more on

The Japanese fisheries catch amount steadily
increased since 1960 and reached a peak (12.7 million

coastal fisheries and aquaculture than ever before.
Though finfish are still the main target ofOcean acidification impacts on Japanese marine fisheries

Fig. 2

Table 1. Japan catch value of marine fisheries andaquaculture in 2017. Values are shown in billion U.S.D

1400

Catch amount (X1000 t)

1200
1000

Inland aquaculture

800

Inland fisheries

600
400

Marine aquaculture
Offshore marine fisheries

Coastal marine fisheries

200
0

57

Distant marine fisheries

Year

Fig. 2. The transition of catch amount of distantmarine fisheries, offshore marine fisheries, coastal
marine fisheries, marine aquaculture, inland fisheries
and inland aquaculture since 1979. The catch amountof distant and offshore marine fisheries steeply
decreased since 1984.

Marinefisheries

Marineaquaculture

Fish

66.7

25.2

Mollusks

9.0

9.5

Crustacean

6.1

0.7

Sea urchin

1.2－

Cephalopod

8.9－

Seaweed

2.0

14.1

Others

1.9

0.1

Total

96.0

49.7

2007). Veliger larvae of the oysters were found to
become smaller in size, and shell mineralization wascompletely inhibited in 45% of the larvae reared
under high CO2/low pH conditions (Kurihara et al.,2007). Recently, a number of studies demonstrated

Japanese marine fisheries, the proportion of othersimilar effects of OA on the larval stage of several

organisms has increased primarily after thedifferent mollusk species including oysters (C.

collapse that occurred after 1982, and now othervirginica, Saccostrea glomerata), scallops (Argopecten

organisms account for about 30% of the total catch

irradians), hard clams (Mercenaria mercenaria),

value. These include mollusks such as scallops andmussels (M. galloprovincialis, M. edullis) andoysters, crustaceans including shrimps, crabs andabalone (Haliotis coccoradiata) (Kurihara et al.,krill, sea urchins, cephalopods and seaweeds (Table

2008a; Tamalge and Gobler, 2009; Parker et al., 2009;1). In particular, many shellfish are now importantGazeau et al., 2010; Byrne et al., 2010). Additionally,aquaculture species and mollusks account for 20%

fertilization rate of scallop eggs (Mimachlamysof the total catch value (about US$9.5 billion). An

asperrima) was found to decline with high CO2/

important concern is that many of these organisms

low pH but not for oysters (Scanes et al., 2014;

are highly susceptible to OA conditions.

Havenhand et al., 2008). Because early life stage
survival is the bottleneck to the recruitment of

1. Impacts on mollusk fisheries

most marine invertebrates, these studies indicatedMollusks are the second largest group ofthat OA could strongly impact the population sizecommercial fisheries organisms next to finfish, andof many commercially important species. OA wasthe catch value accounts for more than US$17 billion

also reported to affect the calcification rate of adult(MAFF, 2019). The most economically importantoysters (C. gigas), mussels (M. edulis), and adultmollusks in Japanese fisheries include scallops,

scallops (Argopecten irradians) (Gazeau et al., 2007;

abalones, oysters and clams. Scallops in Hokkaido

Ries et al., 2009). The immune system of adult C.

and oysters in Miyagi and Seto Inland Sea havegigas was found to be significantly affected by high

the greatest market values for mollusks in Japan.

CO2/low pH, suggesting that OA can increase their

Oysters are mainly cultivated through aquaculture,disease susceptibility (Wang et al., 2016). Synergistic

while scallops are both wild caught in coastal waters

effects of high CO2/low pH and high temperatureand farmed.

were also found in both oysters and scallops.

The effect of OA on mollusks was first

These studies indicated that global warming anddemonstrated in the mineralization of Pacific oyster

ocean acidification could intensify their negative(Crassostrea gigas) larval shells (Kurihara et al.,effects on these organisms (Parker et al., 2009;

58

Haruko KURIHARA
Fig. 3

Schalkhausser et al., 2014). From these studies, most

Planktonic stage

mollusk fisheries including oysters and scallops
have been suggested to be at high risk under OA
conditions. This prediction was confirmed in 2009,
when U.S Pacific Northwest Pacific oyster (C. gigas)

Dispersal period
Impact on early
developmental
stage

Impact onlarval stage

hatcheries experienced a substantial production
failure of C. gigas larvae production (Pacific Coast
Shellfish Growers Association, 2010). This loss wasinterpreted to be related to the fact that the year’s
intense upwelling brought high CO2 deep seawater
up to the surface which acidified the seawaterused at these hatcheries. This has highlighted

Impact on reproduction

Catching individuals
Growth period

the potential extensive effects of OA on oyster
fisheries that may occur all over the world, including
Japan, and the importance of considering adaptive
strategies to mitigate OA impacts.

Impact on
settlement

Impact on growth

Benthos period

Fig. 3. Ocean acidification impacts on the whole life
cycle of a sea urchin. OA can affect several different
life cycle stages and different biological activities.

2. Impacts on sea urchin fisheries
Sea urchins are also commercially important

Paracentrotus lividus, Pseudechinus huttoni,organisms in Japan, where the gonads are eaten

Evechinus chloroticus, Sterechinus neumayeri,

as “sushi” topping and have a catch value of about

Arbacia dufresnei, Centrophanus rodgersii, T.

US$1.2 billion. In Japan, they are mainly caught

gratilla (O’Donnell et al., 2010; Brennand et al., 2010;

around the northern island, Hokkaido, and the main

Clark et al., 2009; Stumpp et al., 2011; Martin et al.,commercial sea urchin species are Strongylocentrotus

2011; Foo et al., 2012; Byrne et al., 2013; Zhan etintermedius and S. nudus, though other sea urchinsal., 2016). Growth and survival of sea urchins weresuch as Pseudocentrotus depressus, Anthocidarisreported to decline with OA in not only planktonic

crassipina and Tripneustes gratilla are also caught.

larval stages but also during juvenile stages

Effects of OA on sea urchins are also well

(Shirayama and Thornton, 2005). Gonad development

studied and represent some of the first studies

and physiology of adult sea urchin, H. pulcherrimus,that demonstrated the effects on early life stages

were also found to decrease when cultured for 9(Kurihara and Shirayama, 2004; Kurihara et al.,months under high CO2/low pH seawater (Kurihara

2004; Kurihara 2008). When eggs of the sea urchin

et al., 2013). Similarly, reduced growth and poor

Hemicentrotus pulcherrimus and Echinometra

gonad production were observed in T. gratilla

mathaei were reared under high CO 2 /low pH(Mos et al., 2016), suggesting that OA can directly

seawater, it was found that the fertilization rate

reduce both the product value and the productivity

and developmental speed of the embryos decreasedof sea urchins. Test thickness and strength were

(Kurihara and Shirayama, 2004; Kurihara et al.,also found to be affected by OA, suggesting less2004). Additionally, the larval skeleton formation

resistance of sea urchin to predation (Byrne et al.,and larval size of sea urchins were found to be2014). The immune system was also found to be

negatively affected by OA, which potentially results

affected by OA, indicating their higher susceptibility

in the decrease of their population size (Kurihara

to pathogens (Leite Figueiredo et al., 2016). Theseand Shirayama, 2004; Kurihara et al., 2004). Larval

studies also suggested that OA can affect several

physiology was also found to be affected by OA

different life stages and different biological activities,

and similar results were found in a number of otherimpacting their population size (Fig. 3).

sea urchin species, including Strongylocentrotus
drobachiensis, S pupuratus, S. franciscanus,
S. intermedius, Heriocidaris erythrogramma,

3. Impacts on crustacean fisheries
There are several crustaceans that are consumed

Ocean acidification impacts on Japanese marine fisheries

59

as a food source in Japan, and the most commercially

occidentalis, and Melicertus plebejus (Wickins, 1984;

important species include Japanese spiny lobsters

Kurihara et al., 2008b; Kikkawa et al., 2008; Ries et(US$64 million), prawns (US$18 million), snow crabs

al., 2009; Dissanayake et al., 2010). Only short-term(US$192 million), Japanese blue crabs (US$23 million)

impacts of extremely high CO2 conditions (3-15%)

and krill (US$13 million). The impacts of OA on

were studied on juvenile Marsupenaeus japonicus,

crustaceans seem to be highly variable among

results of which showed high tolerance to CO 2

species, where some species show strong tolerance(Kikkawa et al., 2008); however, long-term effects

to high CO2/low pH, while some other species show

should also be studied because the effect of 1,000

high sensitivity (Whiteley, 2011; Kroeker et al., 2010).

µatm CO 2 on P. pacificus only became apparent

In terms of commercially important species, Ries

after 30 weeks of exposure. Furthermore, not only

et al. (2009) reported that the calcification of theare commercially important crustaceans as a humanjuvenile American lobster (Homarus americanus)

food source affected by OA, but also trophicallywas not affected by high CO2/low pH conditions.

important prey species such as copepods andHowever, growth rate and mineralization of

Antarctic krill, suggesting critical impacts of OA

carapace of larvae and postlarvae of Americanon entire marine ecosystems (Kurihara et al., 2004;

lobster and European lobster (H. gammarus)

Kawaguchi et al., 2010; Cripps et al., 2015)

have been found to be affected by OA (Arnold
et al., 2009; Keppel et al., 2012). Additionally, the

4. Impacts on cephalopod fisheries

immune response of the Norway lobster (Nephrops

Only a few studies have evaluated the effects ofnorvegicus) has been found to be suppressed by

OA on cephalopods, including octopus and squid.

high CO2/low pH, suggesting a potential increase

While reduced metabolism was reported for jumboof disease susceptibility of lobsters due to OA

squid Dosdicus gigas (Rosa and Seibel, 2008), neither

conditions (Hernroth et al., 2012). Although there

calcification nor growth of the cephalopod Sepia

is still no study evaluating the effects on Japanese

officinalis was affected by high CO2/low pH seawater

spiny lobster (Panulirus japonicus), which is one

and they showed efficient ability to regulate acid-the most commercially important crustaceans inbase status (Gutowaska et al., 2009). However,Japan, these studies suggest that OA can potentiallyhigh CO2/low pH was observed to depress energy

impact the lobster fisheries in Japan. Low seawater

expenditure rate of S. officinalis embryos (Rosa et al.,pH significantly decreases embryonic development2013). Additionally, increased time to hatching andand hatching success on the Florida stone crab

effects on statoliths were found on the commercially(Menippe mercenaria), while the effect on hatchingimportant squid Doryteuthis pealeii, reared under

was highly variable, suggesting that there are somehigh CO 2/low pH conditions, suggesting impacts

differences in tolerance capacity among individuals

of OA on their behavior and survival (Kaplan et(Gravinese, 2018). Negative impacts on growth andal., 2013). A more recent study evaluating thesurvival rate of juvenile Red King Crab (Paralithodes

swimming behavior of paralarval D. pealeii by three-camtschaticus) and snow crab (Chionoecetes bairdi)

dimensional video system did not show clear effects

exposed at high CO 2/low pH condition were alsoof high CO2 conditions on the swimming behavior

observed (Long et al., 2013). Punt et al. (2016)(Zakroff et al., 2018).

conducted a model analysis evaluating the potential
effects of expected future OA conditions on the

5. Impacts on seaweed fisheries

Tanner crab fishery and estimated that catch andSeaweed aquaculture has great commercial

profits would decrease by 50% over the next 20

importance in Japan and the farmgate value ofyears if survival of the crabs is affected by high CO2

red algae, Pyropia spp., collectively called “Nori”

conditions.

in Japanese language, accounts for about US$11.6

Ocean acidification has also been observed tobillion. Undaria pinnafida called “Wakame” andaffect several prawn species, including Palaemon

Japanese kelp such as Saccharina japonica calledpacificus, Penaeus serratus, P. elegans, P. monodon, P.

“Konbu” are also commercially important seaweeds

Haruko KURIHARA

60

harvested in Japan. In contrast with most marine

et al., 2011). Survival rate of Menidia beryllina in

calcifiers, seaweeds are expected to be positively

early life stages was demonstrated to decrease byaffected by OA, because increases in seawater pCO2

73% even at 780 µatm CO2 seawater (Baumann etpotentially increases their photosynthetic rate andal., 2012). Meta-analysis using data obtained fromproductivity. However, the majority of seaweeds

different fishes indicated that high sensitivity on-

are known to use both CO2 and HCO3 for carbon

early life stages were broadly observed particularly

fixation and have evolved a carbon concentratingon pelagic and euryhaline fishes (Cattano et al.,mechanism (CCM) and hence may not be positively2018). The size of otoliths made of CaCO3 was foundaffected by the increase of seawater pCO2 (Kochto increase in several fishes, though it is still not

et al., 2013). For example, although many seagrassclear if this change affects sound detection (Munday

species such as Zostera marina show increased

et al., 2011; Heuer and Grosell, 2014). Furthermore,productivity under OA conditions (Palacios andsensory lateralization, learning ability, predator

Zimmerman, 2007), the photosynthetic rate andavoidance behavior and spatial orientation haveproductivity of the giant kelp Macrosystis pyrefera

been found to be disrupted under high CO2/low pH

did not change with seawater pCO2 (Fernandez etconditions particularly in tropical fishes (Mundayal., 2015). Therefore, seaweed fisheries are suggested

et al., 2009, 2014; Dixson et al., 2015). Moreover,not to be negatively or positively affected by OA,

even though most adult fishes have the capability

though increased temperatures would affect theseto compensate the acid-base balance when reared

species. Meanwhile, although seaweed may haveunder high CO2 conditions, long-term exposure toless capacity of sequestering carbon comparedhigh CO2/low pH conditions are suggested to affectto the seagrass, seaweed farms can potentiallyfish energy budgets because the regulation of pH

work as pCO2 offset by being harvested as humanis energetically costly (Heuer and Grosell, 2016).

resources (Froehlich et al., 2019). Particularly, taking

Furthermore, although low growth rates of fish

into account the market size of seaweed farming inlarvae have been reported (Frommel et al., 2016), no

Japan and other Asian countries, seaweed fisheries

clear effects have been found on swimming speed or

may potentially work as one of the strategies forstandard metabolic rate of most adult fishes (Melzner

sustainable ocean use.

et al., 2009; Lefevre, 2016; Esbaugh, 2018).

6. Impacts on fish fisheries

Potential Management and Solutions

Marine fishes have been thought to be tolerant
to OA because of their high capability to regulateWith the increase of scientific knowledge

acid-base balance and the internal pH at highregarding the effects of high CO2/low pH seawater

pCO2 seawater (Kroeker et al., 2013; Wittmann andon many marine organisms, it has become evident

Pörtner, 2013). Branchial cells are known to actively

that Japanese fisheries are likely greatly impacted

excrete ions through the Na+/K+-ATPase channels

by OA. However, much research is still needed tousing energy to regulate the internal pH (Pörtner

better understand OA in the context of Japaneseand Peck, 2010). However, more recent studies

fisheries including topics such as the present OA

demonstrate that fish in early life stages with lessconditions in Japanese coastal waters, presence ofdeveloped acid-base regulation capabilities can behotspots, potential populations highly vulnerable orhighly vulnerable to OA. For example, Atlantic

more tolerant to OA, the effects of multiple stressherring (Clupea harengus L.) embryos show a decline

exposure on organisms, and many others.

in protein biosynthesis when reared under a highBecause coastal waters are strongly affected by

CO 2/low pH conditions (Franke and Clemmesen,the land through receiving freshwater and organic2011). Additionally, high CO 2/low pH conditions

inputs, seawater carbonate chemistry can be highly

were reported to cause tissue damage on various

spatially and temporally variable compared toorgans including the liver, pancreas and kidneys

open water. Therefore, different locations can haveof Atlantic cod (Gadus morhua) larvae (Frommel

different seawater carbonate chemistry. There is a

Ocean acidification impacts on Japanese marine fisheries

61

fundamental need to know the carbonate chemistry

known regarding OA effects, such as the sea urchinof each location of interest to predict and manage

S. nudus, the Japanese spiny lobster (P. japonicus),

the potential impacts of OA on the local marine

seaweeds and most marine fishes.

fisheries. Nevertheless, locations that have higher

For aquaculture species, existing scientific dataseawater residence times, such as inner bay areas

can be used for better management and solutions

and locations that show higher organic carbon

to mitigate OA impacts. For example, controlling

decomposition, are suggested to have higher risk

seawater pH in hatchery tanks or within fish cages

to OA. This is because remineralization of organic

is possible by adding alkaline solutions or bubbling

matter by bacteria can decrease seawater oxygen

controlled gas. Additionally, avoidance of crowded

concentration and increase CO 2 concentrationfish cages or tanks, particularly during the summer

causing local hypoxia and hypercapnia concurrently.

season, can decrease the risk of hypercapnic

For example, pH and Ω in Tokyo Bay was foundconditions. Because many studies revealed thatto be lower in bottom water than in surface water

the early life stage of most marine organisms arebecause of remineralization of organic carbon

particularly vulnerable to OA (Kurihara, 2008),(Yamamoto-Kawai et al., 2015). Additionally, lowmanagement efforts can be focused on those stages.

pH and low oxygen was found to occur in bottom

Several studies also demonstrated that when adults

water in Shizugawa Bay during the summer

are reared under high CO2, the next generation can(Kurihara et al., unpubl.). This suggests thatshow higher tolerance to CO2. For example, larvaeorganisms living in these locations can be exposedof Sydney rock oyster Saccostrea glomerate spawned

to increased OA and low oxygen simultaneously

by adults cultured under high CO 2 conditions,and may be synergistically affected by theseshow higher growth rate compared to the larvae

stressors. Additionally, such conditions put benthic

spawned by individuals cultured under control

organisms at particularly high risk during summer

conditions (Parker et al., 2012). Additionally, asseasons. These data can be highly informative foralready mentioned, many studies also demonstrated

aquaculture site selection and early detection of OA

that tolerance capacity to OA can vary amongrisks to the marine fisheries. Additionally, reduction

individuals. Therefore, selective breeding of OA

of eutrophication can be effective in reducing theresistant strains could be one potential solution forrisk of both OA and hypoxia.

aquaculture.

Along with environmental factors, further

Finally, even though many scientific studies arebiological data and assessment models for evaluating

now available on the effects of OA, general concern

the effects of OA on commercially important marine

for OA impacts on Japanese fisheries is still limited.

fisheries are essential for better management.

Further scientific efforts addressing the issues

Recent studies have focused on using socioeconomic

caused by OA for use by decision makers andmodels to predict the population of shellfishes,

fisheries and environment management sectors insuch as scallops and red king crab, under different

Japan is essential to develop adaptive strategies andCO 2 emission scenarios (Cooley et al., 2015; Punt

to be prepared for environmental changes in the

et al., 2014). These studies can provide importantnear future.

information for decision makers and fisheries
managers for better fisheries management in dealing

Acknowledgementswith climate change. There is still a lack of biological
data and very little information on the effects of OA

This work was supported by funding from theon the entire life cycle of a species. Furthermore,

Pew Marine Conservation Fellow Program andthere is little data regarding long-term effects on

Japan Society for the Promotion of Science (JSPS)

reproduction of many commercially important

KAKENHI, grant number: 15H04536.

species, which could be the focus of future studies.
Additionally, there are still several commercially
important species where there is no information

Haruko KURIHARA

62
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67

Sustain seafood resources in the U.S. affiliated Pacific islands- status
and strategies
Cheng-Sheng LEE＊

Abstract: Seafood plays important crucial socio-economic roles in daily life of the U.S.-affiliatedPacific Islands (USAPI). USAPI includes American Samoa, the Republic of the Marshall Islands(RMI), the Federated States of Micronesia (FSM), the Commonwealth of the Northern Mariana
Islands (CNMl), Guam, and the Republic of Palau (Palau). With less than 2,558 km2 in total landmass but extended Exclusive Economic Zone (EEZ), the primary source of dietary protein for
Pacific islanders has to come from the Ocean. The per capita seafood consumption exceeded global
average. In 2015, per capita seafood consumption in Oceania was 25.0 kg/year vs 15.5 kg/year
worldwide (FAO, 2018).
Seafood comes from capture fisheries and aquaculture but capture fisheries are the main seafoodsource for the USAPI. Increasing threats from climate change and overfishing have diminishedthe sustainability of yield from capture fisheries. Although there were optimistic reports on
status of tuna stocks (key species in capture fisheries) in the Pacific, foreign fishing companies
have exported majority of their catch to consumers outside the islands. Islanders have relied on
subsistence catch to meet their demand. As the harvest from nearshore fishery declined, they
have to consider secure their seafood supplies from other means such as aquaculture.
USAPI can take the advantages of their superior natural resources, such as pristine water, yearround warm weather, and isolated condition for disease prevention to sustain their seafood sources
via fish farming. However, they have to overcome constraints to aquaculture development such as
small land area, natural hazards for some islands, insufficient knowledge base, shortage of available
capital, distant markets, and poor transportation systems. Except natural hazards for someislands, the other constraints are solvable. A good strategic development plan is essential to reveal
aquaculture potential in the region.
This report discuss the potential threat of climate change to fishery, review the current status
and challenges of aquaculture, and finally present some suggestions on future development.
Key words: seafood, aquaculture, sustainability, climate change, US affiliated Pacific Islands, foodsecurityIntroduction

the USAPI. USAPI include American Samoa, theRepublic of Marshall Islands (RMI), the Federated

Seafood comes from both capture fisheries

States of Micronesia (FSM), the Commonwealth ofand aquaculture. In 2016, capture fisheries andthe Northern Mariana Islands (CNMl), Guam, andaquaculture contributed equally to the total global

the Republic of Palau (Palau). This region, composed

seafood production. For the U.S.-affiliated Pacificof thousands of tiny islands spread between the

Islands (USAPI), capture fisheries remains thelatitudes of 15° N to 14° S and the longitudes ofmain source of seafood supplies. Seafood plays

134° E to 170° W, has less than 2,558 km2 in total

a key important role in supporting livelihood of

landmass. With their Exclusive Economic Zone
2020年12月11日受理（Accepted on December 11, 2020）＊
College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, 1955 East West Road, Ag. Sci. 216, Honolulu, HI 96822
E-mail: chenglee “at” hawaii.edu

Cheng-Sheng LEE

68(EEZ), it extends across an area as large as thetransportation systems. Other than natural hazards

continental United States. This extended EEZ

for some islands, the remaining constraints areis their primary nature resource for living andsolvable. Currently, the Pacific islands is still the

economic development. Their ancestors have relied

least developed region in terms of aquacultureon their surrounding water to support their daily

production worldwide with annual production

living and recreations. Seafood was their primaryaround 24,091 tonnes or less than 0.022% of thesource of dietary protein until strong influences fromtotal worldwide aquaculture production (FAO, 2019,western countries. However, the per capita seafoodwww.fao.org/fishery/statistics/software/fishstati/

consumption still exceeded global average. In 2015,en). A good strategic development plan is essential

per capita seafood consumption in Oceania was 25.0

to reveal aquaculture potential in the region. Withkg/year vs 15.5 kg/year worldwide (FAO, 2018).

the complex political systems in the region, eachWithin Oceania, Pacific Small Island Countries (PICs)

island’s entities must have their own development

had an average national fish consumption ranging

plan. In this region, there are two territories, one

from 55 kg to 110 kg per person per year (Bell et al.,commonwealth and three independent countries.

2009).

This report will discuss the potential threat of

Climate change and overfishing have threatened

climate change to fishery, review the status andthe sustainability of yield from capture fisheries

challenges of aquaculture, and finally present somein USAPI. Although there were some optimistic

suggestions on future development.

reports on future status of tuna stocks in the Pacific,
Food Sources

foreign fishing companies have exported majorityof their catch to consumers outside the islands.
Islanders have relied on subsistence catch to meet

Fish plays an important role in food security intheir demand. As the harvest from nearshore fishery

USAPI. Traditionally, nearshore fishery is the main

declined due to overfishing and pollutions, theysource of seafood for local consumption. Inhabitants

have to consider secure their seafood supplies via

were able to survive on the catch plus limited

aquaculture. In 2016, per capita seafood supply for

agriculture farming products from land. As distant

Oceania was down to 24.3 kg while worldwide per

fishery advanced, government leases out offshore

capita supply increase to 19.7 kg (FAO, 2019).

fishing ground to foreign countries to harvest and

USAPI has superior natural resources forexport to outside of the countries. The fishing rightaquaculture development, such as pristine water,

leasing income played significant portion of total

year-round warm weather, and isolated condition

GDP in RMI and FSM (Table 1). Aids from foreignfor disease prevention. However, they have togovernments were another major income for theovercome constraints to aquaculture development

government (Table 2). As a result, communities havesuch as small land area, natural hazards for somemade a shift from eating traditional seafood items

islands, insufficient knowledge base, shortage

to importing cheap, processed foods. Consequently,of available capital, distant markets, and poor

many residents have suffered widespread health

Table 1. Fisheries Contribution
FSM
FY2014 GDP
Total$$% of GDP
200 mi EEZ (KM2)
Per Capita
Consumption (kg)

318.1
31.8
10.0
2,978,000
72.0-142.0

Marshall

186.7
26.3
14.1
2,131,000
38.9-59.0

Palau

249.085.46
2.2
629,000
84.0-135.0

American
Samoa
711
1.6
0.2
390,000
15.5

Guam

CNMI

1.36

2.12

218,000
20.4-27.2

1,823,000
23.0

Sustain seafood in Pacific Islands

69

Table 2. Comparison of Top Donor Countries’ and Organizations’ Total Aid Spent in the Freely Associated States(2011-2018) (cited from Grossman et al., 2019).
UnitedStatesChina

Australia

Japan

TaiwanMultilateral
Organizations

Federated Statesof Micronesia(FSM)

532.86

86.23

27.8

61.08

Republic of the
Marshall Islands
(RMI)

313.6

31.23

48.9

51.99

16.7
(Asian Development
Bank)

Palau

48.77

24.20

57.26

4.92

10.94
(Asian Development
Bank)

14.97
(World Bank)

Note: All figures are in U.S. dollars (million).
problems. Diabetes, cardiovascular diseases orreef and other habitats for many aquatic species

hypertension significantly downgraded their quality(Bell et al., 2018). Bell et al. (2016) further predictedof life (Carlin et al., 2016; Charlton et al., 2016). It isthat the abundances of tuna would decrease due

essential to revert their daily food diagram back toto the decline in richness of food web. To increase

their tradition to regain their quality life. Increasing

the resilience of USAPI to climate change, agencies

seafood supply via fish farming is one of the solutions

such as US Agency for International Development

to make seafood affordable and sustainable. Reliance(USAID) has created a Pacific-American Climate

on capture fishery as seafood supply is facing

Fund to civil society organizations to reduce long-the challenge of climate change. Climate change

term vulnerabilities associated with climate change.

may cause the migration of main tuna fisheries toSince the vulnerability is a function of potential

other location (Bell et al., 2016), thus, small island

impacts and adaptive capacity. Islanders have tocountries may lose both revenue and seafood. Losing

strengthen their adaptive capacity to modify or

seafood may mean more dependence on less healthychange actual or expected climate stress to reduce

processed foods from abroad and increases medical

the vulnerability and to stay at their islands.

expenses.

USAID’s Pacific-American Climate Fund has
supported three aquaculture projects in Marshall
Climate Change and Fishery

Islands and Federal State of Micronesia to train local
residents to produce seafood and aquatic products

USAPI has three main physiographical types: highin their water and to generate income (Ellis etvolcanic islands, raised limestone islands, and lowal., 2018; Hicks and Murashige, 2018; Zackhras etcoral atolls and is the most vulnerable to climateal., 2018). Although the scale is still small and notchange (Bell et al., 2011). A few high islands in thesignificant, it has potential to expand to a large scaleregion are large enough to influence local weather.

and ultimately enhance the nature fish stocks via

The majorities are the small atolls and are at theaquaculture.

mercy of sporadic rainfall for their fresh water. With
the islands at or below sea level, rising sea level from

Status of Aquaculture

global warming definitely imposes negative impacts
on the livelihood of island habitants. The rising

Adam et al. (2001) presented the status ofwater temperature might also alter the distribution

aquaculture in the Pacific islands. Now, the totalof tuna stock eastward from western part of the

production still did not show any noticeable

Pacific Ocean (Fig. 1; Bell et al., 2016). Besides theincrease after about twenty years. In 2017, the total

redistribution of tropical tuna, global warming andaquaculture production worldwide reached 112

ocean acidification can also cause damages to coralmillion metric tonnes (including aquatic plants) but

70

Cheng-Sheng LEE

Fig. 1.

Fig. 1. Climate change and predict tuna distribution. From Bell et al. (2016).
the estimated contribution from USAIP was only at

cucumber, Pacific threadfin (Moi), milkfish, rabbitfish,

201 metric tonnes (FAO, 2018). In spite of the lower

tilapia, groupers, marine shrimp, and mangrove crab,

productivity from aquaculture, aquaculture has been

etc. (Table 3). Out of those farming species, giant

the focus of technical and development attention

clams, various coral species, ornamental fish, andthroughout the region. Numerous documents,

processed bath sponge are export items currently.

reports and reviews on the accomplishments are

Sea cucumber and black pearl oysters are next twoavailable (Adams et al., 2001). It shows the intention

items with high export potential.

of developing aquaculture remains strong. Almost all

Research Facilities- There are aquacultureisland entities have their aquaculture development

research facilities at University of Guam andplan but did not completely implement for various

community colleges throughout the region forreasons. Most of previous or on-going aquaculture

research and training purpose. The following is a list

projects led by outsiders have focused on technology

of available facilities throughout the region.

transfer and capacity building. Projects have shown1) Palau- Palau Mariculture and Demonstration

impressed outputs but not in actual production.

Center (PMDC or giant clam hatchery) under

Until now, the total aquaculture yield from USAPI

Palau Marine Resources Bureau may be the mostwas still insignificant and could not meet the daily

impressive governmental facility in the region. At

domestic food demand. The followings are thejunction to PMDC, Palau Aquaculture Center funded

highlight of regional aquaculture status.

by Taiwan government is an upgraded marine finfish

Farming species- There are several target farming

hatchery. Palau Community College runs a multi-

species in the region, which includes but not limit

species hatchery located at Ngermetengel Village,

to giant clams, several hard and soft corals, marine

Ngeremlengui State. Biota Palau at Airai Old Dock

ornamental fish, black pearl oyster, bath sponge, sea

Ordomel is a private operation and a branch of Biota

Sustain seafood in Pacific Islands

71

Table 3. List of Species Produced in U.S.-Affiliated Pacific Islands (USAPI)
Commonwealth of theNorthern Mariana Islands(CNMI)

Tilapia, Marine shrimp, Rabbitfish

Guam

Tilapia, Marine shrimp, Milkfish, Freshwater prawn

Palau

Corals, Giant clams, Tilapia, Milkfish, Rabbitfish, Marine shrimp,
Marine ornamental fish, Grouper

Federated States ofMicronesia (FSM)

Corals, Sponges, Giant clams, Sandfish, Black pearl oyster

Republic of the MarshallIslands (RMI)

Corals, Giant clams, Pacific threadfin, Marine ornamental fish,
Grouper

American Samoa

Tilapia, Giant clams

Florida. The company focuses on marine ornamental
fish, coral, clams and other finfish species.

5) RMI- College of Marshall Islands has one
science station with facility for aquaculture research.

2) Guam- Guam Aquaculture Development andAnother finfish hatchery build by Rongelap Atoll

Training Center (GADTC), also known as the Fadian

local government under a grant from USAID has

Hatchery, is under the care of the University ofbeen used for research purpose as well.

Guam (UOG) from 2001 until 2019. Now, a private

Skilled workforce- USAPI has relied on foreigncompany has established a lease agreement withtechnicians to take full charge of farm operation

UOG to operate the hatchery. The UOG Marine

and train local workers. College education and on-

Laboratory can conduct basic and applied research onsite training at funded aquaculture projects are

the biology of tropical marine organisms. Instead of

two key sources for local aquaculture technicians.

aquaculture, the laboratoryʼs research focuses on the

Local trained technicians have shown their abilities

conservation and development of marine resources of

to operate black pearl oyster and sea cucumber

the near-shore waters of Guam and Micronesia.

hatcheries, for example. However, it seemed to be3) FSM- In Pohnpei, College of Micronesia Landa challenge to assist those skillful workers to apply

Grant Program has operated Nett Point hatcherytheir technique in production. With less working

since August 2001 for black pearl oyster and sea

opportunities, it has been a difficult task for themcucumber. This hatchery was installed in an old

to find a good job. Often time those well-trained

warehouse space next to water. At the east sidetechnicians had to find another different job afterof the island, a non-profit organization establishedthe funded projects ended.

Marine and Environmental Research Institute ofCommercial farms- Because of the limitation of

Pohnpei works on several coral species, bath sponge,

land area, over one hectare land-based grow-out

giant clam and ornamental species. In Kosrae, thefarms only located in CNMI, Guam and Palau so far.

Federated States of Micronesia National Aquaculture

Cage culture at open water so far only took place inCenter established in 1991 at Lelu produces giant

Majuro, and Palau. Cage culture has great potential

clam, mangrove crab and others. In Chuuk, theto expand to other islands where do not have

Korea-South Pacific Ocean Research Center (KSORC)

natural hazards, such as hurricanes.

was established on 30 May 2000 on a small island,

There were several commercial ventures taken

Weno Island, located within the Chuuk Lagoon withplace in USAPI. Currently, only a few operations

the long-term goal of promoting ocean research andremain. A hopeful commercial marine shrimp

related marine industries.

farm at CNMI was permanently stopped operation4) CNMI- College of North Mariana had a small

after strong hurricanes damaged the facilities inaquaculture research facility but was destroyed by

2017. Marshall Islands mariculture farm with thea recent hurricane in 2017. They have budget tostrong support from parent company Ocean, Reef

rebuild another one.

and Aquarium (ORA) in Florida is doing well inCheng-Sheng LEE

72

ornamental fish and giant clams. The marine finfish

to determine which islands have friendly investment

cage farm by Rongelap Atoll local government inenvironments. A true partnership between private

Majuro is progressing toward to be a commercial

and public sectors is essential to make sure theoperation. In Palau, milkfish, rabbitfish and mangrovesustainability of the development. Finally, everyone

crabs are cultured for commercial purpose. Up

involved must have the “will” to overcome any

to now, investors from outside have expressedthreats in front of aquaculture development before

interested in starting fish farming in USAPI but yet

we will be able to success (Lee and Awaya, 2003).

to be seen.

The sustainable utilization of the unique natural
resource at EEZ for fisheries development will have
Path for the Future

benefits to the people and economies of the Pacificregion and extend far beyond their economic returns

Accepting USAPI is the most vulnerable toat every level.

climate change and knowing coastal fisheries andReferences

staple food crops will decline due to climate change,
skillful management of the harsh environment
condition and nature resources is the only way toAdams T., Bell J., and Labrosse P., 2001: Current

continually inhabit in USAPI for years to come. View

status of aquaculture in the Pacific Islands,

the success or failure of aquaculture in the region

in “Aquaculture in the Third Millennium,

while we review the status, we can conclude theTechnical Proceedings of the Conference on

pathway to the future success. USAPI has uniqueAquaculture in the Third Millennium” (ed. bysituation different from other countries in their

Subasinghe R. P., Bueno P., Phillips M. J., Houghsocial culture background. It indicates the essential

C., McGladdery S. E., and Arthur J. R.), NACA,

need of community engagement in any aquaculture

Bangkok and FAO, Rome, pp.295-305.

development. The initial aquaculture practice has toBell J. D., Johnson E. J., Ganachaud A. S., Gehrke P. C.,

be simple technique and accessible to all required

Hobday A. J., Hoegh-Guldberg O., Le Borgne R.,

components. Any new practice should respect their

Lehodey P., Lough J. M., Pickering T., Pratchetttraditional rural lifestyles and become a part ofM. S. and Waycott M., 2011: Vulnerability ofcommunity social activities.

Tropical Pacific Fisheries and Aquaculture to

To utilize fully and sustainably the most valuable

Climate Change: Summary for Pacific Island

resource, i.e. EEZ, for food production, skillful

Countries and Territories, Secretariat of theworkforce is the fundamental and essential base.

Pacific Community, Noumea, New Caledonia,

Capacity building has been the center of all previous

386pp.

and on-going aquaculture projects. It seemed to be

Bell J., Kronen M., Vunisea A., Nash W., Keeble G.,

not an issue to find talents and train them. The big

Demmke A., Pontifex S., and Andréfouët S.,

challenges are to retain skillful workforce. It is a

2009: Planning the use of fish for food security

fruitless effort of having skillful workforce but noin the Pacific. Marine Policy, 33(1), 64–76.

established business ventures to receive them. After

Bell J., Taylor M., Amos M., and Andrew N., 2016:

establishment of skillful workforce, appropriate

Climate change and Pacific Island food systems,

farming technology can be developed and applied

CCAFS and CTA, Copenhagen, Denmark andin food production. The appropriate farming

Wageningen, the Netherlands, 40pp.

technology should also consider the adaptation to

Bell J. D., Allain V., Gupta A. S., Johnson J. E.,

the climate change (Wijkström, 2012). And, it is

Hampton J., Hobday A. J., Lehodey P., Lenton A.,

important to have a business mindset to implement

Moore B. R., Pratchett M. S., Senina I., Smith N.,

any farming technology. We need the entrepreneurs

and Williams P., 2018: Climate change impacts,

connect all the above dots together, furthermore

vulnerabilities and adaptations: Western andto the markets and attract capital for operation.

Central Pacific Ocean marine fisheries, in

Investors will be not interested unless they are able

“Impacts of climate change on fisheries andSustain seafood in Pacific Islands

73

aquaculture: synthesis of current knowledge,

lessons from giant clam and sponge farming.

adaptation and mitigation options” (ed. by

Aquaculture Economics & Management, 7(1&2),

Barange M., Bahri T., Beveridge M. C. M.,

125-135.

Cochrane K. L., Funge-Smith S., and Poulain

Wijkström U. N., 2012: Is feeding fish with fish aF.), FAO Fisheries and Aquaculture Technicalviable practice? in “Proceedings of the Global

Paper No. 627. FAO, Rome, pp.305–324.

Conference on Aquaculture 2010, Farming theCarlin M., Mendoza-Walters A., and Ensign K., 2016:

Waters for People and Food” (ed. by Subasinghe

Half an Ocean Away: Health in the US-Affiliated

R. P., Arthur J. R., Bartley D. M., De Silva S. S.,

Pacific Islands. J Public Health Management

Halwart M., Hishamunda N., Mohan C. V., andPractice, 22(5), 492–495.

Sorgeloos P.), FAO, Rome and NACA, Bangkok,

Charlton K. E., Russell J., Gorman E., Hanich Q.,

pp.33–55.

Delisle A., Campbell B., and Bell J., 2016: Fish,

Zackhras M., Deeks P., and Ellis S., 2018: Black

food security and health in Pacific Island

pearl farming as an adaptation in coastal

countries and territories: a systematic literatureclimate change, in “Climate change impacts andreview. BMC Public Health, 16, 285. (doi:adaptation strategies for coastal communities”

10.1186/s12889-016-2953-9)(ed. by Filho W.L.), Springer International

Ellis S., Haws M., Mendiola J., and Hemil M., 2018:

Publishing AG, pp.209-224.

Sustainable Small-scale Mariculture Ventures
as a Comparative Climate Friendly Livelihood

Annotated Bibliography of Key Works

Alternative in Pohnpei, Federated States ofMicronesia, in “Climate change impacts and(1) Adams T., Bell J., and Labrosse P., 2001: Current

adaptation strategies for coastal communities”

status of aquaculture in the Pacific Islands, in(ed. by Filho W. L.), Springer International“Aquaculture in the Third Millennium, TechnicalPublishing AG, pp.31-42.

Proceedings of the Conference on Aquaculture in the

FAO, 2018: The State of World Fisheries andThird Millennium” (ed. by Subasinghe R. P., Bueno P.,

Aquaculture 2018 - Meeting the sustainable

Phillips M. J., Hough C., McGladdery S. E., and Arthur

development goals, FAO, Rome. 227pp.

J. R.), NACA, Bangkok and FAO, Rome, pp.295–305.

FAO, 2019: FAO yearbook. Fishery and Aquaculture
Statistics 2017, FAO, Rome, 109pp.

In the book “Aquaculture in the Third
Millennium” , this chapter “Current status of

Gillett R., 2016: Fisheries in the Economies ofaquaculture in the Pacific Islands” gave an overview

Pacific Island Countries and Territories, Pacificthe status of aquaculture in the Pacific Islands at theCommunity, Noumea Cedex, New Caledonia,

thturn of 20 century. It served as the base to assess

657pp.

any new development in 21st century. Through theGrossman D., Chase M. S., Finin G., Gregson W.,

new wave of international cooperation, it is expect

Hornung J. W., Ma L., Reimer J. R., and Shih A.,

to increase sustainable use of aquatic resources to

2019: America’s Pacific Island Allies- The Freely

meet the goal of food security. Food and Agriculture

Associated States and Chinese Influence. RAND

Organization of the UN (FAO) and Network ofCorporation, Santa Monica, Calif., 99pp.

Aquaculture Centres in Asia-Pacific (NACA) co-

Hicks K., and Murashige R., 2018: Economic

organized this conference in 2000.

resiliencey and food security in the MarshallIslands through Polydactylus sexfilis(2) Lee C. S., and Awaya K., 2003: Viable aquaculture

Aquaculture, in “Climate change impacts anddevelopment in the U.S. affiliated islands – lessons

adaptation strategies for coastal communities”

from giant clam and sponge farming. Aquaculture(ed. by Filho W. L.), Springer International

Economics & Management, 7(1&2), 125-135.

Publishing AG, pp.283-297.

This paper reviewed the farming technology

Lee C. S. and Awaya K., 2003: Viable aquaculture

for giant clam and bath sponges. Then, it used

development in the U.S. affiliated islands –giant clam and bath sponges as an example to74

Cheng-Sheng LEE

discuss the challenges of technology transfer, whichto oceanic fisheries, to coastal fisheries, to freshwater

include biological, technological, environmental,

and estuarine fisheries, to aquaculture, economic andand socioeconomic, to Pacific Islands. Finally, somesocial implications, and adaptations and suggested

recommendation was made for the successfulpolicies. It is a valuable document to have a quicktechnology transfer.

overview of potential climate change impacts andsuggested remedy policy.

(3) FAO, 2018: The State of World Fisheries andAquaculture 2018 - Meeting the sustainable(5) Charlton K. E., Russell J., Gorman E., Hanich Q.,

development goals, FAO, Rome, 227pp.

Delisle A., Campbell B., and Bell J., 2016: Fish, foodAn important of aquaculture and fisheries status

security and health in Pacific Island countries andreport published by FAO every two years. It

territories: a systematic literature review. BMC

highlights the critical importance of fisheries andPublic Health, 16, 285. (doi: 10.1186/s12889-016-

aquaculture for the food, nutrition and employment2953-9)of millions of people, many of whom struggle toThis paper discussed the importance of fish to

maintain reasonable livelihoods. Data and graphics

Pacific Island Countries and territories (PICTs)

presented in this publication are widely used byin both food security and health related concerns

research groups to assess the progress and to

based on the review of 29 studies. However, there

propose future works.

is a paucity of research aimed at assessing how
maintaining and/or improving fish consumption(4) Bell J. D., Johnson E. J., Ganachaud A. S., Gehrke

benefits the diets and health of Pacific Islanders.

P. C., Hobday A. J., Hoegh-Guldberg O., Le Borgne R.,

Instead of fresh seafood, there is an increasing

Lehodey P., Lough J. M., Pickering T., Pratchett M.

demand for packaged imported foods, such as cannedS., and Waycott M., 2011: Vulnerability of Tropicalmeats, instant noodles, cereals, rice, and sugar-

Pacific Fisheries and Aquaculture to Climate

sweetened beverages, with subsequent decreased

Change: Summary for Pacific Island Countries andconsumption of locally-produced plants and animals.

Territories, Secretariat of the Pacific Community,
Noumea, New Caledonia, 386pp.

(6) Gillett R., and Tauati M. I., 2018: Fisheries of the

The book entitled Vulnerability of Tropical

Pacific Islands - Regional and national information,

Paciftc Fisheries and Aquaculture to Climate change

FAO Fisheries and Aquaculture Technical Paper

provides the region with the understanding needed,

No. 625, FAO, Apia, 401pp.

and the adaptations, policies and investments

This paper discusses the important species,

recommended to reduce the likely impacts ofthe status of the resources, and the fisheries

climate change on fisheries and aquaculture. It also

management under offshore fishing and coastal

gives the sector a roadmap for capitalising on the(or nearshore) fishing. This report also provides

opportunities expected to arise from the changing

information on the fisheries in each of the 14

climate. This book is the product of a partnership

independent Pacific Island countries (includingthat started between the Australian Agency for

Federated States of Micronesia, Marshall Islands,

International Development (AusAID) and theand Palau) in the following categories:

Secretariat of the the Pacific Community (SPC),

・Overview and main indicatorsand then grew to embrace contributions from 36

・Production sector

institutions. This summary, which is a companion

・Post-harvest sector

to the book, provides this vital information in an・Socio-economic contribution of the fishery sector

accessible form for each Pacific Island country

・Trends, issues and development

and territory. It is a quick access to understand・Institutional framework

projected changes to surface climate and the ocean,・Legal framework

水 研 機 構 研 報， 第 50 号，75 － 82， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，75－82，2021

75

Towards effective coral community restoration for sustainable fishery of a coral
reef grouper Epinepherus ongus: implications of ecosystem-based management
Atsushi NANAMI＊

Abstract: Spawning aggregations of coral reef fishes are particularly vulnerable to fishing since
only conspecific individuals gather at specific sites in restricted seasons and lunar phases. Since
capturing spawning aggregations leads to significant negative impacts for both local stock andreproductive success, protection of spawning aggregations has been an urgent need recently forcoral reefs all over the world. Furthermore, conservation of habitat in the non-spawning season is
also essential for species that form spawning aggregations since habitat loss at their home groundwould also lead to a reduction in their population. In the present paper, the focus is on a coral reefgrouper species (Epinephelus ongus), that is a very important fishery target and forms spawning
aggregations in an Okinawan coral reef. Effective coral community restoration is discussed toestablish a sustainable fishery of the species. This author’s previous studies have shown: (1) theirhome ground was several kilometers around the spawning ground; (2) their home range wasvery limited around a coral colony during non-spawning periods; (3) juveniles preferentially usedcorals with fine structure (e.g. bottle-brushed acroporid corals) whereas adults were mainly foundat corals with coarse structure (e.g. massive corals and staghorn acroporid corals); and (4) very
precise returning ability was observed after spawning migration (i.e. species would return tothe coral colonies that were used before the spawning migration). These results suggest that: (1)conservation of the coral community around the spawning ground is indispensable; (2) coral species
that are preferentially used by E. ongus should be selected for coral community restoration; (3)the area several kilometers around the spawning ground should be the proposed area for coral
community restoration. Since the spawning ground of the species has already been assigned as amarine protected area, coral community restoration around the spawning ground would be usefulto enhance the E. ongus stock.
Key words: coral community restoration, sustainable fishery, spawning aggregation, white-streaked
grouper, Epinephelus ongusIntroduction

site. However, recent coral degradation has caused
significant negative impacts on populations ofHabitat restoration is one of the essential tools

fisheries target species. Thus, coral community

for fisheries management, since almost all marine

restoration has been urgently needed for sustainableorganisms require appropriate habitat space tofisheries in coral reefs.

complete their life cycle (e.g. settlement, growth

Among the diverse fish species, at least 80

and reproduction). Thus, an ecosystem-based

species have been reported to form spawning

management approach is needed. In coral reefs,

aggregations (Sadovy de Mitcheson and Colin,

numerous fisheries target species rely on the coral2012). Domeier (2012) has defined reef fish spawningcommunities as their refuge space and resting

aggregations as consisting of only conspecific
2020年12月11日受理（Accepted on December 11, 2020）＊
Research Center for Subtropical Fisheries, Seikai National Fisheries Research Institute, Japan Fisheries Research and Education Agency, 148
FukaiOta, Ishigaki, Okinawa 907-0451, Japan
E-mail: nanami “at” fra.affrc.go.jp

Atsushi NANAMI

76

individuals where spawning is highly predictable in

the non-spawning periods. The habitat loss in thetime and space. Spawning aggregations are usuallyhome ground would decrease population size of thefound in restricted seasons and lunar phases at

species in the non-spawning periods and this would

particular sites (Nemeth, 2009). Due to the ecologicaldecrease the magnitude of spawning aggregations,characteristics, spawning aggregations have greateven if the spawning aggregation is protected fromvulnerability to fishing (Sadovy de Mitcheson andfishing (Fig. 1). Thus, habitat restoration (i.e. coral

Erisman, 2012). In addition, spawning aggregation

community restoration) around spawning grounds

fishing could decrease reproductive success of theshould be considered as part of effective spawningspecies, leading to decreased larval supply andaggregation protection. The protection of thesejuvenile settlement. Thus, effective protection ofrestored habitats (e.g. marine protected areas) is anspawning aggregations is needed for sustainable

important additional step to ensure their longevity

fisheries and reproductive success. Recently, marine

and success.

protected areas have been applied to protect
spawning aggregations all over the world.

There are at least two aspects that determine
success of coral community restoration: “How”

However, if habitats around the spawning groundand “Why”. “How” means the techniques used

are degraded, the effects of spawning groundto enhance survival rates or scale up coral

protection would decrease. This is because thecommunities. In contrast, “Why” is related to thespecies that form spawning aggregations inhabit

purpose of restoration (e.g. biodiversity conservation

their home ground around the spawning ground inor sustainable fishery). In the latter case, we should

Fig. 1. Schematic diagram showing the reason why coral community should beconserved or restored around the spawning ground. If little damage to the coral
community around the spawning ground and a large population size of the fish is foundin a non-spawning season (A), then a large spawning aggregation would be found at the
spawning ground during spawning season (B). If coral community degradation occurs
around the spawning ground, the population size of the fish would decrease around thespawning ground (C), and only a small spawning aggregation would be found even if thespawning ground is protected from catch (D).

Fig.1

Coral community restoration for sustainable fishery of a grouper

determine strategies such as “what is the target

77

Definition of ecological terms

species to enhance?”, “what types of corals should

Four ecological terms are defined as follows

be restored?” and “which area is proposed for coral(Fig. 3): (1) home ground: the area that are usedcommunity restoration?” In order to determine theby fishes in non-spawning period; (2) home range:

strategy, we should clarify ecological aspects forthe area that are used by a focal fish individual (i.e.

the target species or target communities before

territory); (3) spawning ground: the area that are

conducting coral community restoration. In theused by fishes in spawning event; (4) microhabitat:

present paper, the focus is on a coral reef grouper inthe habitat that are associated by fishes in fine scale

an Okinawan coral reef. Subsequently, the effective

within several-tens centimeters (e.g. coral species

coral community restoration needed to achieve stockand coral morphology). This is contrary to landscape-enhancement of the grouper is discussed.

level habitat categorization (e.g. reef flat, reef crestand reef slope).

Materials and Methods
Spawning migration distanceStudy species

Migration distance is defined as the distanceWhite-streaked grouper Epinephelus ongus is

between home ground and spawning ground forone of the important fishery targets around thethe focal individual. Clarification of the spawning

Okinawan region and is known to form spawningmigration distance would provide useful information

aggregations (Ohta and Ebisawa, 2015) (Fig. 2A).

to estimate the home ground area around the

The spawning of the species was found to occur

spawning ground. Namely, “how many areas should

during the last-quarter moon in only one month (May)be considered for coral community restorationor two consecutive months (April-May or May-June).

around the spawning ground?” Using a tag-and

In order to protect the spawning aggregation of therelease method, the spawning migration distancespecies, the spawning ground has been protected

was estimated. In total, 1157 E. ongus individuals

during spawning periods since 2010 (Nanami et al.,were tagged and released at their home grounds in2017) (Fig. 2B).

the non-spawning period and 350 were tagged at the

In order to achieve effective spawning aggregation

protected spawning ground in the spawning period.

protection of the species, several ecological traits
should be clarified such as (1) spawning migration

Microhabitat association

distance, (2) microhabitat association and (3)Understanding the microhabitat association would

returning ability after spawning migration of theprovide useful information to clarify what types of

species.

corals should be restored. Microhabitat associations

A

B
Spawning groundprotection area
Iriomote
IslandSekiseilagoonIshigaki
Island10 km

Fig. 2. Spawning aggregation of white-streaked grouper (A). Location of the spawning
ground protection area in Sekisei lagoon, Okinawa, Japan (B). The aerial photograph wasprovided by the International Coral Reef Research and Monitoring Center.

Fig.2

Atsushi NANAMI

78

Home ground
Home
range

Spawning migration

Spawning ground

Home
range
Home range

Home range

Microhabitat (coral colony)Fig. 3. Schematic diagram for four ecological terms (home ground, home range, spawning ground andmicrohabitat). (1) home ground: the area that are used by fishes in non-spawning period; (2) home range: thearea that are used by a focal fish individual (i.e. territory); (3) spawning ground: the area that are used by fishes
in spawning event; (4) microhabitat: the habitat that are associated by fishes in fine scale within several-tens
centimeters (e.g. coral species and coral morphology).

Fig.3

Results

for juvenile and adult white-streaked grouper
were examined at home grounds by underwaterobservations.

Migration distanceFor the fish that were released at the home

Site fidelity and returning ability

grounds, 23 individuals were recaptured at the

Clarifying the site fidelity and returning ability

spawning ground during the spawning periods (Fig.

would be useful to consider if the restored coral

4A). For the individuals that were released at thecommunity would be used throughout the white-spawning ground, six individuals were recaptured

streaked grouper’s lifetime. Seventeen individuals

outside the spawning ground (Fig. 4B) (Nanami etwere captured and an acoustic coded transmitteral., 2015). The estimated migration distances fromwas surgically implanted into the abdominal cavity

the home ground to the spawning ground rangedof each fish. All tagged individuals were promptly

from 2.2 to 8.8 km (Fig. 4C).

released back to the site of their capture at therespective coral colony. In addition, 19 automatedMicrohabitat association

monitoring acoustic receivers were deployed at the

Most juveniles (total length < 14 cm) were found inrelease point. The location of each fish was detectedcorals with fine structure (e.g. bottlebrush Acropora

using a VEMCO Positioning System.

and arborescent Acropora), whereas most adults (total
length > 18 cm) were found in corals with coarse
structure (e.g. staghorn Acropora, massive Porites
and branching Porites) (Fig. 5) (Nanami et al., 2013;
Nanami unpublished data).

Coral community restoration for sustainable fishery of a grouper

A

Max = 6.6 km

B

Max = 8.8 km

Max = 8.8 km

Fig. 4. Estimation of spawning migration distance by tag-and-release. Results of 23 individuals
releasedoutside the spawning ground (A) and 6 individuals released inside the spawningCaggregation (B). The estimated home ground of white-streaked grouper around the protected
spawning ground (C). Redrawn from Nanami et al. (2015). The aerial photographs were
provided by the International Coral Reef Research and Monitoring Center.

B

Fig.4
A

Juvenile

Corals with fine structure

Adult

Corals with coarse structure

Genus Acropora spp. = greater coral bleaching susceptibility
Fig. 5. Microhabitat association of juveniles (A) and adults (B) of the white-streaked grouper.
Both fine-structure and coarse-structure corals included genus Acropora, which has greatercoral bleaching susceptibility and subsequent death.
Fig.5

79

Atsushi NANAMI80

Egg dispersal???

Fig. 6. Schematic diagram showing the strategy to enhance spawning aggregation
protection by coral community restoration. Since the spawning ground has alreadybeen protected, other protective actions at earlier life stages would be effective. If
appropriate, corals are selected and when these coral communities are successfully
restored, fish population size would increase at their home ground resulting in large
spawning aggregations at the protected spawning ground during the spawning
Fig.6The manner in which egg dispersal occurs should be clarified in the future.
season.
Site fidelity and returning ability

species throughout their lifetime. One of the future

Tagged individuals showed high site fidelity tochallenges is to clarify the manner of egg dispersal

their release point (patchy coral substrates). For

from the protected spawning ground (e.g. Almany etreturning ability, 10 individuals were analyzed andal., 2013). If the eggs produced from the protected

8 out of the 10 individuals showed precise returning

ground arrive at a restored coral community, it isafter the spawning migration to the patchy coral

expected that greater settlement at the restored

substrates that were used before the spawningcorals would be exhibited.

migration (Nanami et al., 2018).
Acknowledgments
Discussion
Grateful thanks are expressed to K. Kinjo and M.
The results of the present study showed several

Sunagawa for their field guide and to the staff of thestrategies for coral restoration to enhance spawning

Research Center for Subtropical Fisheries (Seikai

aggregation protection (Fig. 6): (1) conservation

National Fisheries Research Institute) for supportof coral communities around the spawning

during the study.

ground is indispensable; (2) coral species that are
preferentially used by E. ongus should be selected

References

for coral community restoration. In particular,
restoration of genus Acropora is urgently needed

Almany G. R., Hamilton R. J., Bode M., Matawai M.,

due to the greater susceptibility to coral bleaching;

Rotuku T., Saenz-Agudelo P., Planes S., Berumen(3) the area that is c.a. 8.8 kilometers around theM. L., Rhodes K. L., Thorrold S. R., Russ G. R.,

spawning ground should be the proposed area

and Jones G. P., 2013: Dispersal of grouper

for coral community restoration. Since high sitelarvae drives local resource sharing in a coral

fidelity and returning ability after spawning

reef fishery. Current Biol., 23, 626-630.

migration were found, it follows that the successfully

Domeier M. L., 2012: Revisiting spawning

restored coral communities would be used by theaggregations: definitions and challenges, in “ReefCoral community restoration for sustainable fishery of a grouper

81

fish spawning aggregations: biology, research

spawning aggregations: biology, research andand management” (ed. by Sadovy de Mitcheson

management” (ed. by Sadovy de Mitcheson Y.,

Y., and Colin P.L.), Fish and Fisheries Series 35,

and Colin P. L.), Fish and Fisheries Series 35,

Springer Netherlands, pp.1-20.

Springer Netherlands, pp.225-284.

Nanami A., Sato T., Takebe T., Teruya K., andSoyano K., 2013: Microhabitat association in

Annotated Bibliography of Key Works

white-streaked grouper Epinephelus ongus:
importance of Acropora spp.. Mar. Biol., 160,

(1) Almany G. R., Hamilton R. J., Bode M., Matawai M.,

1511-1517.

Rotuku T., Saenz-Agudelo P., Planes S., Berumen M.

Nanami A., Ohta T., and Sato T., 2015: Estimation

L., Rhodes K. L., Thorrold S. R., and Russ G. R., 2013:of spawning migration distance of the white-

Dispersal of grouper larvae drivers local resource

streaked grouper (Epinephelus ongus) in an

sharing in a coral reef fishery. Current Biol., 23,

Okinawan coral reef system using conventional

626-630.

tag-and-release. Environ. Biol. Fish., 98, 13871397.

This study examined the dispersal distance oflarvae from the spawning ground for a coral reef

Nanami A., Sato T., Kawabata Y., and Okuyama J.,

grouper, Plectropomus areolatus. The parentage

2017: Spawning aggregation of white-streaked

analysis revealed that 50% of larvae settled within 14

grouper Epinephelus ongus: spatial distribution

km of the spawning ground. The noteworthy point

and annual variation in the fish density within aof the study is that spawning ground protection

spawning ground., PeerJ, 5, e3000.

would be effective at a local scale (within several to

Nanami A., Mitamura H., Sato T., Yamaguchi T.,

ten kilometers) for larval dispersal.

Yamamoto K., Kawabe R., Soyano K., AraiN., and Kawabata Y., 2018: Diel variation in(2) Nanami A., Sato T., Takebe T., Teruya K., andhome range size and precise returning ability

Soyano K., 2013: Microhabitat association in white-

after spawning migration of coral reef grouper

streaked grouper Epinephelus ongus: importance of

Epinephelus ongus: implications for effective

Acropora spp.. Mar. Biol., 160, 1511-1517.

marine protected area design. Mar. Ecol. Prog.
Ser., 606, 119-132.

This study quantified microhabitat associations
for juvenile and adult white-streaked grouper. For

Nemeth R. S., 2009: Dynamics of reef fish andjuveniles, most individuals showed a significant

decapod crustacean spawning aggregations:

positive use of bottlebrush Acropora. For adults,

underlying mechanisms, habitat linkages, andmost individuals showed a significant positive use

trophic interactions, in “Ecological connectivityof staghorn Acropora. A habitat choice experiment,

among tropical coastal ecosystems” (ed. byusing pre-settlement individuals, revealed that both

Nagelkerken I.), Springer, Dordrecht, pp.73-134.

bottlebrush Acropora and staghorn Acropora were

Ohta I., and Ebisawa A., 2015: Reproductive biology

used as settlement sites, whereas coral rubble wasand spawning aggregation fishing of the white-rarely used as a settlement site.

streaked grouper, Epinephelus ongus, associatedwith seasonal and lunar cycles. Environ. Biol.

(3) Nanami A., Ohta T., and Sato T., 2015: Estimation

Fishes., 98, 1555-1570.

of spawning migration distance of the white-streaked

Sadovy de Mitcheson Y., and Colin P. L. (eds),

grouper (Epinephelus ongus) in an Okinawan coral

2012: Reef fish spawning aggregations: biology,

reef system using conventional tag-and-release.

research and management. Fish and Fisheries

Environ. Biol. Fish., 98, 1387-1397.

Series 35, Springer Netherlands, 622pp.

Using a tag and release method, this study

Sadovy de Mitcheson Y., and Erisman B., 2012:

estimated the migration distance and the degree

Fishery and biological implications of fishingof unified movement of white-streaked grouper

aggregations, and the social and economic

associated with the spawning migration. In total,

importance of aggregating fishes, in “Reef fish

1157 E. ongus individuals were tagged and released

82

Atsushi NANAMIat their home grounds in the non-spawning period(5) Nanami A., Mitamura H., Sato T., Yamaguchi T.,

and 350 were tagged at a known spawning groundYamamoto K., Kawabe R., Soyano K., Arai N., andin the spawning period. The estimated migration

Kawabata Y., 2018: Diel variation in home range

distances from the home ground to the spawning

size and precise returning ability after spawning

ground ranged from 2.2 to 8.8 km.

migration of a coral reef grouper Epinephelus ongus:
implications for effective marine protected area

(4) Nanami A., Sato T., Kawabata Y., and Okuyama

design. Mar. Ecol. Prog. Ser., 606, 119-132.

J., 2017: Spawning aggregation of white-streaked

This study examined the diel variation in homegrouper Epinephelus ongus: spatial distribution

range size and the degree of precision for returningand annual variation in the fish density within aability of white-streaked grouper by acoustic

spawning ground. PeerJ, 5, e3000.

telemetry. Seventeen individuals were studied,This study revealed 1) spatial variations in theand nighttime home range sizes were over 5-times

density of E. ongus at the spawning ground, 2) the

greater than the respective daytime home ranges.

relationship between fish density and environmental

Returning ability for 10 individuals that showedvariables, 3) inter-annual variations in the spawningclear spawning migration behaviour was also

aggregation, 4) the proportion of males to females

analyzed and 8 out of the 10 individuals showedat the spawning ground for several days pre-and

precise returning after the spawning migration to

post-spawning and 5) the relationship between

the patchy coral substrates that were used before

male density and female density at the protected

the spawning migration.

spawning ground, based on observations over five
years at an Okinawan coral reef.

水 研 機 構 研 報， 第 50 号，83 － 89， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，83－89，2021

83

Scaling up coral restoration to meet the demands of acollapsing ecosystem
Tali VARDI＊



Abstract: Coral reefs feed millions of people on earth and are critical to the livelihoods of millions
more. They house approximately a quarter of the ocean’s biodiversity and are critical to fisheries
worldwide. Their importance and fragility to humans cannot be overstated. According to climate
models, in about thirty years half of all coral reefs are predicted to disappear due to bleachingcaused by unprecedented ocean warming (Bindoff et al., 2019). If political and economic forces
begin to seriously address emissions, corals will still need help maintaining enough abundanceand diversity to rebuild reefs by the end of the century when ocean temperatures may begin tostabilize. With the tacit assumption that emissions will be curbed, an explosion of coral restoration
and research into coral “interventions has taken place over the past two years. Coral aquaculture
is a pivot point of any advancement in scaling-up restoration or implementing a new intervention- as any and all applications involve the delicate care and propagation of corals in the water oron land. NOAA has taken a lead role in coordinating coral restoration efforts throughout the US
and the globe, primarily via the Coral Restoration Consortium and by commissioning the National
Academies of Sciences to review coral interventions. This paper highlights recent restoration andintervention successes in the U.S. and globally, and describes NOAA’s proposed research andaction plan on coral interventions.
Key words: coral, restoration, larval propagation, coral restoration, coral bleachingBackgroundnatural recovery processes (e.g. marine protectedareas) to active interventions (e.g. coral gardening)

Coral reef restoration is a new and rapidly

which promote adaptation and resilience to changing

evolving field, made necessary by the calamitousconditions (Rinkevich 2005, 2008, 2019; Young et al.,degradation of coral reefs worldwide (Bostrom-

2012; Possingham et al., 2015; Bostrom-Einarsson etEinarsson et al., 2020). The primary threat to coralal., 2020).

reef ecosystems is ocean warming, and current
climate change trajectories which predict a rise

The Coral Restoration Consortiumin global temperature of 2ºC, also predict a loss of
99% of the world’s reefs (Bindoff et al., 2019). The

As the field of coral reef restoration and theincreasing severity and frequency of climate-change

number of practitioners grows rapidly, so does

related coral bleaching events and tropical storms

the need to share the successes and failures, andare limiting the ability of coral reefs to recoverto ensure the latest science reaches restoration

naturally between disturbances (Fabricius et al.,practitioners working underwater. To meet this2017; Hughes et al., 2019). With this rapidly changing,need, the Coral Restoration Consortium (CRC) was

unprecedented scale of decline, there has been

started by NOAA in 2017 to facilitate cooperation

an increasing shift in management priorities fromand communication among coral reef restorationa reliance on passive interventions that facilitate

practitioners, managers, researchers, and educators
2020年12月11日受理（Accepted on December 11, 2020）＊
ECS for NOAA Fisheries Office of Science and Technology, 1315 East West Highway, Silver Spring, Maryland 20910
E-mail: tali.vardi “at” noaa.gov

Tali VARDI

84

in order to support scientific and practical ingenuity

corals to weather the next 50-100 years.

in the field. At its inception the CRC focused on
the Caribbean and most of the leadership was from

2. S cale-up larval propagation for its effective

the Caribbean. Within the past year, as reefs haveintegration in coral reef restoration efforts, with an

struggled and a growing number of locations engage

emphasis on recruit health, growth, and survival.

in restoration, the CRC has expanded globally.

Larval recruitment from sexually assisted coral

In December 2018, the CRC hosted the first everreef restoration is essential for improving theinternational conference on coral reef restoration

genetic diversity of small coral populations. Recently,

and shortly afterwards voted in new Steering

important advances have been made in coral in vitro

Committee Members from diverse tropical regions.

fertilization, as well as husbandry of coral larvae

In September 2019, we adopted three Regional(reviewed in Randall et al., 2020). However, larvalGroups (Eastern Tropical Pacific, Latin America,

propagation research has focused on broadcast

and Australia). Japan is a notable exception and wespawning species and post-settlement mortality

would welcome a Japanese Regional Group!

continues to be high. To increase the scale andefficiency of larval propagation we need to expand

Priorities for Coral Restoration

the use of existing larval propagation technologies(more species, more places), develop new

Here we present the six priorities as defined by
Coral Restoration Consortium to help guide andtechnologies, and develop criteria for maximizing
genetic diversity.

promote the scaling-up of coral reef restoration
efforts across disciplines and localities. These

3. D evelop guidance that promotes a holistic

priorities were not developed to the exclusion of

approach to coral reef ecosystem restoration.

the priorities developed by other organizations.

The CRC is developing a roadmap to help the

The task of conserving and restoring coral reefs isrestoration community evolve from restoring single

immense. No one organization nor a consortium of

species of corals to restoring a functioning coral

organizations will solve these issues alone. Our goal

reef ecosystem that will be resilient to current andin articulating these priorities is to encourage others

future biophysical challenges.

to join, either formally or via parallel efforts, so that
we are working towards common goals.

4. D evelop guidance to ensure restoration of
threatened coral species takes place within a

1. Increase restoration efficiency, focusing on scale

comprehensive population genetics management

and cost-effectiveness of deployment.

context.

A large majority of coral reef restoration projects

In many places around the world, critical reef-

fall under the “coral gardening” umbrella (Bostrom-building corals are suffering population declines

Einarsson et al., 2020). This technique involves

requiring strategic and thoughtful population

taking coral fragments (often grown in a nursery)

management plans that can allow these species toand transplanting them onto a degraded reef. The

be used effectively in restoration programs. To do

CRC takes pride in tackling restoration issues from

this, careful consideration is needed to maximize

multiple angles. While the CRC provides guidance

any remaining genetic diversity of degradedbased on the latest science, we also have a firm

coral populations to allow for successful sexual

understanding that most of the restoration occurring

reproduction, adaptation, and recovery (Baums et al.,in the world today is the relatively simple technique2019). Sound guidance is needed to ensure populationof coral gardening. Although this technique alone

management plans are developed with appropriate

will not save the world’s coral reefs - it can be done

genetic (including epigenetic), propagation,

more or less effectively, and more or less efficiently.

husbandry, and environmental considerations, andBy improving the efficiency of the most common

an understanding of new reef states (Rinkevich,method, we could vastly increase the potential for2020).

Scaling up coral restoration

5. Develop and promote the use of standardized

85

documenting the successes and challenges of reef

terms and metrics for coral reef restoration.

restoration. The large area images can illustrate

Although terrestrial habitat restoration has been

the ultimate metric of reef health at an appropriate

conducted for decades, coral reef restoration is aecological scale. They document species composition,

relatively new field. A lack of common definitions

coral cover, and growth of corals, and allow a re-and standardized metrics slows the transmission

examination of past data. The webinar relied onof ideas and the adoption of new techniques, while

expertise from several academics and featured

limiting comparability across projects (Bostrom-case studies by two reef restoration practitioners.

Einsarsson et al., 2020). To increase the scale and

It is archived on the Reef Resilience website. The

efficiency of coral reef restoration, we must be able

Monitoring Working Group has also prepared Coral

to communicate effectively across regions, fields ofRestoration Monitoring Guidelines (Goergen et al.,study, and public and private sector stakeholder2020) as well as a beta version of a Restorationgroups with varying levels of technical expertise.

Database.

This entails (1) identifying commonly used terms,

The Genetics Working Group published two recent

comparing existing definitions, and establishingpapers:standard definitions when possible (while also

beyond biomarker discovery (Parkinson et al., 2020),

acknowledging differing or conflicting definitions);

and Considerations for maximizing the adaptiveand (2) developing and promulgating standard

potential of restored coral populations in the western

metrics for evaluating the success of coral reef

Atlantic (Baums et al., 2019). The latter is a succinct

restoration efforts.

guide to maximizing genetic diversity using various

Molecular tools for coral reef restoration:coral aquaculture techniques for restoration. Despite
6. Develop new and synthesize existing resources

differences in species and environmental conditions,to guide and support coral reef restoration

restoration is happening throughout the globe.

practitioners working in diverse geographic

Although this paper uses a Caribbean example, thelocations.

recommendations serve as a helpful starting point

While there is a clear need to increase thefor coral aquaculture and restoration elsewhere.

efficiency of current methods, develop new

The paper explicitly recommends the number oftechniques and approaches, and formalize the best

genets per area that should be planted to maintain

available science, none of this has utility if theor maximize diversity. Because bleaching, coral

information does not reach the hands of people doing

gardening, and assisted evolution have the potential

the work. Practitioners need knowledge exchanges,

to reduce genetic diversity, having clear guidelines

resources to build capacity, and access and trainingthat managers and practitioners can understand isto new technological tools.

paramount for proper aquaculture and coral reef
restoration techniques.

Recent Accomplishments of the Coral Restoration

Members of the Land-based and Larval

Consortium

Propagation Working Groups created the firsttrans-regional crosses of an endangered coral

Most of the work that the Coral Restoration

population using cryopreservation (Hagedorn etConsortium does is via its Working Groups. Hereal., 2018). Rescuing genetically depauperate coral

I will outline a few recent accomplishments. The

populations by increasing genetic diversity from

Monitoring Working Group developed a robust

nearby populations, is one of the most feasible coralwebinar on Photomosaics in coordination with theinterventions proposed. This study demonstrated forReef Resilience Network. The webinar presents thethe first time that viable juveniles of an endangered

state of the science, the benefits of monitoring reefs

coral can be created by artificially inseminating cryo-with this technology, low-budget as well as high-preserved sperm from one population, with eggs ofresolution options for implementation, and resources

another regionally-distinct population. This project

for adoption. Photo-mosaics are an ideal method forhighlighted the importance and need for additional

Tali VARDI

86

expertise in coral larval rearing and early life-stage

global efforts to maximize results. Under Action (1),aquaculture. Currently, there are only a handful ofthe interventions that NOAA chose to prioritize

such experts worldwide. As corals continue to be

fit the following six objectives (taken directly fromthreatened with extinction primarily due to climate

Vardi et al., 2020):

change, these techniques will become increasingly

1. Increase Diversity of Coral Populations. Coral

necessary for the long-term persistence of coral

populations are becoming increasingly small

reefs.

and fragmented, leading to depensatory effects
that further limit spawning and recruitment.
US National Academies Review of Coral

Increasing the stress tolerance or simply

Interventions

the genetic diversity of small, fragmented
populations by importing corals fromIn 2018, NOAA commissioned a two-part study

populations in different parts of the species’

by the U.S. National Academies of Sciences,

range or even from within a population, may

Engineering, and Medicine to examine ecologicalbe some of the least risky and most effective

and genetic interventions that have the potential

intervention strategies. The interventions

to increase the resilience of coral reefs. Most ofthat would help accomplish this objective

these techniques rely on coral aquaculture in one

are: Assisted Gene Flow, Outcross Between

form or another. The Research Review describes

Populations, Supportive Breeding, andthe benefits, potential scale of application, current

Cryopreservation.

feasibility, risks, limitations, and infrastructure

2. Improve techniques to support interventions.

needs for 23 intervention types (NASEM, 2019a).

Techniques to support interventions include:

The Decision Framework outlines an adaptive

identifying stress tolerant coral colonies

management strategy by which interventions canor genes (“Managed Selection”), expanding

be evaluated against each other, additional research

cryopreservation capabilities to captureneeds, and recommendations for the Caribbean

current genetic variation for future research(NASEM, 2019b). Research on coral interventions

and restoration, and harnessing the diversity

is progressing very rapidly and these reports

and abundance of coral spawning. Gamete andthoroughly capture the current state of the sciencelarval capture and seeding research is being led

and make recommendations about how the fieldby institutions in Japan, Australia, and SECORE

should move forward in the near future. Given theinternational.

accelerating pace of threats to reef ecosystems,

3. Develop a framework for coral epidemiology.

it is clear that the coral conservation community

Disease is ravaging Caribbean corals andwill need effective, timely interventions, judiciously

worsening climate conditions are likely toapplied; and that coordinated bodies of scientists,

increase the frequency and severity of coral

governmental officials, and other stakeholders,disease outbreaks worldwide. The research,

will have to decide which blend of conventional

veterinarian, management, and restoration

management and interventions will maximize theircommunity needs an epidemiology framework

local reef’s ability to persist considering budget, local

for coral-disease intervention, as well as

buy-in and policy.

research and development of therapies andBased on these reports, NOAA created an actiondelivery mechanisms.

plan to guide how the agency will approach coral

4. Stress-harden corals (“Pre-exposure”). Multiple

interventions in the next one to three years (Vardi

lab experiments and field observation have

et al., 2020). Four primary actions are identified: (1)demonstrated that corals can increase theirresearch and test priority interventions (2) develop

resilience to temperature and ocean acidification

local or regional structured decision support. (3)stress under certain conditions.

review policy implications of coral interventions. (4)5. Manipulate algal symbionts to improve thermal

invest in infrastructure, research, and coordinate

tolerance (“Algal Symbiont Manipulations”).

Scaling up coral restoration

87

Coral bleaching is expected to increase inpotential of restored coral populations in thefrequency over the next decades. Algal

western Atlantic. Ecol. Appl., 29(8), e01978. (doi:symbionts vary in their thermotolerance.

10.1002/eap.1978)

Interventions that take advantage of thisBostom-Einarsson L., Babcock R. C., Bayraktarov E.,

variability have experimentally increased the

Ceccarelli D., Cook N., Ferse S. C. A., Hancock

thermotolerance of the coral holobiont.

B., Harrison P., Hein M., Shaver S., Smith A.,

6. A s s e s s f e a s i b i l i t y o f e n v i r o n m e n t a l

Suggett D., Stewart-Sincliar P.J., Vardi T.,

interventions. Environmental interventions

and McLeod I. M., 2020: Coral restoration

are manipulations to the physical or chemical- A systematic review of current methods,

environment to reduce or prevent bleaching,successes, failures, and future directions. PLoS

or reduce acidification. They can be geared toONE, 15(1), e0226631. (doi.org/10.1371/journal.

protect high-value sites such as nurseries orpone.0226631)

frequent tourist destinations. Examples include

Bindoff N. L., Cheung W. W. L., Kairo J. G., Arístegui“Shading” corals from incident light, “Mixing ofJ., Guinder V. A., Hallberg R., Hilmi N., Jiao

Cool Water”, and changing the alkalinity of reefN., Karim M. S., Levin L., O’Donoghue S.,

waters by restoring nearby plant communities

Purca Cuicapusa S. R., Rinkevich B., Suga T.,(“Seagrass Meadows and Macroalgal Beds”).

Tagliabue A., and Williamson P., 2019: Changing
Ocean, Marine Ecosystems, and Dependent

Conclusion

Communities, in “IPCC Special Report on the

Due to precipitous ocean warming, restoration(ed. by Pörtner H. -O., Roberts D. C., Masson-(including intervention) is a necessary bridge toDelmotte V. , Zhai P., Tignor M., Poloczanska E.,

stave the demise of tropical coral reef ecosystems.

Mintenbeck K., Alegría A., Nicolai M., Okem A.,

Here I have described how NOAA and the Coral

Petzold J., Rama B., and Weyer N. M.), in press.

Restoration Consortium are approaching this topic.

Fabricius K. E., Noonan S. H. C., Abrego D.,

However, the following cannot be overstated -H a r r i n g t o n L . , a n d D e ’a t h G . , 2 0 1 7 : L o w

these interventions are fruitless without addressing

recruitment due to altered settlement

basic local and regional reef conditions such as

substrata as primary constraint for coral

sedimentation, eutrophication, and over-fishing ascommunities under ocean acidification. Proc.

well as global climate change. Restoration should

Biol. Sci., 284(1862), 20171536. (doi: 10.1098/

Ocean and Cryosphere in a Changing Climate

always be conducted within broader resilience-based

rspb.2017.1536)

management strategies in coral reef ecosystems andGoergen E. A., Schopmeyer S., Moulding A., Moura

ideally within effectively managed marine protectedA., Kramer P., and Viehman T. S., 2020: Coral

areas that reduce and control background factors

reef restoration monitoring guide: Methodsof coral reef degradation. Coral reef protection andto evaluate restoration success from local torestoration requires local management efforts asecosystem scales, NOAA Tech. Memo. NOS

well as climate change mitigation if we want to

NCCOS 279, NOAA, Silver Spring, MD, 145pp.

witness coral reef recovery and the restoration of(doi:10.25923/xndz-h538)

reef ecosystem services.

Hagedorn M., Page C. A., O’Neil K., Flores D.
M., Tichy L., Chamberland V. F., Lager C.,

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and Resilience of Coral Reefs, The National

Atlantic. Bull. Mar. Sci., 88(4), 1075-1098.

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Medicine, 2019b: A Decision Framework forInterventions to Increase the Persistence(1) National Academies of Sciences, Engineering, andand Resilience of Coral Reefs, The National

Medicine, 2019: A Research Review of Interventions

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to Increase the Persistence and Resilience of Coral

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Reefs, The National Academies Press, Washington,

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DC, 245pp.

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beyond biomarker discovery. Conserv. Lett.,and Medicine, 2019: A Decision Framework for13(1), e12687.

Interventions to Increase the Persistence andPossingham H. P., Bode M., Klein C. J., 2015: Optimalconservation outcomes require both restorationand protection. PLoS Biol., 13(1), e1002052. (doi.
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Resilience of Coral Reefs, The National Academies
Press, Washington, DC, 212pp.
NOAA commissioned the U.S. National Academies
to review the science behind next generation coral

Randall C., Negri A., Quigley K., Foster T., Ricardo

restoration techniques. Most of these techniquesG., Webster N., Bay L., Harrison P., Babcock R.,

rely on coral aquaculture in one form or another.

Heyward A., 2020: Sexual production of corals

The Research Review groups 23 interventions

for reef restoration in the Anthropocene. Mar.

types (e.g. assisted evolution, marine shading to

Ecol. Prog. Ser., 635, 203–232.

prevent bleaching, genetic engineering) into four

Rinkevich B., 2005: Conservation of coral reefs

categories and describes the benefits, potential scale

through active restoration measures: Recent

of application, current feasibility, risks, limitations,approaches and last decade of progress.

and infrastructure needs for each. The Decision

Environ. Sci. Technol., 39(12), 4333-4342.

Framework outlines an adaptive management

Rinkevich B., 2008: Management of coral reefs: We

strategy by which interventions can be evaluatedhave gone wrong when neglecting active reef

against each other, additional research needs, andrestoration. Mar. Pollut. Bull., 56(11), 1821-

recommendations for the Caribbean. Research on

1824.

coral interventions is progressing very rapidly andRinkevich B., 2019: The active reef restorationthese reports thoroughly capture the current state oftoolbox is a vehicle for coral resilience andthe science and make some solid recommendations

adaptation in a changing world. J. Mar. Sci.

about how the field should move forward in the nearEng., 7(7), 201.

future.

Rinkevich B., 2020: Ecological engineering
approaches in coral reef restoration. ICES J.

(3) Hagedorn M., Page C. A., O’Neil K., Flores D. M.,

Mar. Sci., fsaa022. (doi.org/10.1093/icesjms/

Tichy L., Chamberland V. F., Lager C., Zuchowicz

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N., Lohr K., Blackburn H., Vardi T., Moore J., Moore

Pacific Ocean were analyzed to delineate key

T., Vermeij M. J. A., and Marhaver K. L., 2018:

drivers and suggest appropriate management at

Successful demonstration of assisted gene flow

three different levels: protection, recovery, andin the threatened coral Acropora palmata acrosstransformation. Over 50% of surveyed reefs would

genetically-isolated Caribbean populations using

benefit from recovery which includes some form of

cryopreserved sperm. BioRxiv. (doi: https://doi.

restoration in addition to traditional management,org/10.1101/492447)

and, most importantly, reduction in ocean warming.

Rescuing genetically depauperate coral

Using comprehensive studies like this and the Global

populations by increasing genetic diversity from

Coral Reef Monitoring Network’s reports, should

nearby populations, is one of the most feasible coral

help nations prioritize coral research, restoration,interventions proposed. This study demonstrated forand management efforts.

the first time that viable juveniles of an endangered
coral can be created by artificially inseminating cryo-(5) Baums I. B., Baker A. C., Davies S. W., Grottoli

preserved sperm from one population, with eggs ofA. G., Kenkel C. D., Kitchen S. A., Kuffner I.

another regionally-distinct population. This project

B., LaJeunesse T. C., Matz M. V., Miller M.

highlighted the importance and need for additionalW., Parkinson J. E., and Shantz A. A., 2019:

expertise in coral larval rearing and early life-stage

Considerations for maximizing the adaptive potential

aquaculture. Currently, there are only a handful ofof restored coral populations in the western Atlantic.

such experts worldwide. As corals continue to be

Ecol. Appl., 29(8), e01978. (doi: 10.1002/eap.1978)

threatened with extinction primarily due to climate

This paper is a succinct guide to maximizingchange, these techniques will become increasingly

genetic diversity using various coral aquaculturenecessary for the long-term persistence of coraltechniques for restoration. Despite differences in

reefs which feed hundreds of millions of people andspecies and environmental conditions, restoration

provide habitat to 25% of marine fisheries globally.

is happening throughout the globe. Although
this paper uses a Caribbean example, the(4) Darling E. S., McClanahan T. R., Maina J., Gurney

recommendations serve as a helpful starting point

G. G., Graham N. A. J., Januchowski-Hartley F.,

for coral aquaculture and restoration elsewhere.

Cinner J. E., Mora C., Hicks C. C., Maire E., Puotinen

The paper explicitly recommends the number ofM., Skirving W. J., Adjeroud M., Ahmadia G., Arthur

genets per area that should be planted to maintainR., Bauman A. G., Beger M., Berumen M. L., Bigotor maximize diversity. Because bleaching, coral

L., Bouwmeester J., and 60 others, 2019: Social–gardening, and assisted evolution have the potential

environmental drivers inform strategic management

to reduce genetic diversity, having clear guidelines

of coral reefs in the Anthropocene. Nat. Ecol. Evol.,that managers and practitioners can understand is3, 1341-1350.

paramount for proper aquaculture and coral reef

Data from more than 2500 reefs across the Indo-

restoration techniques.

水 研 機 構 研 報， 第 50 号，91 － 100， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，91－100，2021

91

Sustainable large-scale coral restoration by establishing“artificial spawning hotspots”
Go SUZUKI＊Abstract: Coral reefs have degraded over time and, recently, severe bleaching events caused
further damage to coral communities worldwide from 2015 to 2017. Because coral reefs are an
important habitat for coastal fish and invertebrates in tropical coasts, the decline of coral reefs
also results in decreased fishery productivity. Therefore, for sustainable use of these resources, it
is important to conserve coral communities that act as fishing grounds and fish nurseries. Coraltransplantation is known as an effective restoration method. However, large-scale transplantation
requires a great deal of labor and the transplanted corals are vulnerable to extermination by onlya single disturbance such as bleaching or an outbreak of crown-of-thorns starfish (COTS). Hence,enhanced annual coral larval recruitment is needed for sustainable large-scale restoration.
Two key factors are crucial for sustainable coral restoration by enhancing reproduction. The
first factor is establishment and maintenance of “artificial spawning hotspots” that consist ofdensely populated conspecific adult colonies. The second factor is improvement of early life
survivability by collecting eggs and sperms at spawning and rearing larvae until settlement. For“artificial spawning hotspots,” safeguards are required against predation by COTS and the use of
shading is desirable against bleaching during the high seawater temperature season. In addition, a
special gamete and larval collector, termed “larval cradle”, was developed to consistently perform
bundle collection, fertilization, and larval rearing. Eventually, it is suggested that a set of simple
methods and techniques are used for sustainable large-scale coral restoration.
Key words: larval supply, Acropora, fish nursery, larval cradle, artificial substrate
Current Coral Reefs Status and Crisis

seawater temperature where corals lose symbiotic
algae (Fujise et al., 2014). Such corals become white

Coral reefs are large biogenic structures thatin color and this phenomenon is called “bleaching.”

develop in shallow waters in tropical and subtropicalSome researchers have described bleaching of corals

regions. Most of the modern coral reefs have formed

as an adaptive behavior to environmental change,

atop stacks of dead corals created several thousand

known as “adaptive bleaching” (Kinzie et al., 2001).

years ago (Braithwaite et al., 2000; Kayanne et al.,

In any case, if the ambient temperature is 1℃ higher

2002; Hongo and Kayanne, 2009). Recently, several

than normal for longer than 1 month, many corals

disturbance factors frequently endanger coral

die after bleaching. Some taxonomic groups such

reefs (De’ath et al., 2012). One of those factors is

as genus Acropora, Montipora, and Seriatopora arecoral bleaching. Corals have symbiotic microalgae,

susceptible to bleaching (Marshall and Baird, 2000),

called “zooxanthellae”, which largely contribute tothough these groups still dominate most of the Indo-

coral growth by providing nutrients obtained by

Pacific reefs. Global warming may cause an increase

photosynthesis to the host coral (Tanaka et al., 2018).

in seawater temperature and some predictions

This symbiotic relationship can collapse due to highforecast that the frequency of large-scale bleaching
2020年12月11日受理（Accepted on December 11, 2020）＊
Research Center for Subtropical Fisheries, Seikai National Fisheries Research Institute, Japan Fisheries Research and Education Agency, 148
Fukai Ota, Ishigaki, Okinawa, 907-0451, Japan
E-mail: gosuzu “at” affrc.go.jp

Go SUZUKI

92
events will be higher than in past centuries.

coral reproductive output.

Another crisis is outbreak of crown of thorns

As mentioned above, most disturbances have large

starfish (COTS) (Moran et al., 1992). COTS is a large

impacts on specific taxa such as Acropora. Although

starfish armed with poisonous spines that predates

Acropora species are fast growing corals, it still takes

corals, especially Acropora and Montipora species

at least 3 years (5 years on average) to grow to a

(Pratchett et al., 2009). Generally, the population

mature size. Furthermore, the older the corals grow,2

density of COTS is 1-2 individuals per 100 m . Oncethe more eggs they spawn that contribute to larvalan outbreak occurs, the density increases by 100

supply. If most corals die from each disturbancetimes, and several hectares of corals can be eaten

within 10 years, robust reproduction (i.e. adequate

up within a few months (Suzuki et al., 2012). Factors

larval supply) lasts only a few years and this could

that cause a COTS outbreak are not clear, but it isalso gradually reduce coral populations.

probable that high nutrients contribute to outbreaks
because COTS larval survival was drastically

Impact on Fisheries Resources

improved by increased phytoplankton density (i.e.
the prey of COTS larvae) that resulted from highFisheries resources in coral reefs have been anutrient concentrations in laboratory experiments

primary source of protein for people in tropical(nutrient hypothesis) (Fabricius et al., 2010).

r egio ns (B ur ke et a l ., 2 0 11 ). Ma ny fish and

The third crisis is physical damage to corals

invertebrates directly or indirectly depend on coralcaused by large typhoons (cyclones or hurricanes).

communities. For example, juveniles of one of theSome corals that have weak skeletal structures,

dominant groupers, Epinephelus ongus, selectively

such as branching corals, are easily and severely

settle in the bottle-brush coral (Acropora species)

damaged by large typhoons that typically occur

habitat (Nanami et al., 2013). Most parrotfishes

once every several years.

utilize the space among coral branches (mainly

It is important to understand the extent ofbranching Acropora species) as a sleeping bed andanthropogenic impacts on coral reef destruction.

coral recruitment is enhanced by fish grazing (RussFor bleaching, if the cause of global warming is

et al., 2015). Some cuttlefish lay eggs in the space

the accumulation of greenhouse gases such as CO2

among coral branches (mainly branching Porites

primarily discharged by human activities, bleachingspecies). In addition, small fish such as damselfish

is thus a consequence of anthropogenic activities.

and cardinalfish live associated with corals, which

However, it is difficult to distinguish the roles

feed large fish such as groupers (Shpigel andbetween global warming and the natural cycle of theFishelson, 1989; Nakai et al., 2001). Importantly,

global environment (e.g. the frequency of El Niño)

recent studies suggested that organic matteron the occurrence of bleaching (Claar et al., 2018).

produced by symbiotic algae through photosynthesis

In addition, it is probable that eutrophication and/or

contributed to the whole coral reef ecosystem asincrease of bacteria in the water column accelerate

primary production. Therefore, it is highly probabledamage to corals during bleaching (Fabricius etthat the decrease of corals is linked to the loss ofal., 2013). For outbreak of COTS, if the nutrient

fisheries resources (Wilson et al., 2006). In terms

hypothesis as stated above is true, the frequencyof reproduction, “spawning aggregation” is one ofof COTS outbreak is expected to increase due tothe representative behaviors in the reproduction

anthropogenic impact. Similarly, anthropogenic

for some reef fishes such as groupers (Domeier andimpacts may indirectly increase the size of typhoons

Colin, 1997; Nanami et al, 2017). Considering that

because global warming may be a contributing

such behavior could also be affected by the decreasecause (Balaguru et al., 2016). Although the extent

of corals, the loss of fisheries resources is likely veryof anthropogenic impacts on these crises remains

serious with long-term consequences.

poorly understood, coral populations are seriously
damaged by a higher frequency of disturbances,
especially in the long term, because they decrease

Coral restoration by creating artificial spawning hotspots

Previous Studies on Coral Restoration

932019). However, it is possible that natural slicks may
not form or be detected due to inclement weather

Corals reproduce sexually and asexually.

conditions. In addition, natural spawning and“Fragmentation” is a process of asexual

fertilization may decrease after disturbances because

reproduction, in which some branches of corals

these events reduce the population density of adultregrow as a new colony by adhering to the seafloor

corals.

once they snapped off. Corals have various methods

Methods for outplanting are very importantof sexual reproduction including mass spawning (bothbecause all seedlings (also called fragments) are

gonochoric and hermaphroditic) and larval brooding

required to be outplanted on the seafloor. Random(Baird et al., 2009). Corals produce swimming planula

outplanting to copy natural coral communities maylarvae that settle on the seafloor and metamorphosebe manageable on a small scale, similar to gardening.

to juvenile corals.

However, for large-scale restoration, there is risk

One of the simplest methods for coral restorationthat all the required cost and effort will be wasted

is transplantation of coral fragments that can beby a single disturbance. Therefore, the key toaccomplished using different techniques such as

restoration is the concept called “artificial spawning

direct attachment of coral fragments (fallen branches

hotspots” (Zayasu and Suzuki, 2019).

are more effective) onto the seafloor, or attachment
on artificial plates in aquaria followed by fixation on

Artificial Spawning Hotspots

the seafloor. The first report of the coral fragment
attachment method was from Guam in the 1970s

The basic ideas for sustainable large-scale coral(Birkeland et al., 1979) and this method has advanced

restoration are: 1) creating a high density of matureeach year since. In Okinawa, a total of 100,000 orcolonies of a single species to enhance fertilization

more fragments were transplanted to approximately

and collection of gametes, and 2) sustaining larval

3 hectares of damaged reef over a 6-year period

supply by protecting these high-density populations.

from 2011 to 2016 (Okinawa Prefectural Government,

First, selection of the target species is important2017).

for establishment of artificial spawning hotspots.

The next step to the transplantation is

Approximately 800 coral species with different

seedling production by sexual reproduction. Coral

reproductive characteristics are known in thecommunities made by transplantation of fragments

world; some are broadcast spawners and someare likely to have low genetic diversity because

are brooders (Baird et al., 2009). Also, within themost are clones of parental strains. Clones cannot

broadcast spawners, some are gonochoric and some

reproduce sexually in most coral species. In addition,

are hermaphroditic.

generally availability of parent strains is limited.

The target corals for artificial spawning hotspots

In contrast, we can produce many seedlings (i.e.

are Acropora species, a representative genus ofgenets) by collecting and fertilizing gametes, rearing

hermaphroditic broadcast spawners. Acropora is alarvae, and settling them on substratum.

dominant group in Indo-Pacific reefs and one of the

One of the most efficient methods for gamete

most diverse genera, which forms various habitats

collection is through collection of the “slick”. Inwith different colony morphologies among species

tropical regions, approximately 100 coral species(Wallace, 1999). Within Acropora, the branching

simultaneously spawn gametes on the same night inspecies are an especially important target, as stateda “mass spawning” (Harrison et al., 1984). Substantial

above, in that they contribute to fisheries resources.

numbers of fertilized and unfertilized eggs are

Because the coral polyps of branching coralgathered at the sea surface the following morning;

distribute less densely than those of tabular andthis is called a “coral slick.” By collecting these slicks

corymbose species (Suzuki unpubl. data), the number

and rearing them, many larvae are obtained at once.

of eggs spawned each year is lower, suggesting that

Recently, a large-scale slick collection method wastheir larval supply is also small. It takes longer for

developed using a large vessel (Doropoulos et al.,these species to recover through natural recruitment

94

Go SUZUKI

from local extinction due to severe disturbance.

of the “larval cradle” (Suzuki et al., 2020). LarvalHence, the efficacy of artificial spawning hotspots

rearing techniques using aquaria have already been

would be higher for the branching Acropora species.

developed using fertilized eggs collected in the

Indeed, the number of Acropora recruits isfield from adult corals or reared in a tank (Omori

clearly different among reef habitats. From the

et al., 2004); however, there were no methods forresults of long-term monitoring of Acropora recruits

completing the entire process in the field. Acropora

around Ishigaki Island, the number of recruits wascorals release egg-sperm bundles and burst open at

extraordinarily high in 2014 in the reef slope after

the sea surface, then the gametes are fertilized withthe local extinction in 2011 due to a COTS outbreak.

the gametes released from other colonies (i.e. almost

The coral cover visually increased from 2017 where

no self-fertilization) (Willis et al., 1997). Utilizing

the cohort grew to a visible size (Suzuki unpubl.

this characteristic, a device that catches releaseddata). However, there was not high recruitment inbundles with a large net and holds them until they

the lagoon area over a 10-year period and no coral

are competent larvae (called the larval cradle) wasrecovery was found (Suzuki unpubl. data). Artificial

invented.

spawning hotspots should be established in suchplaces where natural recruitment is always low.

A completed version of the larval cradle consistedof a cylinder with a diameter of 1.7-m and a height

In the concept of artificial spawning hotspots, itof 4.25 m made of 30 µm mesh nylon net (Fig. 1a),is necessary to complete seedling production (fromwhich achieved the collection and rearing of several

gamete collection to larval settlement on artificial

million coral larvae with more than 90% fertilization

substrate) in the sea, which led to the development

and survival rates. The size of the larval cradle was

(a)

(b)

(c)

Fig. 1. (a) Setting of the larval cradle with a dedicated float. Size of the cylindrical cradle was 1.7-m diameter and
4-m height below sea level (0.25-m height above sea level). (b) Artificial substrate of short square tube (i.e. squarehollow section, SHS) for Acropora settlers. Scale bar represents 4 cm. (c) Method of placing the SHSs into thecradle (SHSs were put in net bags and hung from an EVA float).

Fig.1

Coral restoration by creating artificial spawning hotspots

95

designed to be easy to handle for a small groupby buffering photoinhibition (Coelho et al., 2017).

of people (e.g. fishermen or leisure divers) while

In addition, moving the corals to deeper locations

ensuring the production of a significant number

could also reduce light intensity. Another candidateof larvae. A larger sized larval cradle was notcountermeasure against bleaching is reducing

preferred because it would break more easily during

starvation of the corals. It is highly probable thatstormy weather.

the direct cause of death for bleaching corals is

Next, the larvae produced by the larval cradle

starvation by losing nutritional supplementation

should be settled on substrates. Post-settlementfrom symbiotic algae (Borell and Bischof, 2008).

mortality of coral juveniles is very high. This

Hence, if any nutrition could be artificially suppliedmortality is primarily caused by unintentional

during bleaching, mass mortality events may be

predation by fish grazing on epibenthic fauna andavoided.

algae (Baria et al., 2010). To prevent fish grazing on

A countermeasure that addresses COTS outbreaks

coral settlers while keeping moderate light intensity,

is easier relative to countermeasures that addressa lattice-shaped plate was contrived that resulted inbleaching. Because COTS cannot climb on thin rods,

good survival of coral juveniles (Suzuki et al., 2011).

50-cm bottom-raised racks with, reinforcing steel

The short square tube (i.e. square hollow section,rods, is sufficient to prevent COTS damage. In fact,

SHS) substrate was further developed from theonly corals on bottom-raised racks survived thelattice-shaped plate (Fig. 1b). The SHS substrate

COTS outbreak in 2010-2011 in the Urasoko Bay,was like a single cell of the lattice-shaped plate,

Ishigaki Island, where 99% of Acropora corals within

which allows us to outplant easily after settled

the bay were eaten by COTS (Suzuki unpubl. data).

juvenile corals grow up. By hanging the SHSs into

By using these countermeasures for corals in

the cradle from a buoy (Fig. 1c), completion of theartificial spawning hotspots, the larval supply fromsettlement process in the sea could be achieved.

the hotspots could be several hundred times higher

Settlement of Acropora larvae at a moderate density

than that of a wild Acropora population experiencing

on SHS was also attained by contriving the number

the same environmental conditions (Fig. 2).

of SHSs and the timing and method of placing themCalculation of the total larval supply in a longer span

into the cradle (Suzuki et al., 2020). The survival rate

showed an even larger gap between the hotspot andof the settled corals on SHS was usually 10-20% inwild population. That is, the larval supply from thethe suitable environment (3-15 m in depth, moderate

hotspots could be stable annually for a long period,

current, little sediment, etc.), although it varied due

while the gradual deterioration of wild populations isto the environmental conditions (Suzuki et al., 2013).

predicted over 50-100 years (IPCC, 2018).

Methods for protection of adult corals from

There may be concern with species diversity of

disturbances, the second factor for creating artificial

target corals for restoration. However, only 5-10

spawning hotspots, have also been developed

branching Acropora species dominate the lagoon(Suzuki et al., unpubl. data). Bleaching and COTS

area in Indo-Pacific reefs, covering more than 50%

outbreak are the most serious disturbancesof the total coral area, even though more than 100

for adult Acropora corals, and, therefore, thecoral species were recorded in the area. In otherprocesses of these disturbances were focused forwords, the larval supply from the artificial spawning

the development of protection methods. One of

hotspots of 5-10 Acropora species is comparable

the available countermeasures against bleaching

to that from the wild coral community in terms ofis shading of corals. It is known that seawater

species diversity. In addition, the phase shift fromtemperature rise of approximately 1℃ causes

corals to macroalgae in a damaged reef is consideredphotoinhibition in the photosynthetic process andto delay the recovering of coral communities aftergenerates excess reactive oxygen species that

disturbances (Kuffner et al., 2006; Bozec et al., 2019).

obstruct the symbiosis with zooxantellate algae

Rapid restoration of dominant species could be(Warner et al., 1999). Thus, shading corals during

effective to avoid or reverse such a phase shift tohigh temperature periods could reduce the mortality

macroalgae.

Go SUZUKI

96

Coral larvae
Larval cradle

Low fertilization rateSparse settlement

High fertilization and survival rates
Extensive settlement

Larval supply
Long distance

Damaged reef

Artificial spawning hotspots

Bleaching and/or COTS outbreakCountermeasuresfor bleaching

Larval supply

for COTS outbreakProtection of artificial spawning hotspots

Further damaged reef

Fig. 2. Image of artificial spawning hotspots concept. Different colors represent different species of Acropora
corals.
Direct Larval Seeding

m2 was found 3 years after the seeding (dela Cruzand Harrison, 2017). An enclosing net was used to

Direct seeding of mass larvae acquired from larval

prevent dispersal of the larval seeding in that study;

cradles or natural slicks would be a shortcut tohowever, it is difficult to apply this method on alarge-scale coral restoration (Heyward et al., 2002).

larger scale (e.g. more than 1 hectare). Ultimately,

However, at present, direct larval seeding on thethe direct seeding of an overwhelming number

seafloor remains impractical due to low settlementof larvae with high density that is comparable toand survival rates of the seeded larvae (e.g. Edwards

natural recruitment would be effective in large scale

et al., 2015). In the Philippines, approximately 0.4

restoration even if the larvae were seeded without

million larvae were directly seeded to several

an enclosing net. Studies to determine suitable2

tens of m of damaged reef and 2.3 colonies per

seeding methods, including the necessary minimum

Coral restoration by creating artificial spawning hotspots

97

density, are currently in progress. In addition, there

Bozec Y. -M., Doropoulos C., Roff G., and Mumby P.

are some places where larval seeding has no effect.

J., 2019: Transient grazing and the dynamics

In the sandy bottom, covering most of the lagoon,of an unanticipated coral–algal phase shift.

few settlement substrata such as rock reefs are

Ecosystems, 22, 296-311.

available. It is expected that outplanting or seeding

Braithwaite C. J. R., Montaggioni L. F., Camoin G.

of corals settled on the artificial plates is moreF., Dalmasso H., Dullo W. C., and Mangini A.,

effective in such places (Chamberland et al., 2017).

2000: Origins and development of Holocene coral

In conclusion, the methods and techniques

reefs: a revisited model based on reef boreholes

reviewed in this paper can be effectively used forin the Seychelles, Indian Ocean. Int. J. Earth

gamete collection, larval rearing, seedling production,

Sciences, 89, 431-445.

direct larval seeding, maintenance of adult corals

Burke L., Reytar K., Spalding M., and Perry A.,

for larval supply, and protection of adult corals from2011: Reefs at Risk Revisited. World Resources

disturbances in the field (i.e. without land facilities).

Institute (WRI), Washington, D.C., 130pp.

Using these simple methods together shows promise
to successfully achieve large-scale coral restoration.

Chamberland V. F., Petersen D., Guest J. R., PetersenU., Brittsan M., and Vermeij M. J. A., 2017:
New seeding approach reduces costs and time

Acknowledgement

to outplant sexually propagated corals for reef
restoration. Sci. Rep., 7, 18076.

Some parts of this study resulted from Coral

Claar D. C., Szostek L., McDevitt-Irwin J. M., Schanze

Reef Restoration and Conservation efforts underJ. J., and Baum J. K., 2018: Global patterns andthe Severe Environmental Conditions project of the

impacts of El Niño events on coral reefs: A

Fisheries Agency, Japan.

meta-analysis. PLoS ONE, 13, e0190957.
Coelho V. R., Fenner D., Caruso C., Bayles B. R.,

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水 研 機 構 研 報， 第 50 号，101 － 113， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，101－113，2021

101

The influence of climate and environment on the growth and survivalof Pacific oyster seed in US West Coast estuaries
Brett DUMBAULD＊1, Evan DURLAND＊2, Konstantin DIVILOV＊2, Kelly MUETHING＊2,
Anna BOLM＊2, Ylva DURLAND＊2, and Brooke MCINTYRE＊2

Abstract: Pacific oysters Crassostrea gigas were introduced to the US west coast in the early 1900’s
and were initially raised directly from seed (juveniles that had set naturally on cultch shell) imported
from Japan. Oysters regularly spawned and became “naturalized” in only several selected West
coast locations, such as Willapa Bay, Washington, where conditions allowed for both adult oyster
spawning and larval survival, retention and settlement. The shellfish industry relied on “naturalor wild” caught seed from these locations or continued seed imports until the advent of hatchery
technology in the late 1970’s. Recent larval mortality events in hatcheries have been linked directly
to changes in seawater chemistry with high pCO2 conditions and acidified water associated withseasonal upwelling along the U.S. West Coast. These conditions may have also resulted in reduced
natural oyster sets in Willapa Bay, but estuarine gradients in water chemistry and temperature
add complexity making this more difficult to discern. Shellfish hatcheries have adapted to these
conditions by measuring seawater carbonate chemistry, buffering incoming water, and adjusting thetiming of larval production cycles. While there appear to be larval fitness traits that are genotypedependent, the potential for breeding programs to improve OA specific traits is only currently
receiving attention and remains uncertain. Experiments have rarely been conducted that distinguishsuccess at important physiological transitions in the larval life history and potentially also at thejuvenile seed stage when these oysters are out-planted to estuaries with variable conditions.
We review results from recent experiments where survival of oyster larvae raised from crossesof “wild” parental broodstock collected in Willapa Bay was compared to that of larvae raised fromcontrolled crosses with improved lines created by the Molluscan Broodstock Program (MBP) at
the Hatfield Marine Science Center (HMSC), Oregon State University. The MBP breeding program
was designed to enhance valuable field traits including growth and survival of juvenile and adultoysters, but to date has not explicitly addressed larval traits. Nonetheless, MBP larvae produced
more than twice the number of settled spat compared with wild larvae under commercial hatcheryconditions (with buffered seawater, pH ~8.3 and Ωarag >2). This advantage also occurred, but to alesser degree, under unfavorable high pCO2) conditions (pH ~7 and Ωarag <1). Separate experiments
were conducted to evaluate survival of juvenile seed along the estuarine gradient and inside andoutside eelgrass (Zostera marina), a marine plant which has the ability to modify local seawaterchemistry. Growth and survival of juvenile oyster seed varied along the estuarine gradient withfastest growth, but lower survival occurring near the estuary mouth and there was no apparent
effect of seed source. Results from experiments where MBP seed was planted both inside andoutside eelgrass along these same gradients, suggest that this plant can reduce seed growth,
especially at locations away from the estuary mouth.
Keywords: Pacific oyster, juvenile, growth, estuary, climate
2020年12月11日受理（Accepted on December 11, 2020）＊1
US Department of Agriculture, Agriculture Research Service, Hatfield Marine Science Center, Newport, OR 97365, USA＊2
Oregon State University, Fisheries and Wildlife Dept., Hatfield Marine Science Center, Newport, OR 97365, USA
E-mail: brett.dumbauld “at” usda.gov

102

Brett DUMBAULD, Evan DURLAND, Konstantin DIVILOV, Kelly MUETHING,
Anna BOLM, Ylva DURLAND, and Brooke MCINTYRE
Introduction

seed sets in estuaries where naturalized oyster
populations reproduce. Willapa Bay, which is one of

The Pacific oyster Crassostrea gigas was

the single largest cultured oyster production sites inintroduced to estuaries along the US West Coast

the US and the third largest estuary on the US west

in the early 1900’s and largely replaced the native

2
coast has only a 358 km signature, which roughly

oyster Ostrea lurida, which had been extensively

equals the size of the James River sub-estuary infished commercially and mostly overharvested or

Chesapeake Bay, the largest estuary on the US

succumbed to other factors including pollution (Blake

East Coast (11,600 km2). Estuaries like Willapa Bay

and Ermgassen, 2015; Dumbauld et al., 2011; Polson

also experience less riverine influence during theand Zacherl, 2009; Steele, 1964). Pacific oysters weresummer months and have shorter residence times

cultured and harvested from leased and privately

than estuaries where oysters are present on eastern

owned estuarine tidelands and the industry eitheredges of continents (Hickey and Banas, 2003).

relied on juvenile seed oysters shipped from Japan

Gradients in water chemistry and temperature areor placed cultch shell in several areas where

greatly influenced by short term events that notthis oyster had naturalized and conditions were

only influence larvae, but also timing and magnitudeconducive for them to spawn (e.g. Dabob and Willapa

of spawning events and retention of larvae in these

Bay in Washington State, USA and/or Ladysmith

estuaries making it more difficult to discern reasons

Harbor and Pendrell Sound in British Columbia,

for failures in larval seed set (Hales et al., 2017;

Canada) (Quayle, 1988). The advent of local hatchery

Ruesink et al., 2018).

production of oyster larvae and oyster seed in the

Improved larval survival and growth underlate 1970’s closed the culture life cycle separatinghigh small 10 day post metamorphosed oyster

the two life history phases and changed the naturespat were CO2 conditions appear to be genotype-of the industry again (Chew, 1984).

dependent fitness traits (Frieder et al., 2017; Pan

The nearshore coastal ocean along the US west

et al., 2018; Sunday et al., 2011), but the potential

coast is part of the California current ecosystem,

for breeding programs to improve these traits has

an eastern boundary upwelling system. Oysters

not yet received much attention. A collaborative

cultured in these systems are thus subject toeffort between scientists at Oregon State University

seasonal events where cold, nutrient rich, high pCO2

and the US West Coast shellfish industry known

water is transported to the surface and episodic

as the Molluscan Broodstock Program (MBP) was

intrusions into estuaries can significantly lower pH

initiated in 1996 to enhance juvenile oyster survival

and the aragonite saturation state especially nearand growth (de Melo et al., 2016; de Melo et al.,the estuary mouth (Feely et al., 2010; Hauri et al.,2018; Langdon et al., 2003). This selective breeding2013). Shellfish hatcheries operating in this region

program uses family-based mating designs andhave experienced significantly reduced rates ofbi-parental crosses to maintain genetic diversity

larval growth and survival during these upwelling

and limit effects of inbreeding. Although seven

events (Barton et al., 2012). In response to thesegenerations of larval cohorts have been reared inchallenges, commercial hatcheries now measure

the hatchery, the program is targeted to improveseawater carbonate chemistry, strategically timeyield of juveniles and adults on farms and there

larval production cycles, and chemically buffer

has been no direct attempt to select for larval

incoming seawater in order to maintain optimal

performance traits. Nonetheless, commercial shellfish

carbonate chemistry conditions (Barton et al., 2015).

hatcheries that partner with MBP reported that

While there are important exceptions like thelarvae spawned from MBP broodstock survived andSalish Sea in Washington State, most U.S. West coastgrew better when compared to wild counterparts,

estuaries are small relative to the spatial extent ofespecially during periods of strong upwelling, so

the nearby open coastline, so these upwelling events

experiments were conducted to discern whether

and changes in seawater chemistry can also result inunintentional selection for this trait had occurred.

larval oyster mortality and reduced or failed oyster

Results demonstrated that larvae from MBP

Influence of climate on Pacific oyster seed growth and survival

103

broodstock produced from 37% to 50% more spatdissolved oxygen, low salinity, high temperature)than larvae from “wild” broodstock collected fromwere less evident.

a naturalized population in Willapa Bay (Durland

Finally, there continues to be interest in evaluating

et al., 2019). This advantage occurred under boththe potential role of seagrasses as refugia from thecommercial hatchery conditions (with bufferedeffects of altered water carbonate chemistry in partseawater, pH ~8.3 and Ω arag >2) and to a lesser

because these important estuarine plants may also

degree, under unfavorable high pCO2 conditions (pHgrow more rapidly under elevated CO2 and increase~7 and Ωarag <1). This suggests that improvements

carbon sequestration (Washington State Blue Ribbon

in larval performance had occurred despite no

Panel on Ocean Acidification, 2012; Cullen-Unsworth

intentional selection. Further analysis of single

and Unsworth, 2016; Kelly et al., 2011). Seagrasses

nucleotide polymorphisms (SNPs) from pooled larval

uptake CO 2 while photosynthesizing during the

DNA indicated that larvae from wild stocks had

day and therefore have the potential to modulate

more than twice the number of loci affected bycarbonate chemistry at least at very local scales,

acidified culture conditions, compared to larvae frombut they also respire at night and die back during

MBP stocks (Durland, 2019). Functional analysis

the winter in most temperate estuaries potentiallyrevealed that the predicted genes associated withincreasing pCO2 (Hendriks et al., 2014). A laboratorychanges under OA conditions were linked to theevaluation of the interaction between juvenile

structure and function of cellular membranes (Deoysters, the seagrass Zostera marina (hereafterWit et al., 2018), but the affected loci were largely

eelgrass) and seawater carbon chemistry showed

exclusive to each parental stock with little overlap,that enhanced daytime seawater pH in the presence

suggesting that development of universal markers

of eelgrass did not counteract the negative effects

and selecting for OA resistance in Pacific oysters

of increased pCO2 when both oysters and eelgrass

will be complicated.

were present (Groner et al., 2018). However studies

The effect of altered water chemistry on

conducted in estuaries suggest that growth andjuvenile oysters once they settle naturally or

survival of juvenile oysters can be enhanced inare transplanted from hatcheries and planted

eelgrass (Lowe et al., 2019; Smith, 2016). This effecton farms for grow-out are less studied. Delayed

was site specific and therefore difficult to attributecarry-over effects on juvenile growth due to larval

to water chemistry and distinguish from effects of

exposure (Gobler and Talmage, 2013; Hettinger

other factors like reduced water flow in eelgrass

et al., 2013) and cross-generational effects when

which potentially results in less suspended sediment

breeding adults are exposed to elevated CO2 havein the water and enhanced phytoplankton foodbeen documented (Parker et al., 2015; Parker etintake.

al., 2012), but distinguishing the effects of elevatedHere we present preliminary results from two

CO2 and reduced aragonite saturation from otherexperiments where juvenile oyster seed was planted

environmental factors in estuaries is difficult.

inside and outside eelgrass at several locations along

Hollarsmith et al. (2020) documented the influence

the axis of two US West coast estuaries whereof seasonal processes on both water chemistry

we expected gradients in water chemistry andand temperature along the estuarine gradient inenvironmental conditions to differ.

Tomales Bay, CA. They found that both juvenile
Pacific and native oyster growth and survival was

Methods

more influenced by freshwater runoff during thewet season (though limited in this estuary) than by

We conducted experiments in two estuaries alongcarbonate chemistry during the summer upwelling

the US West Coast. Netarts Bay, Oregon, USA (45°25’

season. Growth was highest during the upwellingN, 123°56’ W) is a small tidally flushed estuary, withseason and appeared to be related to higher

over ~75% of its total volume turning over every

phytoplankton concentrations in the water column

tidal cycle (6.22 hours) (Fig. 1). It has a relatively

especially near mid-bay where other stressors (lowsmall (36.3 km) watershed whose summertime

Brett DUMBAULD, Evan DURLAND, Konstantin DIVILOV, Kelly MUETHING,
Anna BOLM, Ylva DURLAND, and Brooke MCINTYRE

104

freshwater contribution to flushing is negligible(Barton et al., 2012) where a continuously-monitoring(Glanzmann et al., 1971; Kentula and McIntire, 1986).

carbonate chemistry analyzer has been in place

Patterns in carbonate chemistry were therefore

since 2010 (Barton et al., 2015). Though still dwarfed

expected to be almost entirely driven by conditions

in size by estuaries along the US East coast as notedon the coastal shelf, perhaps at both the marine

above, Willapa Bay, Washington USA (46°32’ N, 123°

end near the estuary mouth and at the riverine

59’ W) is the third largest estuary along this coast. It

end during the summer when this experiment was

has a much larger watershed than Netarts Bay (2,857

conducted. Netarts Bay is also home to the Whiskeykm2) (Fig. 2), but is still strongly tidally-influenced,

Creek Shellfish Hatchery, the largest independent

especially in the summer when river flow is virtually

producer of oyster seed in the Pacific Northwest

non-existent. Though roughly half the water volume

15 Site 1

Eelgrass
OutsideInside105015Whiskey Creek
Shellfish Hatchery

Site 2

1

Shell Area (cm2)

10
5

2

0
15 Site 3

3

10

4

5
0
15 Site 410
5
0 June July Aug Sept Oct Nov

MonthFig. 1. Results of the 2015 experiment in Netarts Bay, Oregon, USA. Oysters were

Fig.1.
Resultsof thein (right).
Netarts
Bay, Oregon,
USA.
Oysters were
deployedin Mayat 42015
sites experimentshown in map
Averageshell area
measurementsof deployed in±SE)in eelgrass(green)and outside
open measurements
habitat (orange) isofshownoystersMay
at 4(cmsites
shown
in maphabitat(right).
Averageshell areaoysters (cm2 ± SE)2

for three time points in July August and November (left).

in eelgrass habitat (green) and outside open habitat (orange) is shown for three time points inJuly August and November (left).

Influence of climate on Pacific oyster seed growth and survival

105

is exchanged on every tide and replaced withand other factors near the Naselle River (Ruesink

new ocean water, the water at the southern end

et al., 2018; Ruesink et al., 2015), so we expectedof the estuary has a 3 to 5 week residence period

a potentially different gradient and influence of(Banas et al., 2004; Banas et al., 2007). It is the single

carbonate water chemistry on oyster survival andlargest producer of oysters on the US West coast

growth in this estuary than Netarts Bay.

and often in the US. Carbonate chemistry has been

Experimental plantings of juvenile oyster seedshown to be influenced by upwelling near the ocean(spat) were made in two separate years to contrast

endpoint at the estuary mouth, but also by alkalinity

growing conditions inside and outside of eelgrass at

30
25Site 1

2018

Eelgrass

North River

OutsideInside

20
15
10
5
0
30
25

Willapa River

Site 31

20
15Shell Area (cm2)

Palix River

2

10
5
0
30
25

Site 5

20

3

15
10

5

5

6

0
30 Site 625

4Naselle River

20
15
10
5
0

4 5 6 7 8 9 10 11 12

MonthFig. 2. Results of the 2018 experiment in Willapa Bay, Washington, USA. Oysters were deployed in
May at six sites (map on right). Average shell area measurements of oysters (cm ±SE) in eelgrass
deployed
May atsixoutsidesites (mapright).
Averageshell forarea
measurementsof Julyoysters(cm2andhabitatin(green)andopen onhabitat(orange)is shown
threetime points inAugustOctober.
± SE)
in eelgrass habitat (green) and outside open habitat (orange) is shown for three timeFig.2. Results of the 2018 experiment in Willapa Bay, Washington, USA. Oysters
were2

points in July August and October.

106

Brett DUMBAULD, Evan DURLAND, Konstantin DIVILOV, Kelly MUETHING,
Anna BOLM, Ylva DURLAND, and Brooke MCINTYRE

several sites along the estuarine gradient in theseceramic tiles (5cm×5cm) in this experiment. Tiles

two estuaries. Our methods differed slightly andwere labeled with an individual plastic identification

were briefly as follows:

tag and then held for 24 hours in tanks before
being transported and deployed at 6 sites along theNetarts Bay 2015

estuarine gradient in Willapa Bay, Wa (Fig. 2) on

Aged Pacific oyster shell was used as cultch

5/20/2018. Tiles were attached to PVC stakes at twofor this experiment. Five shells were labeled withpositions: 20 cm above the sediment and just above

an individual plastic identification tag and placed

the sediment surface. Two tiles were attached backin each of 32 small mesh bags (2.5 cm mesh) andto back at each position: one with spat from an MBP

these placed in a setting tank at the Whiskey Creek

cohort and one with spat from broodstock collected

Shellfish hatchery on 5/11/2015. Eyed oyster larvae

from a “wild” population in Willapa Bay. Three(MBP Pod, cohort 26) were placed in the tank andstakes with tiles were deployed within eelgrass andallowed to set on these shells following standard

three outside eelgrass in bare habitat at the same

hatchery practices. Bags were removed from thetidal elevation at each site. Poles with tiles weretanks and deployed at 4 sites along the estuarine

placed approximately 10 m apart and each pole had

gradient in Netarts Bay (Fig. 1) on 5/20/2015.

copper tape wrapped around it at the sediment

Four shell bags were deployed within eelgrasssurface in order to prevent access by oyster drills.

and four outside eelgrass in bare habitat at the

Tiles were visited and photographed at roughlysame tidal elevation. Shell bags were attached tomonthly intervals thereafter and returned to the

PVC pipes so they were suspended approximately

lab for final measurements on 10/30/2018. Fouling

20 cm above the sediment. Poles with bags wereorganisms were removed from areas surrounding

placed approximately 5 m apart and each pole had

each oyster using a toothbrush before photographs

copper tape wrapped around it at the sedimentwere taken at each time point. Photographs

surface in order to prevent access by oyster

from each time point were examined and oyster

drill predators. Oyster shells were removed frommeasurements (length and width) made using Image

the bags and photographed at roughly monthly

J (v1.49®). Temperature was recorded continuouslyintervals thereafter and returned to the lab for final

at 15 min intervals using Hobo® data loggers

measurements on 9/30/2015. Temperature was

deployed in two habitat treatments at each site

recorded continuously at 15 min intervals using

and YSI® sondes were deployed at two locations

Hobo® data loggers. Photographs from each time

(Nahcotta and Palix) for 24 hours in August. At the

point were examined and oyster measurements

conclusion of the experiment oysters were counted,(length and width) made using Image J (v1.49®) on

measured (length and width in cm) with calipers,

size calibrated images. Oyster cultch shell labels andscraped from the tile and both dry and wet meat

shape enabled each individual oyster to be labeled

and shell weight measured.

and recorded so that they could be identified andcounted in subsequent photos. An assessment of

Data Analysis

fouling which became an obvious potential factor

Data from both experiments were analyzed using(% cover, mostly barnacles) was also made. At thegeneral linear and linear mixed effect models in R

conclusion of the experiment oysters were counted,(R Core Team, 2019). Here we present data for shellmeasured (length, width in cm) with calipers,

size (length×width) examined over the summer

removed from the cultch shell and both dry and wet

deployment period. Because we were able to track

meat and shell weight measurements made.

individual oysters in both experiments, we thenevaluated oyster growth for discrete time intervals

Willapa Bay 2018

for which we had the most complete data: 1) an

After being set as singles in the MBP hatchery

initial interval fairly close to deployment whenat HMSC, five oyster spat were cemented

oysters first experienced estuarine conditions and(cyanoacrylate glue) to the rough side of small2) over the entire period or at the end of the period

Influence of climate on Pacific oyster seed growth and survival

107

when we collected the oysters and made additional

outside eelgrass than within eelgrass at the two

measurements (e.g. tissue and shell mass). For

southernmost locations in Netarts Bay in 2015 andthe 2015 Netarts Bay experiment there were tworeaching the largest size at site 4 located furthest

categorical factors of interest represented in thisfrom the estuary mouth (Fig. 1). The opposite

model: eelgrass (present or absent) and location (4pattern was observed at the two sites closer tosites). We also examined and added the proportion

the estuary entrance, where oysters grew to aof each cultch shell covered with fouling organisms

larger size within eelgrass resulting in significant(mostly barnacles) as a factor in this model for theinteraction between location and eelgrass factors

initial period where we collected this information.

in models for final shell size, but not for normalized

Models for the second 2018 Willapa Bay experiment

growth from August to November (Table 1). Oyster

included the same primary categorical treatments:

mortality across the whole season was highest at

eelgrass (present or absent) and location (6 sites),

locations away from the ocean, but there was nobut also included height (20cm above sediment, on

discernable trend attributable to eelgrass presence.

bottom) and seed source (MBP, Wild). Growth was

Fouling on the cultch shells, mostly by barnacles,

evaluated as a normalized daily average = final

was apparent at the first sampling effort in July,size (area in mm 2) – initial size (area in mm2) /highest at sites closest to mouth and higher outsidedays in interval / initial size. Complete models with

eelgrass than inside eelgrass (Fig. 3). Fouling wasinteraction terms were initially examined, the mostnot a significant factor however when included in theimportant random component determined if needed,

models for size or growth at this time point (Table

and then the most parsimonius model selected by

1). Smith (2016) conducted experiments at site 1 incomparing full and nested models with log-likelihood

Netarts Bay during the same year, but repeatedly

tests and comparing Akaike information criterion

deployed small 10 day post metamorphosed oyster(AIC).

spat monthly from March – October and monitored
for short term growth and mortality. Smith (2016)
Results and Discussion

documented a significant effect of eelgrass on in- situ
carbonate chemistry, but only observed significant

Oyster growth differed along the estuarine

growth and survival advantages for oyster spat

gradient with oysters reaching a larger size

placed in eelgrass during May. Survival of these

Table 1. Results of linear mixed effect models (“nlme” package, Pinheiro et al. 2019) used to examine the effect of
habitat and site on shell area and growth of oysters in Netarts Bay in 2015. Individual cultch shells were used as
random intercept terms in each model
Full Model
Response
Shell area (Nov)factor
site

df

F

p

3,92

27.41

<0.001df

Fpeelgrass

1,92

19.56

<0.001site*eel

1,92

11.30

<0.001

3,94

14.50

<0.001

3,94

13.34

<0.001

1,94

9.40

0.003

1,94

9.0

0.004

Normalized growth (Aug – Nov) site
eelgrass
Shell area (July)

Final Modelsite*eel

1,94

0.26

0.856

site

3,84

43.45

<0.001

3,89

42.31

<0.001eelgrass

1,84

14.64

0.002

1,89

3.76

<0.001barnacles

1,84

3.68

0.058

site*eel

3,84

0.41

0.744

barn*eel

1,84

0.31

0.576

Brett DUMBAULD, Evan DURLAND, Konstantin DIVILOV, Kelly MUETHING,
Anna BOLM, Ylva DURLAND, and Brooke MCINTYRE

108

account (Koweek et al., 2018).

small spat planted in subsequent months appeared
to correlate with an overall declining trend in daily

Though experimental design and deployments

pCO 2 minima. These findings agree with otherwere slightly different than those in the 2015 Netarts

research and suggest that additional variables likeexperiment, oyster growth also differed along thewater flow and tidal exposure must be taken into

estuarine gradient in Willapa Bay in 2018. Oysters
consistently reached a larger size at locations closest

100

to the estuary mouth in this estuary (Sites 1-3) (Fig.

Eelgrass2), but few oysters survived at Sites 1 and 2 due atleast in part to experimental problems (early losses

N

Barnacle Cover80of glued oysters from tiles and lost stakes). OystersYobtained a larger size outside of eelgrass than withineelgrass at these locations, but there was significant

60

interaction between location and eelgrass in models
for both shell area and normalized growth (Table402), because no eelgrass effect was observed at
locations away from the mouth (Sites 4-6) (Fig. 2).
We also deployed oysters from two parental sources

20

0(selected MBP family and “wild”) at two heights
above the sediment in this experiment. While there
1

2

3

was no significant effect of oyster source, there4

was significant interaction between height above

Site

sediment and location (Table 2). Oysters grew larger

Fig. 3. Comparison of average percent barnacle
cover measured on shells deployed in eelgrass (greenbars) and outside eelgrass (brown bars) at each sitein Netarts Bay during July 2015.

20 cm above the bottom at locations near the estuary
mouth and the opposite effect was observed atsites 5 and 6. Previous authors also found enhanced

Fig.3. Comparison of average percent barnacle cover measured on shells deployed in eelgrass(green bars) and outside eelgrass (brown bars) at each site in Netarts Bay during July 2015.

Table 2. Results of general linear models used to examine the effect of habitat (eelgrass, open), seed source (MBP,
Wild), position (off bottom, on bottom), and site on shell area and growth of oysters in Willapa Bay in 2018. Onlysites 3-6 used in these models due to lack of sufficient data at sites 1-2
Full Model
Response
Shell area (August)factordf

Final Model

F

p

df

F

p

site

3

9.31

<0.0013

9.47<0.001eelgrass

1

3.59

0.061

1

3.66

0.058

position

1

2.48

0.118

1

2.52

0.115

source

1

3.57

0.061

1

3.63

0.059

site*eel

3

3.97

0.010

3

4.04

0.009

site*pos

3

3.41

0.020

3

3.47

0.018

site*source

3

0.35

0.789

Normalized growth (July – Aug) eelgrass

1

0.001

0.970

1

0.001

0.970

site

3

3.19

0.026

3

3.15

0.026

position

1

0.49

0.487

1

0.49

0.487

source

1

1.24

0.267

site*eel

3

2.45

0.067

3

2.53

0.060

site*pos

3

3.02

0.033

3

3.02

0.044

site*source

3

1.25

0.295

Influence of climate on Pacific oyster seed growth and survival

109

growth of Pacific oysters near or close to the mouth

survival might also reflect predator abundance orof this estuary (Ruesink et al., 2003). Lowe et al. (2019)

even abundance of bacteria and disease as stressors

also documented little difference in oyster growthin these two habitats rather than water chemistry.

between eelgrass and open habitat treatments at

In single 24 hour records, we observed higher levels

the same riverine endpoint (site 6). They foundof food (measured as relative chlorophyll a) withinsignificantly larger oysters in eelgrass than outside

eelgrass at two of the stations along this gradient

this habitat at their site closest to the ocean. Whilein Willapa Bay and more dramatic fluctuation at

we observed the opposite trend, this agrees with ourthe location closest to the estuary mouth where weobservations for Netarts Bay above. These authors

also observed colder average temperatures over theand others (Hollarsmith et al., 2020) mostly attribute

entire experimental period and greater fluctuations

trends in oyster growth to the concentration ofand differences in pH (Fig. 4). This location (site 1) is

phytoplankton as food and found that trends in

however potentially also more affected by freshwaterA

Site 3Site 1

B

C

Fig. 4. Twenty-four hour records for A) water depth – following the tide, B) pH , and c) relative chlorophylla, at intertidal sites 1 (Palix River) and 3 (Nahcotta) where oysters were deployed in Willapa Bay. Note theFig.4. Twenty-four hour records for A) water depth – following the tide, B) pH , and c) relative
fluctuating values of both parameters at site 1, but differences in pH in eelgrass at low water at night at thissite
and consistently
higher chlorophyll
a in eelgrass
both
locations. where oysters were deployedchlorophylla, at intertidal
sites 1 (Palix
River) atand3 (Nahcotta)

in Willapa Bay. Note the fluctuating values of both parameters at site 1, but differences in pH
in eelgrass at low water at night at this site and consistently higher chlorophyll a in eelgrass
at both locations.

Brett DUMBAULD, Evan DURLAND, Konstantin DIVILOV, Kelly MUETHING,
Anna BOLM, Ylva DURLAND, and Brooke MCINTYRE

110

from the Palix River as potentially evidenced by apatterns of primary production in Willapa Bay,drop in pH and chlorophyll a at low tide. We have

Washington, USA. Mar. Ecol. Prog. Ser., 341,

not yet directly related this environmental data

123-139.

directly to oyster growth and survival, in part

Barton A., Hales B., Waldbusser G. G., Langdon

because we recognize that the density and spatial

C., and Feely R. A., 2012: The Pacific oyster,

configuration of eelgrass will also affect water flow

Crassostrea gigas, shows negative correlationand food supply so static measurements and simple

to naturally elevated carbon dioxide levels:

site/treatment characterizations and point in time

Implications for near-term ocean acidification

estimates of these water chemistry parameters need
to be refined.

effects. Limnol. Oceanogr., 57(3), 698-710.
Barton A., Waldbusser G. G., Feely R. A., Weisberg

Our preliminary conclusions are:

S. B., Newton J. A., Hales B., Cudd S., Eudeline▪O yster larvae have clearly been shown to be

B., Langdon C. J., Jefferds I., King T., Suhrbiersensitive to water chemistry including low

A., and McLauglin K., 2015: Impacts of coastal

aragonite saturation state, but other conditions are

acidification on the Pacific Northwest shellfish

important and genotype/broodstock may play an

industry and adaptation strategies implemented

important role in initial spat settlement and size.

in response. Oceanography, 28(2), 146-159.

▪Location or planting site for juvenile spat (seed)

Blake B., and Ermgassen P. S. E. Z., 2015: Thewithin the estuary is important. Juvenile oyster

history and decline of Ostrea lurida in Willapa

seed generally grows faster near the mouth ofBay, Washington. J. Shellfish Res., 34(2), 273-

US West Coast estuaries, but there are importantfiner scale, within estuary spatial and temporal
gradients.

280.
Chew K. K., 1984: Recent advances in the cultivationof molluscs in the Pacific United States and▪The effect of eelgrass on oyster seed also depends

Canada. Aquaculture, 39(1-4), 69-81.

on planting site. We suspect this is due to several

Cullen-Unsworth L. C., and Unsworth R. K. F., 2016:

interacting factors including water flow, food

Strategies to enhance the resilience of the

supply, and perhaps settlement and competition

worldʼs seagrass meadows. J. Appl. Ecol., 53,

with other invertebrates, and not water

967-972.

temperature or modifications to water chemistry.

de Melo C. M. R., Durland E., and Langdon C., 2016:

However, this data and relationship remains to

Improvements in desirable traits of the Pacificbe collected and further examined at appropriate

oyster, Crassostrea gigas, as a result of fivespatial and temporal resolution.

generations of selection on the West Coast, USA.

▪Juvenile oyster growth is generally faster above

Aquaculture, 460, 105-115.

the sediment surface, but this varies along thede Melo C. M. R., Morvezen R., Durland E., andestuarine gradient as well. This is also likely

Langdon C., 2018: Genetic by environment

due to food supply and perhaps other factors

interactions for harvest traits of the Pacificinfluencing oyster feeding behavior including

oyster Crassostrea gigas (Thunberg) acrosssediment load but needs to be further explored indifferent environments on the West Coast, USA.

these estuaries.

J. Shellfish Res., 37(1), 49-61.
De Wit P., Durland E., Ventura A., and Langdon
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This is a review of the history and science

R Core Team, 2019: R: A language and environment

underpinning the effects of changing seawater

for statistical computing, R Foundation forchemistry on bivalve shellfish larvae and the impacts

Statistical Computing, Vienna, Austria. http://that have already taken place to the commercial

www.R-project.org/

shellfish aquaculture industry on the US West Coast.

Ruesink J. L., Roegner G. C., Dumbauld B. R., Newton

Multiple authors contributed to this review which

J. A., and Armstrong D. A., 2003: Contributions

addresses a broad audience but covers the leadingof coastal and watershed energy sources toresearch on direct effects to bivalve larvae as wellsecondary production in a Northeastern Pacificas monitoring seawater conditions and adapting toestuary. Estuaries, 26(4B), 1079-1093.

these changes.

Ruesink J. L., Yang S., and Trimble A. C., 2015:
Variability in carbon availability and eelgrass(2) Durland E., Waldbusser G., and Langdon C., 2019:(Zostera marina) biometrics along an estuarine

Comparison of larval development in domesticatedgradient in Willapa Bay, WA, USA. Estuaries

and naturalized stocks of the Pacific oyster

Coast., 38(6), 1908-1917.

Crassostrea gigas exposed to high pCO2 conditions.

Ruesink J. L., Sarich A., and Trimble A. C., 2018:

Mar. Ecol. Prog. Ser., 621, 107-125.

Influence of climate on Pacific oyster seed growth and survival

113

Two replicated experiments were conducted(4) Lowe A. T., Kobelt J., Horwith M., and Ruesink

where Pacific oyster larvae from “wild” parent

J., 2019: Ability of eelgrass to alter oyster growthcrosses and those from selected stocks (OSU

and physiology is spatially limited and offset by

Molluscan Broodstock Program) were raised andincreasing predation risk. Estuaries Coasts, 42, 743-

set under favorable hatchery conditions (buffered754.

seawater, pH ~7.8 and Ω arag ~2) and unfavorable

The authors planted juvenile seed oysters and(pH ~7.4 and Ω arag ~1) conditions. Early larvalmeasured water chemistry inside and outside of

development was inhibited by acidified seawater andeelgrass (Zostera marina) at several locations in

it affected the timing, but not magnitude of larval

Willapa Bay and in the Salish Sea in Washington

mortality. The effect on metamorphosis (setting)state. They demostrate that while eelgrass modified

was variable, but MBP larvae produced more andcarbonate chemistry, it did not appear to be thelarger spat in ambient and high pCO2 seawater,

primary variable influencing juvenile oyster growth

respectively.

and survival and, instead, predation was higher
and thus survival lower inside eelgrass beds where(3) Hales B., Suhrbier A., Waldbusser, G. G., Feely R.

surviving oysters also grew slower.

A., and Newton J. A., 2017: The carbonate chemistryof the “Fattening Line,” Willapa Bay, 2011-2014.

(5) Ruesink J. L., Roegner G. C., Dumbauld B.

Estuaries Coasts, 40, 173-186.

R., Newton J. A., and Armstrong D. A., 2003:

The authors present detailed data on seawater

Contributions of coastal and watershed energy

chemistry (especially pCO2 and aragonite saturation

sources to secondary production in a Northeasternstate) for Willapa Bay, Washington where PacificPacific estuary. Estuaries, 26(4B), 1079-1093.

oysters have been the mainstay of the oyster

These authors measured growth ofjuvenile

aquaculture industry for almost 100 years andPacific oyster seed deployed at numerous locations

there is a long term record of spawning and setting.

throughout Willapa Bay, and used stable isotopes

They reconstruct this record for a longer historicalto distinguish marine versus terrestrial energy

period and their data suggest that recent conditions

sources. Results demonstrated a very distinct alongprovide a smaller window of optimal conditions

estuary gradient with oysters growing faster near(low aragonite saturation state and warm enoughthe mouth and slower at greater distance fromtemperatures for oyster spawning) than occurred

the mouth and away from the mouth. Oysters also

historically. While they did not sample larvae anddisplayed distinct stable isotope ratios that reflected

therefore can’t confirm effects, they substantiate

a strong marine signal near the mouth (-18 δ C) andthe complexity of measuring these effects anda stronger terrestrial signature at distance -22 δ13C).

attributing them to a single cause in an estuary

Oysters grown just off the bottom grew slower thanwhere conditions are variable.

those off-bottom at any given tidal elevation.

13

水 研 機 構 研 報， 第 50 号，115 － 121， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，115－121，2021

115

Comparative study of the impact of environmental changes on oyster
culture between USA and Japan, as collaborative research under UJNR
Natsuki HASEGAWA＊1, Brett R. DUMBAULD＊2, Masakazu HORI＊3, Satoshi WATANABE＊4,
Michael RUST＊5, and Zachary FORSTER＊6

Abstract: Several oyster species are cultured globally, and the Pacific oyster, Crassostrea gigas, is a
widely cultured species in both the USA and Japan. In Japan, aquaculture production of the Pacificoyster is decreasing slightly for multiple reasons including large die-offs of adults during andafter the reproductive season due to a delay in the reproductive season and poor post-spawning
recovery, poor wild spat collection, and a labor shortage for both operation of aquaculture andpost-harvest processing. These problems may be aggravated by environmental changes such as
global warming and oligotrophication around Japan’s coastal areas. The Japan Fisheries Researchand Education Agency is investigating the causes of the die-off during the reproductive season andattempting to establish countermeasures. The Pacific oyster is native to Japan and was introduced
to the USA for aquaculture in the early 1900’s. Nonetheless, there are large differences in theculture system, habitats and environmental conditions between the two countries. A comparative
study was initiated to evaluate oyster reproduction in the two countries in order to understandthe effects of habitat and the environment on future success of aquaculture given predictedenvironmental changes. Oyster culture experiments were conducted in intertidal and subtidal
zones inside and outside of seagrass habitat in Hiroshima Bay in Japan and Willapa Bay in USA
during the reproductive season (March to June 2019 and February to July 2019, respectively). We
focused on elucidating the effects of habitat and the environment on the energy allocation of theoyster between reproduction and somatic growth.
Key words: Pacific oyster Crassostrea gigas, aquaculture, die-off, energy allocation, reproduction
Background

Pacific oysters have the second highest production
volume after Crassostrea virginica (American cupped

Oyster species are cultured globally, and theoyster, 112 × 103 ton yr-1) in the USA (FAO, Fishery

Pacific oyster, Crassostrea gigas, is the most widely

Statistical Collections). The Pacific oyster has its

cultured species in the USA, Japan, and world.

origins in East Asia and has been cultivated for

Aquaculture production of the Pacific oyster (Pacificcenturies. It has been cultured on the west coast ofcupped oyster in FAO, Cultured Aquatic Species

the USA since the 1920s, and in France since 1966.

Information Programme) globally exceeds 572 ×

It has been widely introduced as an alternative

3

-1

3

10 ton yr ; Japan’s production was 174 × 10 ton

species to indigenous oysters that had been severely

yr-1 in 2017, and the production in USA was 26 ×

depleted by overfishing and disease, and to create

3

10 ton yr-1(FAO, Fishery Statistical Collections).

new industries.

2020年12月11日受理（Accepted on December 11, 2020）＊1
Hokkaido National Fisheries Research Institute, Kushiro Laboratory, 116 Katsurakoi, Kushiro, Hokkaido, 085-0802, Japan＊2
US Department of Agriculture, Agriculture Research Service, Hatfield Marine Science Center, Newport, OR 97365, USA＊3
National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5, Maruishi, Hatsukaichi, Hiroshima, 739-0452, Japan＊4
National Research Institute of Aquaculture, 422-1 Nakatsuhamaura, Minamiise, Mie, 516-0193, Japan＊5
NOAA Office of Aquaculture, 1315 East-West Highway, Silver Spring MD. 20910, USA＊6
Washington Department of Fish and Wildlife, Ocean Park, WA. 98640, USA
E-mail: hasena “at” affrc.go.jp

116

Natsuki HASEGAWA, Brett R. DUMBAULD, Masakazu HORI, Satoshi WATANABE,
Michael RUST, and Zachary FORSTER

There are two large traditional production areas

large die-offs in years with high water temperature

in Japan: Hiroshima and Miyagi Prefectures, andand small amounts of rainfall, especially during

there are many small production areas all overthe spawning season when metabolic activities are

Japan. Production areas are distributed from thedevoted to gamete production (Akashige et al.,temperate zone (western part of Japan, Kyushu)

2005). Thus, excessive energy allocation to gonad

to the cold-temperate-zone (northern Island ofdevelopment is speculated to be one of the major

Hokkaido). In Japan, wild spats are collected withcauses of the large die-offs.

scallop shells hung in open water areas and arewidely used for aquaculture. Suspended culture from

Comparative Culturing Experiments

rafts or floating longlines in offshore area are thein the USA and Japan

main culture system. Most of the harvested oysters
are shucked for commercial distribution.

Elucidating the effects of habitat, environment andIn the USA, hatchery produced spats are used asculture system on the energy allocation of the Pacificwidely as wild spats. Bottom and off-bottom culture

oyster between reproduction and somatic growthin the intertidal area (tidal flats where seagrass

would contribute to an understanding of the reasons

beds may be present) are the main culture systems.

oysters experience challenges during the spawning

Pacific oyster culture is mainly operated in theseason under climate change and would support

northwestern part of the USA, primarily in the Stateestablishment of countermeasures. For this purpose,of Washington.

comparative oyster culture experiments were

In Japan, production of the oyster is decreasing

conducted in high oyster production areas in thegradually for multiple reasons. In addition to

USA and Japan, where there are large differences

insufficient wild spat collection and a labor shortagein the culture system, habitats and environmental

for both operation of aquaculture and post-harvest

conditions. This study was developed and conducted

processing, large die-offs of adults during andas collaborative research through the UJNR

after the summer reproductive season is a majorbilateral. The Japan site examined was Hatsukaichiproblem. This problem may be aggravated by

in Hiroshima Bay, a part of Seto-Inland-Sea (HI), andenvironmental changes such as global warming

the USA sites were Nahcotta (NA) and Bay Centerand oligotrophication of coastal waters. The Japan(BC) in Willapa Bay, Washington State (Fig. 1). In

Fisheries Research and Education Agency isthese sites, mesh bags including ten tagged oysters

investigating the causes of the die-off during the(oyster bag) were set up in tidal flats at intertidal

reproductive season and attempting to establish

and sub-tidal levels (Fig. 2). Oyster bags were alsocountermeasures (Hasegawa and Sakami, 2019).

set up inside seagrass beds at the experimental

Summer die-off of the oyster has also been atidal flats to evaluate the effect of seagrass, which

recognized problem on the west coast of the USA

are common around oyster culture tidal flats in thesince the mid 1950ʼs, and some of these incidences

USA. In addition to the experiments around tidal

are related to stress during the reproductive season

flats, oyster bags were also suspended in docks andcombined with environmental factors (Cheney etkept submerged throughout the experiments toal., 2000). However, occurrence of high mortality is

mimic suspended culture in the subtidal zone, whichless frequent in the USA than Japan. Koganezawa

is a common oyster culture system in Japan.

and Goto (1972) reported that oysters from relatively

Gametogenic development of the Pacific oyster

oligotrophic waters were characterized by a smaller

progresses at ambient temperature above 10℃ andgrowth rate and reduced amount of spawning

reaches the peak in sexual maturation at 600℃・days

eggs than oysters from eutrophic waters in Japan.

heat summation (i.e. cumulative daily temperature

Production of a large amount of eggs associatedexceeding 10℃; Mann, 1979 and Oizumi et al., 1971).

with some environmental conditions that prolong

Oyster culture experiments were conducted from

spawning may increase mortality risk. Akashige etMarch to June 2019 in Japan and from Februaryal. (2006) concluded that there was a high risk of

to July 2019 in the USA, which corresponds to thePacific Oce

N46.5

NA

×

Comparative study of oyster culture between USA and Japan
N46.4

W124.1

W124.0W123.9
(a)

10 km

N46.7

N34.3

BC

×

N46.6

117

10 km

(b)

HI

× Itsukushima(Miyajima)

N34.2

N46.5

Pacific Ocean

Willapa Bay
Hiroshima Bay

NA

×

N34.1
E132.3

N46.4

E132.4E132.5

Fig. 110 km

Fig. 1. Study sites for oyster culture experiments in Willapa Bay, USA (a) and
(b)
10 km
Hiroshima
Bay, Japan (b).
N34.3

HI

× Itsukushima(a)(Miyajima)

N34.2

Intertidal

Hiroshima Bay
Unvegetated. Top

Seagrass. Top

N34.1

Unvegetated.
Bot
E132.3E132.4

Suspend in dock
(Subtidal)

Seagrass. Bot

E132.50.3 m

Fig. 1

1.0 m(b)Intertidal
Unvegetated. Top
Unvegetated. Bot

Seagrass. Top (Intertidal)

Suspend from raft
(Subtidal)

0.3 m
0.3 m

1.0 m
Seagrass. Bot (Subtidal)

Fig. 2. Design of oyster culture experiments in Willapa Bay, USA (a) and Hiroshima
Bay, Japan (b). Oyster bags, which were mesh bags including ten tagged oysters in
each, were set around the tidal flat.

Fig. 2Natsuki HASEGAWA, Brett R. DUMBAULD, Masakazu HORI, Satoshi WATANABE,
Michael RUST, and Zachary FORSTER

118

Initial (12th Mar)

Water Temperature
(℃)

30
25

NA.Eelgrass

20
15

50 mm

End

10
5
600 2/1

Cumulative Temperature
(Σ ℃)

(a)

BC.Eelgrass

3/3

4/2

5/2

6/1

7/2

8/1

500
400

Initial

300
200
100
0

2/1 Feb 3/3 Mar 4/2 Apr 5/2 May 6/1 Jun 7/2 Jul

8/1

50 mm

Water Temperature(℃)

30
Hiroshima Pref.

20
15

End (12th Jun)

End

10
5
600 2/1

Cumulative Temperature
(Σ ℃)

(b)

Suspended

25

3/3

4/2

5/2

6/1

7/2

8/1

500

Fig. 4. Example of tagged oysters at the beginningand end of oyster culturing experiments in Willapa
Bay, USA.

400
300

experiments. At the beginning of the experiments,

Initial200

the total wet weight inclusive of shell and shellheight were measured, and the oysters were tagged

100
0

2/1 Feb 3/3 Mar 4/2 Apr 5/2

May 6/1

Jun 7/2 Jul 8/1

Fig.
Fig. 33. Change of daily mean water temperature(during submerged times) and cumulative
temperature at oyster culture experiments in
Willapa Bay, USA (a) and Hiroshima Bay, Japan (b).
Water temperature before and after the culture
experiments obtained from Hiroshima PrefecturalFig. 4
Fisheries and Marine Technology Center.

for individual identification (Fig. 4). Initial total wet
weight of oysters (shell + tissue + seawater) ranged
from 77.6 to 274.6 g and from 61.9 to 162.4 g for the
USA and Japan experiments, respectively. At theend of the experiments, oysters were measured
for total wet weight and shell height and were also
dissected. After measuring the whole tissue wetweight, the tissue was separated into the outersomatic parts (mantle, gill, and adductor muscle),
inner somatic parts, and reproductive parts.

periods between beginning and peak (just before

Vertical histological sections of the inner somatic

spawning) of sexual maturation. Peak in sexualand reproductive parts were prepared (Fig. 5).

maturation was one month later in the USA thanOuter somatic parts and one side of the inner parts

Japan, but duration until sexual maturity (600℃・

were weighed and stored frozen at −20℃. Frozen

days) was similar between USA and Japan: about 4

samples were then freeze-dried and homogenized formonths (Fig. 3).

nutrient component analysis (crude carbohydrate,

Oysters for the USA experiments were harvested

protein and fat). The other side of the inner parts

from tidal flats at the NA site in Willapa Bay, andwere weighed and fixed in Davidson’s fixative,

market-size suspended oysters were purchased

dehydrated in tissue dehydration solutions andfrom oyster farmers in Hiroshima Bay for the Japanembedded in paraffin. The paraffin blocks wereComparative study of oyster culture between USA and Japan

119

Outer somatic part (mantle, gill, adductor muscle)• Weighing• Proximate composition
Inner somatic and reproductive part

Separating

Cutting at vertical section• Weighing• Proximate composition• Weighing• Histological image analyses

Fig. 5. Workflow of oyster sampling for evaluating energy allocation to the gonads.
protein content by multiplying the conventional
conversion factor of 6.25. Crude fat content wasdetermined by the modified gravimetric method(Ichihara et al., 2011) based on the method originallydescribed by Bligh and Dyer (1959). For total lipid
extraction, tert-butyl methyl ether was used insteadof chloroform. Energy contents of each tissue partwere calculated as the sum of the energy content ineach nutrient component estimated by the following
factors: carbohydrate (17.2 kJ/g DW), protein(23.9 kJ/g DW) and lipid (39.8 kJ/g DW) (Ansell,1974). Finally, energy allocation to the gonads wascollectively estimated with the obtained data.

Fig. 5

Relative growth rate (RGR) of shell height during
culture experiments was calculated as follows:
RGR =Fig. 6. Example of tissue section of oyster innerparts (male) stained with hematoxylin and eosin.
sliced at 5–8 μm thickness and stained withhematoxylin and eosin (Fig. 6). Sectional area ratio

Ln (Shell heightend)－Ln (Shell heightinitial)
Culture period (day)Condition index (CI) was calculated as follows;
CI =

TSW
TTW－SLW

×102

of gonad to total inner part were histologicallydetermined and volume ratio, which are substitutes

where TSW is the tissue weight (gWW), TTW isfor weight ratio, were calculated. Total carbohydrate

the total weight with shell (filled with water, gWW)

content of dried sample was determined using theand SLW is the shell weight (gWW).

phenol-sulfuric acid method (Dubois et al., 1956). The

This is a progress report of the UJNR collaborative

amount of total nitrogen in samples was measured

study. At the moment, the culture experiments andusing an elemental analyzer (Flash EA1112, Thermo-

sample analysis have been finished, and collected

Finnigan) and nitrogen content was converted todata are being organized and analyzed. The results

Natsuki HASEGAWA, Brett R. DUMBAULD, Masakazu HORI, Satoshi WATANABE,
Michael RUST, and Zachary FORSTER120

are planned to be published in future reports.

FAO: Fishery statistical collections, Global
aquaculture production, FAO Fisheries andAcknowledgements

Aquaculture Department, Rome. http://www.
fao.org/fishery/statistics/global-aquaculture-

We are grateful to A. J. Trimble, A. Bolm, andproduction/en, (Cited 31 March 2020)

B. McIntyre for assistance with field experiments

Hasegawa N., and Sakami. T., 2019: Change inand Y. Fuchigami, S. Tanaka, and T. Kawabuchi forwild spat collection of oysters in Japan andassistance with analysis. This study was supported

new wild spat collection method for single-

by the international joint research project of Japan

seeds. Aquaculture Business, 56(5), 14-17. (inFisheries Research and Education Agency.

Japanese)
Ichihara K., Yoneda K., Takahashi A., Hoshino N.,

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Akashige S., Hirata Y., Takayama K., and Soramoto K.,

Lipids, 46, 297-306.

2005: Seasonal change in oxygen consumption

Koganezawa A., and Goto K., 1972: Ecologicalrates and filtration rates of the cultured Pacificstudies at a seed oyster production area-Ⅰ.

oyster Crassostrea gigas. Nippon Suisan Gakkai

Characteristics of mother oyster populations in

Shi, 71, 762-767. (in Japanese with English

Sendai. Bay. Nippon Suisan Gakkai Shi, 38, 1-8.

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Akashige S., Hirata Y., Takatsuji Y., and Aida S.,

Mann R., 1979; Some biochemical and physiological

2006: Occurrence of mass mortality in oyster

aspects of growth and gametogenesis inculture with relation to seawater temperature

Crassostrea gigas and Ostrea edulis grown atand rainfalls in Hiroshima. Bulletin of thesustained elevated temperatures. J. Mar. Biolog.

Hiroshima Prefecture Fisheries and Marine

Assoc. U.K., 59, 95 -110.

Technology Center, 1, 9-13. (in Japanese)

Oizumi S., Ito S., Koganezawa A., Sakai S., Sato

Ansell A. D., 1974: Seasonal changes in biochemical

R., and Kanno H., 1971: Techniques of oyster

composition of the bivalve Lima hians from theculture, in “Aquaculture in. shallow seas:

Clyde Sea Area. Mar. Biol., 27, 115–122.

progress in shallow sea culture (Revised

Bligh E. G. and Dyer W. J., 1959: A rapid method of
total lipid extraction and purification. Canadian J.

edition)” (ed. by Imai T.), Koseisha Koseikaku,
Tokyo, pp 153–189. (in Japanese)

Biochem. Physiol., 37, 911–917.
Cheney D. P., MacDonald B. F., and Elston R. A.,

Annotated Bibliography of Key Works

2000: Summer mortality of Pacific oysters,
Crassostrea gigas (Thunberg): Initial findings(1) Akashige, S., Hirata Y., Takatsuji Y., and Aida S.,

on multiple environmental stressors in Puget

2006: Occurrence of mass mortality in oyster cultureSound, Washington, 1998. J. Shellfish Res., 19,with relation to seawater temperature and rainfalls

353-359.

in Hiroshima. Bulletin of the Hiroshima Prefecture

Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A.,

Fisheries and Marine Technology Center, 1, 9-13.

and Smith F., 1956: Colorimetric method for

Mass mortality in the Pacific oyster, Crassostrea

determination of sugars and related substances.

gigas occurred in 1979, 1994, 2001 and 2002, andAnal. Chem., 28, 350–356.

relationships with surface water temperature or

FAO: Cultured Aquatic Species Information

rainfall in Hiroshima Bay were analyzed using a

Programme, Crassostrea gigas (Thunberg,

dataset from 1970 to 2004. Mass mortality occurred1793), FAO Fisheries and Aquaculture

in years with high water temperature (i.e. theDepartment, Rome. http://www.fao.org/fishery/

number of days over 20℃ was greater than theculturedspecies/Crassostrea_gigas/en (Cited 31

mean+SD) and small rainfall (i.e. cumulative total

March 2020)

rainfall from July to October was smaller than theComparative study of oyster culture between USA and Japan121

mean－SD) except in 2001. Spat introduced fromwhich was lower than the value expected by the

Miyagi Prefecture experienced extraordinary mass

formula applied during the non-spawning season.

mortality in 2001, which has a colder climate than

These results clearly showed that feeding activities

Hiroshima Prefecture. Therefore, there is high risk

declined during the spawning season, but oxygenof mass mortality of local oysters in years with highdemand (weight base and temperature dependent)

water temperature and small rainfall.

was constant throughout the year. Oysters would
be especially vulnerable during the spawning season(2) Akashige, S., Hirata Y., Takayama K., andwhen metabolic activities would be devoted toSoramoto K., 2005: Seasonal change in oxygen

gamete production.

consumption rates and filtration rates of the cultured
Pacific oyster Crassostrea gigas. Nippon Suisan(3) Cheney, D.P., MacDonald B.F., and Elston

Gakkaishi, 71(5), 762-767.

R.A., 2000: Summer mortality of Pacific oysters,

The oxygen consumption rate (OCR) and filtration

Crassostrea gigas (Thunberg): Initial findings onrate (FR) of cultured Pacific oysters, Crassostrea

multiple environmental stressors in Puget Sound,gigas, of different sizes were measured in still water

Washington, 1998. Journal of Shellfish Research, 19,

systems at different seawater temperatures in353-359.

various seasons. The OCR had clear relationships

Summer mortality has been a recognized problem

with water temperature (t ℃) and dry body flesh

for oyster aquaculture on the west coast of the USA

weight (Wd g) expressed by the formula: OCR (mgsince the mid 1950ʼs, and these authors related someO2 hr-1 ind-1) = (0.072t－0.64) Wd0.75 and there was noof these incidences to stress during the reproductive

difference among seasons. The FR was expressed byseason combined with environmental factors.

the formula: FR (L/h/ind) = (0.70t－6.6) Wd in non-

Nonetheless triploid oysters experienced higher

spawning seasons. However, in the spawning season,mortality than diploids.

the FR was expressed by the formula: FR = 4.9 Wd,水 研 機 構 研 報， 第 50 号，123 － 133， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，123－133，2021

123

Oyster aquaculture using seagrass beds as a climate change
countermeasure
Masakazu HORI＊1, Masami HAMAGUCHI＊1, Masaaki SATO＊2, Réjean TREMBLAY＊3,
Alana CORREIA－MARTINS＊3, Valerie DEROLEZ＊4, Marion RICHARD＊4, and Franck LAGARDE＊4

Abstract: In the framework of the Sustainable Development Goals (SDGs) led by the United
Nations, coastal management methods are required to achieve both sustainable food productionand environmental conservation as a climate change countermeasure. Oyster farming is an
important food production method now being developed in coastal areas around the world.
Recently, climate change has caused several negative effects on oyster aquaculture such as poor
spat collection due to oligotrophication, ocean acidification, and poor spat growth and survival
due to frequent anoxic events derived from high seawater temperature. The oysters cultivated inmany regions of the world are intertidal species inhabiting intertidal zones such as sandy/muddy
tidal flats and estuaries, where seagrass beds are often distributed in adjacent lower intertidal
and subtidal areas. Seagrass vegetation is one of the most important ecosystems functioning as acountermeasure for global climate change. Not only does it mitigate greenhouse gas emissions by
sequestration and storage of blue carbon derived from atmospheric CO2, but it also functions as
an adaptation measure providing a buffering function against ocean acidification and water quality
improvement.
Based on the concept of aquaculture supported by natural ecosystem interactions between
oysters and seagrass beds, our project examined whether aquaculture techniques that take into
account both mitigation and adaptation to climate change are effective for both sustainable useof coastal areas and environmental conservation. We conducted field experiments in both the
French Mediterranean Sea and the Seto Inland Sea of Japan to clarify the effect of eelgrass bedson (1) natural oyster spat collection and (2) growth and survival of oyster spat. The results of ourexperiments revealed that spat recruitment was significantly higher in areas without eelgrass
distribution, while spat growth and survival rate after the settlement were significantly higher
in eelgrass beds even when anoxic events occurred in the study areas. Therefore, our results
indicate a possibility that seagrass vegetation contributes to sustainability of oyster aquaculture by
mitigating environmental degradation during cultivation.
Keywords: oyster aquaculture, Zostera, Crassostrea gigas, blue carbon ecosystem, integrated coastalmanagement

2020年12月11日受理（Accepted on December 11, 2020）＊1
National Research Institute of Fisheries and Environment of Inland Sea, Japan Fisheries Research and Education Agency, 2-17-5 Maruishi,
Hatsukaichi, Hiroshima 739-0452, Japan＊2
National Research Institute of Fisheries Engineering, Japan Fisheries Research and Education Agency, 7620-7 Hasaki, Kamisu, Ibaraki 3140408, Japan＊3
Institut des Sciences de la mer de Rimouski (ISMER), Université du Québec à Rimouski, 310 Allée des Ursulines, Rimouski, QC G5L3A1,
Canada＊4
MARBEC Univ. Montpellier, CNRS, Ifremer, IRD, 34200 Sete, France
E-mail: mhori “at” affrc.go.jp

124

Masakazu HORI, Masami HAMAGUCHI, Masaaki SATO, Réjean TREMBLAY,
Alana CORREIA-MARTINS, Valerie DEROLEZ, Marion RICHARD, and Franck LAGARDEIntroduction

production. Therefore, the blue carbon ecosystem
is a typical ecosystem providing co-benefits to bothAs the impacts of climate change on humansocieties and ecosystems intensify, various measuressustainable food production and climate change
mitigation.

to address climate change are being implemented

Oyster farming is an important food production

in various fields across the world. The Paris

method now being developed in coastal areas around

Agreement adopted in 2015 by the UN Conference

the world. Recently, climate change has causedof the Parties to the United Nations Framework

several negative effects on oyster aquaculture such

Convention on Climate Change (UNFCCC) states

as poor spat collection due to oligotrophication,that efforts will be made to keep global averageocean acidification, and poor spat growth andtemperature rise well below 1.5°C compared tosurvival due to frequent high seawater temperature

before the Industrial Revolution. To achieve theand anoxic events (Hori et al., 2018; Lagarde et al.,climate target, it is recommended that the balance

2018, 2020). The oysters are originally intertidal

between greenhouse gas emissions and absorption

species inhabiting intertidal zones such as sandy/

be reduced to zero. However, the CO2 emissions

muddy tidal flats and estuaries, where seagrassthat are currently in operation and the CO 2

beds are often distributed in adjacent lower

emissions that are committed by fossil-fuel energy

intertidal and subtidal areas. Not only can seagrass

infrastructure were estimated at more than 800 Gt

vegetation mitigate greenhouse gas emissions

in 2018, which is already far beyond the targets ofthrough sequestration and storage of blue carbon

the Paris Agreement to keep the temperature risederived from atmospheric CO2, but it also functions

below 1.5°C (Tong et al., 2019). Therefore, in orderas an adaptation measure buffering against ocean

to meet the Paris Agreement climate goals, it will beacidification and improving water quality (Larkum etnecessary to retire these infrastructures as soon asal., 2006; Duarte et al., 2013; Groner et al., 2018).

possible, as well as increase CO2 sequestration sinks.

Based on the concept of returning to traditional

Moreover, climate change is triggering food crises

aquaculture using the natural ecosystem interactions

in various regions of the world, and the measures

between oysters and seagrass beds (Hori et al.,for sustainable food production are also urgently

2018), our project is now demonstrating whether

needed. It is generally recognized that there is aaquaculture techniques that take into account bothtrade-off between current food production systems

mitigation and adaptation to climate change are

and climate change mitigation measures (Elmqvist eteffective for both sustainable use of coastal areasal., 2013). For example, deforestation in terrestrial

and environmental conservation. In this study, we

ecosystems is accelerating to create pastures andpresent the results from field experiments to clarify

other agricultural fields to increase food production

the effect of eelgrass beds on the sustainability of(Schiermeier 2019). Therefore, some countermeasuresoyster aquaculture in both the French Mediterranean

are needed to harmonize sustainable food production

Sea and the Seto Inland Sea of Japan. The seagrasswith climate change mitigation (Bommarco et al.,contribution to oyster production was divided into2013).

two processes in relation to oyster life cycle stages:

Shallow coastal ecosystems are now highlighted as

recruitment processes from the larval stage to spat

one of the measures to mitigate the effects of climate

settlement, and post-recruitment processes withchange (Hoegh-Guldberg et al., 2019). In particular,

spat growth. A series of in situ experiments were

blue carbon ecosystems such as mangroves, salt

conducted in Japan and France (1) to demonstrate

marshes and seagrass vegetation are importantthe larval recruitment variability in shellfish farming

ecosystems currently being considered as a newareas in the presence or absence of eelgrass beds,

and effective atmospheric CO 2 sink (Kuwae andand (2) to reveal the effects of eelgrass vegetation

Hori, 2018). In addition, the blue carbon ecosystemon the survival and growth of oyster spats. To ouris known as one of the most productive ecosystems

knowledge, there has been no prior case study

with high biodiversity resulting in higher fooddirectly demonstrating the effect of oyster-seagrassOyster aquaculture with seagrass beds

125

interactions on ecosystem functioning, althoughflourishing in many areas of the Seto Inland Sea.

there has been some prior modelling research on

The production in the Hiroshima Bay and all areas

material cycling in a coastal ecosystem includingof the Seto Inland Sea accounts for about 60% andoyster and seagrass beds (e.g. Kishi and Oshima,

80% of the national production of oysters in Japan,2008).

respectively. Recent oligotrophication due to legal
restrictions on nutrient input from the watershed
Methods

has resulted in eelgrass recovery in the Seto Inland
Sea over the last decade. It has been estimatedStudy sitesthat the area of seagrass meadows has increased

The Seto Inland Sea (coordinates at its centre:
34.1667 N, 133.3333 E) in Japan and the Thau lagoon

from 6000 ha to about 10,000 ha in 2011 (Hori and
Tarutani, 2015).

(coordinates at its centre: 43.41 N, 3.6241 E) in

The Thau lagoon is the largest lagoon located on

France were chosen as study areas for this research

the southern French coast in the Mediterranean(Fig. 1). The Seto Inland Sea is located at the

Sea. The lagoon is famous for oyster farming using

southwestern part of the main island of the Japanese

non-native Pacific oyster spats attached on ropesarchipelago. Rafted aquaculture using natural spats

containing a specific cement. The ropes with theof the native Pacific oyster Crassostrea gigas isspats are hung from oyster tables established in

(b)

(a)

100 km5 km

Oono channel

Figu
Fig. 1. The two comparative study sites, including a) Seto Inland Sea, including the Hiroshima Bay
and Oono channel, and b) Thau Lagoon near the Gulf of Lion, Mediterranean Sea. These maps wererevised from Hori et al. (2018).

126

Masakazu HORI, Masami HAMAGUCHI, Masaaki SATO, Réjean TREMBLAY,
Alana CORREIA-MARTINS, Valerie DEROLEZ, Marion RICHARD, and Franck LAGARDE

the nearshore zone. About 10% of the French

Zostera spp. sites (Fig. 3b) (Lagarde et al., 2020). Allnational production of oysters is cultivated there,

collectors were sampled after 2 weeks of immersion

representing the largest oyster farming area in theto assess pediveligers and postlarvae abundance andMediterranean Sea. It has been suggested that theafter 4 weeks of immersion to assess oyster spatrecovery of eelgrass beds is still proceeding, and thatabundance.

now the area of seagrass distribution extends up

In Japan, oyster spat collectors were deployed onfrom 2 ha to 800 ha (Hori, personal communication

the seagrass beds and also hung from the oysterwith Syndicat mixte du bassin de Thau). The

rafts in the Oono channel of the Hiroshima Bay inexpansion of eelgrass meadows was observed even

July 2017 (Fig. 1). Only the collectors in seagrasswithin oyster farming areas in June 2016.

beds were exposed daily to the atmosphere due totidal movement. All collectors were sampled after 4

1. Larval recruitment variability in shellfish farming

weeks of immersion to assess oyster spat abundance.

area
In Thau lagoon, settlement and recruitment of

2. Effect of eelgrass vegetation on oyster survival

oysters were monitored at six contrasting stations

and growthin August 2017 from east to west, in the absence or

Our hypothesis was that seagrass beds could

presence of Zostera meadow and shellfish farming

maintain or improve the safety and environmental

in the Thau Lagoon (Fig. 1). The oyster collectors

sanitary conditions for oysters, especially reduction

were deployed as described by Lagarde et al. (2017;of harmful microbiomes (e.g. Lamb et al., 2017;2019) (Fig. 2). The collectors were deployed in the

Groner et al., 2018). To clarify the effects ofwater column (Fig. 3a) inside/outside shellfish farmseagrass vegetation on microbiome in both oyster

sites and also hung from the oyster tables above

and ambient sea waters, a series of census andand below the canopy in Zostera meadows insideexperiments were carried out in the Thau Lagoon

Fig. 2. The six sampling sites scattered from east to west inside Thau Lagoon
with three conditions (ISFZ; Inside Shellfish Farming Zone, OSFZ: Outside Shellfish
Farming zone, Zost: Zostera spp. beds). Shaded squares located at northwest side ofthe lagoon show oyster tables. This map was revised from Lagarde et al. (2020).

Figure 2

Oyster aquaculture with seagrass beds

127

in 2017 and 2018 in the presence and absence

seagrass vegetation on the sanitary condition of theof Zostera meadows (Fig. 2: Bouzigues ISFZ,

oysters. Analyses of this data and the results from

Bouzigues ZOST, Montpénèdre ZOST, Montpénèdre

Thau lagoon are ongoing.

ISFZ) for analyzing environmental DNA and
Result and Discussion

oyster microbiome. The analyses are still ongoing,
therefore the results are not shown in this paper.

1. Larval recruitment variability in shellfish farming

In parallel, the effect of Zostera meadows on growtharea

and survival of juvenile oysters after 3 monthsof growing (September-December) was tested

In France, the abundance of young settlers wasinside and outside eelgrass beds at Bouzigues andlower within the canopy of eelgrass beds than above

Montpénèdre in the Thau Lagoon in 2018 (Fig. 2).

the eelgrass canopy at both Zostera sites (Fig. 4).

In Japan, we established a field experiment in

The best sites for settlement were the OSFZ sites

Hiroshima Bay to clarify the contribution of eelgrass(Listel and Meze: outside of the oyster farming andbeds to the survival and growth of oyster spats as aseagrass areas), which confirm those of Lagarde etfeasibility study of oyster-seagrass interactions (Horial. (2017; 2018; 2019), where there is a combination

et al., 2018). We established an experimental areaof high level hydrodynamic connectivity and(5 m×5 m) in the lower intertidal area on the tidal

favorable trophic supply. These results indicate

flat with seagrass vegetation, and set a raft floatingthat eelgrass beds are unlikely to be a preferredon the sea surface 200 m offshore from the tidal flat,site for oyster settlement over other nearby sites

hanging a replicate of three cages at a depth of 2

in term of abundance. However, between the sites

m from the sea surface using vinylon ropes. Thirty

with seagrass vegetation, the abundance of youngspats of each of three native species (C. gigas, C.

settlers was much higher in the Bouzigues site

nippona and C. sikamea), which were hatched from

where the abundance of eelgrass vegetation was

the same lot, were put into the cages on the tidal

also higher than that in the Montpénèdre site. (Hori,flat, and the other half of the spats of each species

unpublished data). Although eelgrass vegetation

were put into the cages hanging from the raft. The

does not increase natural spat recruitment, thereexperiment was conducted for two months frommay be no negative effect on the recruitment.

November 2016 to January 2017. In addition, we took

In Japan, the mean abundance of recruited spats

environmental DNA and microbiome samples fromwas lower in the eelgrass beds than that hungthe oysters and ambient waters in both tidal flatfrom the raft without eelgrass vegetation (Fig.

with seagrass and the raft to clarify the effects of5), although the difference was not statisticallySurfacebuoy
Surfacebuoy

2.0 2mm

Traction
Traction
Sling
SilingFloating
flag
Floating flag
Rope

Rope
RopePPО 12 mm

(c)

(b)

sub-surfacebuoy
Sub-surface
buoy

2.0 m 1.1m

2.02m
m

Oystertable

PVC HP
Pipe
Coupelle
0.6m
Coupelle PVC
Collectors
Collectors
Traction sling

Bottom
Bottom surface

Oystertable

Surface
1.1 m

2m 1.1 m

AnchorClamp
Silingeyes
Bottomsurface

Surface
Water column
collector
Eelgrass bed
collector
Eelgrass beds

1.1 m

(a)

BottomsurfaceAnchorFig. 3. Oyster spats collectors deployed in each site of Thau lagoon. In the site of Outside Shellfish Farming Zone
(OSFZ), mooring system (a: left) was used. In the site of Inside Shellfish Farming Zone (ISFZ) without eelgrassbeds (b: middle) and the site of inside shellfish farming zone with eelgrass beds (ZOST, c: right), collectors werehung from the oyster table, and deployed above eelgrass canopy (water column) and below the eelgrass canopy.
These illustrations were revised from Lagarde et al. (2020).

Figure 3修正

Masakazu HORI, Masami HAMAGUCHI, Masaaki SATO, Réjean TREMBLAY,
Alana CORREIA-MARTINS, Valerie DEROLEZ, Marion RICHARD, and Franck LAGARDE

log10(1 + young_settlers coupelle-1)
0.5 1.0 1.5 2.0 2.5

128

Sampling Sites

Inside oyster
farming zone
without
eelgrass bed

Inside oyster
farming zonewitheelgrass bed

Outside oyster
farming zone

Inside oyster
farming zone
without
eelgrass bed

Inside oyster
farming zonewitheelgrass bed

No. recruited spats・collector-1

Fig. 4. The abundance of young settlers (pediveligers and postlarvae) on each coupellecollector (mean ± SE) in each site. ISFZ: Inside Shellfish Farming Zone, OSFZ: Outside
Shellfish Farming Zone, ZOST_ZOST: collectors inside eelgrass bed, ZOST_WC: collectors
in water column above eelgrass bed.

2. Effect of eelgrass vegetation on oyster survival

700

and growth600

Although eelgrass vegetation has no significant

500

positive effect on oyster recruitment, the results ofspats cultivation experiments exhibited that eelgrass400

beds seemed to develop a better environment for300

spat survival and growth in both France and Japan.
In France, the survival rate of oyster spats after

200

three months of cultivation was higher in the sites

1000with eelgrass beds (Fig. 6a). Moreover, the survival

Seagrass

Raft

Fig. 5. The abundance of recruited spats oneach collector (mean ± SD) on both eelgrassbeds and oyster raft.

rate was higher in the Bouzigues site with dense
eelgrass beds than the Montpénèdre site with sparse
eelgrass beds, suggesting that more abundanteelgrass vegetation could be a better environment
for oyster spats. The spats also exhibited better
growth in shell length and fresh weight in the sitewith eelgrass beds than the site without eelgrasssignificant (ANOVA: F=3.573, p>0.05). Natural oyster

Figure 5

beds after three-months cultivation (Fig. 7a).

spats are generally collected in the intertidal zone

In the experiment conducted in Hiroshima Bay,

where native spats of C. gigas most frequently settle

the survival rate of oyster spats was higher in the(FRA, 2016), and young settlers reach the intertidal

tidal ground with eelgrass beds than that hungzone during high tide. The depth of the collectors

from the raft, except for C. nippona (iwagaki) spatshung from the raft is similar to the depth of the(Fig. 6b). The tidal flat may not be a good habitat

intertidal zone during high tide.

for the iwagaki oyster because this species naturally

Figure 4Oyster aquaculture with seagrass beds

(a)

129

100

Fraction of survival (%)

80
60With eelgrass
Without eelgrass

40
20

(b)

Fraction of survival (%)

0

All sites mean

Bouzigues site

Montpénèdre site

All species mean

Pacific oyster(C. gigas)

Iwagaki oyster(C. nippona)

1
100
0.8
80
0.6
60
0.4
40
0.2
20
0

Kumamoto oyster(C. sikamea)

Figure 6

Fig. 6. The survival rate of oyster spats in a) Thau lagoon and b) Hiroshima Bay. In Thaulagoon, the biomass (shoot density x shoot height) of eelgrass in Bouzigues site was muchlarger than that in Montpénèdre site.

Molluscous weight (gDW)/Total weight (gDW)

60.0
50.0
40.0
30.0
20.0
10.0
0.0

0.21
0.18
0.15
0.12

With eelgrass
Without eelgrass

0.09
0.06
0.03
0.00

0.25

60.0
50.0

Molluscous weight (gDW)/Total weight (gDW)

Longest part of shell length (mm)(b)

Longest part of shell length (mm)

(a)

40.0
30.0
20.0
10.0
0.0

0.20
0.15
0.10
0.05
0.00

C. gigas

C. nippona

C. sikamea

C. gigas

C. nippona C. sikamea

Figure 7

Fig. 7. The difference in shell length and relative meat weight between the site with eelgrass beds and thesite without eelgrass beds as an index of growth rate in a) Thau lagoon and b) Hiroshima Bay. The results in
Hiroshima Bay was originally referred from Hori et al. (2018).

130

Masakazu HORI, Masami HAMAGUCHI, Masaaki SATO, Réjean TREMBLAY,
Alana CORREIA-MARTINS, Valerie DEROLEZ, Marion RICHARD, and Franck LAGARDE

inhabits subtidal rocky shore without exposure to

50% of the relative abundance of potential bacterial

the atmosphere. The growth rate based on relative

pathogens capable of causing disease in humans

fresh weight per total weight after two months wasand marine organisms. In our ongoing analyses ofalso better in the tidal flat with seagrass beds (Fig.

the environmental DNA and microbiome from the7b), although the shell length of all three species

oysters and ambient waters, some of the results

were shorter in the tidal flat with eelgrass. Oyster

support the previous report by Lamb et al. (2017),

spats in the cages deployed on the tidal flat were

although more detailed analyses are needed.

frequently exposed to heavy wave action, such as

Anoxia decreases the growth of oysters evenfishing boat wake during the low tide period, andwhen it is not lethal, because oysters keep their

the tip of the shells were often broken by clashingshells closed in an anoxic environment. In fact,

each other in the cage (Hori, personal observation).

serious anoxia occurred around oyster farmingThis may be a reason that the shell length wasareas in Thau lagoon in August 2018 (Author’s

shortened in all spats in the tidal flat site.

personal observation). The exceptional climatic

At least two possible hypotheses can be raised for

conditions in 2018 led to a heat wave and summer

seagrass vegetation providing better environment toanoxia in Thau lagoon, causing massive mortality

oyster spats: trophic support and the improvementof oysters and mussels (4000 t, correspondingof the sanitary conditions. As a trophic support

to 41% of the annual production). Even in thishypothesis, Hori et al. (2018) suggested that oyster

condition, the survival rate of oyster spats in thespats cultivated in the tidal flat could be usedsite with more abundant eelgrass vegetation wasfor pelagic production and benthic production inapproximately 90%, suggesting a possibility thateelgrass beds based on the result of carbon andeelgrass vegetation mitigated anoxic environment innitrogen stable isotope analyses. Even iwagaki oyster

the study site. To unravel this mitigative function,

spats with poor survival exhibited better growthmore surveys and analyses are needed, such as thein relative fresh weight, thus trophic function ofrelationship between mortality and dissolved oxygen

eelgrass beds may have some contribution to theconcentration in detail.

spat growth. These results are also supported in

In conclusion, seagrass vegetation has potentiallystudy of Barbier et al. (2017) showing that bivalve

positive effects on oysters as reported in Morimoto

recruitment abundance is lower in Zostera marina

et al. (2017). Our results directly revealed thatbeds than others benthic habitat, like subtidal coarse

eelgrass beds can contribute to some processes

sand or maerl beds (Corallinophycidae, Rhodophyta).

of oyster aquaculture such as spat cultivation.

However, they observed higher accumulation of

Therefore, oyster aquaculture using oyster-

fatty acids in the digestive glands of juveniles settled

seagrass interactions could be an effective coastal

in Z. marina, suggesting higher food availability.

management option to achieve both sustainable

However, Crepidula fornicata banks appear to be

food production and environmental conservation

the favorable habitat for bivalve recruitment (higher

as a climate change countermeasure. For example,

recruit abundance and diversity) with individuals

oyster-eelgrass interactions may support high water

having fatter digestive glands.

transparency and better sanitary conditions, which

The other hypothesis is that seagrass beds could

are also beneficial for recreational uses. Larger

maintain or improve the safety and environmental

eelgrass beds can absorb more carbon dioxide

sanitary conditions for oysters (e.g. Lamb et al.,from the atmosphere and store them as organic

2017; Groner et al., 2018). There are some known

carbon (Kuwae and Hori, 2018), which can mitigate

negative environmental factors. Some lethal viruses

ocean acidification and, moreover, offset the carbon

and bacterial pathogens capable of causing mortality

emissions from oyster aquaculture and recreationalof oysters are well known, and anoxia events areactivities. Such local offset systems for carbon

a growing threat related to seawater temperatureemissions can contribute towards solutions forrise during the summer. Lamb et al. (2017) reported

climate change in relation to the framework of thethat the presence of seagrass vegetation reduced

Sustainable Development Goals (SDGs) led by theOyster aquaculture with seagrass beds

131

a high pCO2 ocean. Ecology, 99, 1802-1814.

United Nations.

Hoegh-Guldberg. O., et al., 2019: The Ocean as a
Acknowledgement

Solution to Climate Change: Five Opportunities
for Action. Report. Washington, DC, World

We would like to thank the committee of 47 th
Scientific symposium of the UJNR Aquaculture

Resources Institute. Available online at http://
www.oceanpanel.org/climate.

Panel for giving us this opportunity to present our

Hori M., and Tarutani K., 2015: Changes in the

study. F.L. and M.H. thank FRA and Ifremer for

distribution of seagrass vegetation with relation

the MoU allowing scientific exchanges. F.L. thanks

to the possible regime shift from pelagic-

Gabriel Devicq and Nabila Guenineche for their

dominant to benthic-dominant system in Seto

technical contributions and involvement during field

Inland Sea, in “Issues of oligotrophication inwork, and F.L. and R.T. thanks the RECHAGLO

ocean and lakes” (ed. by Yamamoto T., andinternational research group co-funded by Ifremer

Hanazato T.), Chijinshokan press, Tokyo, pp.

and MPO for the financial support of exchanges

129-148. (in Japanese)with Canada. Finally, F.L., V.D., M.R., M.S. and M.H.

Hori M., Hamaoka H., Hirota M., Lagarde F., Vaz S.,

thank JSPS and Campus France/Ministry of Foreign

Hamaguchi M., Hori J., and Makino M., 2018:

Affairs for funding the scientific exchange between

Application of the coastal ecosystem complex

France and Japan.

c o nc e pt t o w a r d int e gr a t e d ma na ge ment
for sustainable coastal fisheries under
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environments influence size at metamorphosis andC., Hong C., Qin Y., and Davis S. J., 2019:

recruitment performance of Pacific oysters. Mar.

Committed emissions from existing energy

Ecol. Prog. Ser., 602, 135-153.

infrastructure jeopardize 1.5℃ climate target.
Nature, 572, 373-377.

Reproduction and recruitment of benthic
invertebrates are influenced by climate and by theecological structure of marine ecosystems, along with

Annotated Bibliography of Key Works

local anthropogenic pressures such as eutrophicationor oligotrophication. Using the Pacific oyster,(1) Hori M., Hamaoka H., Hirota M., Lagarde F., Vaz S.,

Crassostrea gigas, as a biological model, we tested

Hamaguchi M., Hori J., Makino M., 2018: Application

the hypothesis that the variability in prodissoconchof the coastal ecosystem complex concept toward

II (PII) size (i.e. size at metamorphosis) depends on

Oyster aquaculture with seagrass beds

133

ecological functioning. Settlement and recruitment

the trophic functioning of the ecosystem. Smaller PII

were assessed at 5 sampling sites in the French

sizes were significantly correlated with the highest

Mediterranean shellfish farmed Thau lagoon

phytoplankton biomass, while larger PII sizes wereduring the main summer recruitment events in 3

positively correlated with mixotrophic cryptophyte

consecutive years (2012-2014). Hydrobiological andabundance. We found an inverse relationshipplanktonic analyses were conducted at 3 sampling

between PII size and survival after metamorphosis,

sites. Our results showed that recruitment wasshowing that recruitment success was associatedextremely heterogeneous, ranging from 0 to 260±with smaller PII sizes. Regional climate conditions−2

throughout the ecosystem and was

and local trophic functioning appear to be key factors

linked with variability in PII size, which ranged

in metamorphosis and consequently contribute tofrom 180 to 296 μm. The annual temporal pattern ofrecruitment heterogeneity. Further studies should

PII sizes appeared to be controlled by temperature

be performed in other ecosystems following an

during the settlement period, whereas the spatial

oligotrophication trajectory to generalize this result.

27 SE ind. dm

pattern depended on phytoplankton biomass and on

水 研 機 構 研 報， 第 50 号，135 － 139， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，135－139，2021

135

Kelp, Saccharina spp, population genetics in New England, US, for
guiding a breeding program of thermally resilient strains
Simona AUGYTE＊1, Jean-Luc JANNINK＊2, Xiaowei MAO＊2, 3, Mao HUANG＊2,
Kelly ROBBINS＊2, Matt HARE＊4, Schery UMANZOR＊1, Michael MARTY-RIVERA＊1,
Yaoguang LI＊1, Scott LINDELL＊5, David BAILEY＊5, Charles YARISH＊1

Abstract: The cold-water sugar kelp, Saccharina latissima has a circumboreal distribution
and in the Northwest Atlantic is at its southern distributional limits in Long Island Sound.
An understanding of genetic diversity of natural kelp populations is critical for making
recommendations for breeding and cultivation efforts of the growing seaweed aquaculture sector
in the US. An important component of the ARPA-E’s MARINER project is selectively breeding
Saccharina spp. in order to improve overall productivity for biofuels, feeds and food.
Historical records indicate the presence of regional kelp ecotypes based on physiologicaltolerance, specifically temperature. We made collections of 15 wild Saccharina spp. populations via
SCUBA along the New England coast. Microscopic gametophytes were isolated and the parental
populations were used to make over 500 hybrid crosses that were planted at several farm locations
over several years. We then used genome-wide single nucleotide polymorphism data to explore
the genetic structure of the kelp throughout this region. An assessment of the sequence diversity
revealed distinct genetic variation between the Gulf of Maine and Southern New England (FST >0.25), confirming that Cape Cod acts as a barrier to S. latissima gene flow. Furthermore, based on
the analysis of molecular variance (AMOVA), we found the largest variance (58%) within sites. We
also observed admixture among three ancestral populations and isolation by distance. Future steps
for this project include skim sequencing the haploid microscopic gametophytes to identify trait
heritability, phenotypic diversity observed for both morphological traits and tissue composition,and genomic selection. Furthermore, in the future, we plan to place our sequence data into a larger
context to include samples from sites in the east Atlantic and Pacific Oceans.
Key words: Saccharina, population genetics, breeding, thermal resilienceIntroduction

important in global food security. Worldwide,
seaweed aquaculture is an important component

For centuries, humans have harvested differentof total marine aquaculture production, and isseaweeds for food, medicinal purposes, feed, andexperiencing exponential growth in the last 50 years

more recently as raw materials for industrial(Kim et al., 2014; Augyte et al., 2017; Langdon etprocesses (Augyte et al., 2018; Grebe et al., 2019;

al., 2019; FAO, 2020). Compared to Asian countries

Kim, et al., 2019)⁠ . Over the years, with humanincluding China, Korea, Japan, Indonesia andpopulation rise, seaweeds have become increasingly

the Philippines, seaweed aquaculture is a young

2020年12月11日受理（Accepted on December 11, 2020）＊1
Dept. of Ecology and Evolutionary Biology, University of Connecticut, 1 University Place, Stamford, CT 06901, USA＊2
Oregon State University, Fisheries and Wildlife Dept., Hatfield Marine Science Center, Newport, OR 97365, USA＊3
Key Laboratory of Vertebrate Evolution and Human Origins, Institute of Vertebrate Paleontology and Paleoanthropology, Chinese Academyof Sciences, Beijing 100044, China＊4
Department of Natural Resources and the Environment, Cornell University, Ithaca, NY 14853, USA＊5
Applied Ocean Physics and Engineering Department, Woods Hole Oceanographic Institution, Woods Hole MA 02543, USA
E-mail: simona.augyte “at” uconn.edu

136

Simona AUGYTE, Jean-Luc JANNINK, Xiaowei MAO, Mao HUANG, Kelly ROBBINS, Matt HARE, Schery UMANZOR,
Michael MARTY-RIVERA, Yaoguang LI, Scott LINDELL, David BAILEY, Charles YARISH

industry in the United States of America (Kim et al.,environmental variables with morphology. Samples

2019). It is estimated that the U.S. Farmed seaweed

were collected at peak reproduction and sorus

production in the year 2019 was 249,000 – 272,000

material was isolated and meiospores were released

kg wet weight with 80% of this production coming

to cultivate the microscopic stage of the life cycle,

from Saccharina latissima or sugar kelp (Piconi et al.,specifically the male and female gametophytes.

2020). It is estimated that edible seaweed production

These gametophytes were used to make over 500

will double in the next five years and will increasehybrid crosses that were out planted at several

by four times by 2035 (Piconi et al., 2020).

aquaculture farms over two years. Parental

Various studies have addressed the gametophyte

sporophytic blade material was collected for DNA

growth, propagation, and thermal tolerance, as well

extraction and were used for genotyping at theas ecosystem benefits of seaweed cultivation in the

Diversity Arrays Technology (DArT) facility inopen – ocean (Egan and Yarish, 1988; Kim et al.,Canberra, Australia. Reduced genomic representation

2014; Augyte et al., 2017; 2018; Wade et al., 2020)⁠,

was generated using restriction enzymes andhowever, a lot remains to be done to achieve a deep

Ilumina HiSeq2500 and then reads were processed

understanding of kelp ecophysiology and applicationand single nucleotide polymorphisms (SNPs) were

to cultivation. To meet the growing demand forcalled using proprietary DArT analytical pipelines

increased seaweed aquaculture production, the(Kilian et al., 2012). Genome-wide SNP data coupled

MARINER program funded by the U.S. Dept. ofwith AMOVA (Analysis of Molecular Variance),

Energy is focused on increasing production for

FST, admixture, isolation by distance and PCoA

biofuels, animal feed and human food. As interest in(Principal Component Analysis) were used to explore

commercially viable species continues, it is therefore

the genetic structure of kelp throughout the region.

critical to have baseline information regarding theunderlying genetics of the wild populations to guide

Resultssustainable practices. Furthermore, an importantcomponent of breeding superior cultivars is selecting

Large morphological variation was observed

kelp strains that are high yielding, and disease andacross all 15 locations. For example, adult kelp blade

thermally tolerant. The purpose of this study was

lengths ranged from 84.5 cm – 227 cm; blade widths

to assess the genetic structure of wild S. latissima

from 3.4 mm – 41.4 mm; blade thickness ranged

populations in Northwest Atlantic, from the southernfrom 0.8 – 2.28 mm; stipe diameter from 2.17 mm -range of its distribution, throughout Southern New

14.43 mm; and stipe length from 4.8 cm – 122.7 cm.

England to the Gulf of Maine. The results of thisThe populations within the Gulf of Maine showed

study will inform managers and conservation groups

significantly higher genetic diversity compared toon genetic hotspot areas of diversity, connectivity

populations in the southern region, in the Southernand gene flow. Finally, this and future research can

New England area. Results further indicate thatguide breeding and cultivation efforts of wild S.

roughly half (56%) of the total variation exists within

latissima by identifying phenotypic variation andlocations (AMOVA, p-value<0.001). Isolation by

hybrid vigor.

distance, indicated by positive correlation between
genetic and geographic distances, was observed
Materials and Methods

for both the Gulf of Maine (r=0.47, p-value=0.002)
and Long Island Sound (r=0.94, p-value=0.125). The

Collections of 15 wild Saccharina spp. populations

pair-wise genomic F ST between the two regions

via SCUBA along the coast of New England, in

was >0.25 and was supported by the PCoA plots.

the Northwest Atlantic were made. Commercially

Despite the isolation by distance pattern in each

important morphometric measurements were done

region, ancestry analyses indicate a complex history

to characterize phenotypic variation specificallyof historical gene flow both within and between theon blade length, width, and thickness and stipe

regions stemming from three ancestral populations.

length and thickness. Tests were run to correlateSaccharina population genetics in New England, Northwest Atlantic

Discussion

137

DE-AR0000915 and DE-AR0000911. We acknowledge
all of the contributions made by M. Chambers andHistorical biogeographic reconstructions reveal

his team at the University of New Hampshire andthat complex kelp originated in the northeast

the volunteers at Wood Hole Oceanographic InstitutePacific and diversified over time by colonizing

and the University of Connecticut, Stamford, who

new habitats (Starko et al., 2019)⁠. The kelp in thehelped during the two breeding seasons of thisNorthwest Atlantic were colonized post-glaciallydomestication program.

via oceanographic flow through the Arctic (Nielsen
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Grebe, G. S., Byron C. J., St. Gelais A., Kotowicz D. M.,of the population structure of S. latissima, but also

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Blois H., Caig V., Heller-Uszynska K., Jaccoud

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Energy, ARPAe MARINER project contract number

Field scale evaluation of seaweed aquaculture

138

Simona AUGYTE, Jean-Luc JANNINK, Xiaowei MAO, Mao HUANG, Kelly ROBBINS, Matt HARE, Schery UMANZOR,
Michael MARTY-RIVERA, Yaoguang LI, Scott LINDELL, David BAILEY, Charles YARISH
as a nutrient bioextraction strategy in Long

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in a marine environment. It differs from agriculture(doi: 10.1111/jpy.12428)

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Neiva J., Paulino C., Nielsen M. M., Krause-Jensen D.,

fertilization or freshwater, which are resources

Saunders G. W., Assis J., Bárbara I., Tamigneauxthat may ultimately constrain the expansion ofÉ., Gouveia L., Aires T., Marbà N., Bruhn A.,

agriculture. Seaweeds are converted into a variety ofPearson G. A., and Serrão E. A., 2018: Glacial

goods, such as food and nutritional supplements forvicariance drives phylogeographic diversification

humans and livestock, fertilizer, unique biochemicalin the amphi-boreal kelp Saccharina latissimi.

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Starko S., Soto Gomez M., Darby H., Demes K.

necessitates establishing product acceptability,W., Kawai H., Yotsukura N., Lindstrom S. C.,

creating a sustained market and then balancing

Keeling P. J., Graham S. W., and Martone P. T.,

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comprehensive understanding of the role thatC., and Alberto F., 2020: Macroalgal germplasm

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the ecosystem services offered by marine farming

Wilson K. L., Skinner M. A., and Lotze H. K., 2019:activities. This will result in a better understandingSaccharina population genetics in New England, Northwest Atlantic

139

of how an ecosystem approach to aquaculture(Laminariales, Phaeophyceae). Algae, 34, 153-162.

incorporates the role and need for both the goods(doi.org/10.4490/algae.2019.34.5.12)

and services these macroalgae will provide.

Anthropogenic disturbances, including coastal
habitat modification and climate change are(2) Augyte S., Umanzor S., Yarish C., and Lindell

threatening the stability of kelp beds, one of theS., 2018: Enhancing marine ecosystem services via

most diverse and productive marine ecosystems. To

kelp aquaculture. ISAP newsletter, December, 10–14.

test the effect of temperature and irradiance on the(https://docs.wixstatic.com/ugd/e9f50b_f3edc9d188f2

microscopic gametophyte and juvenile sporophyte

48a18061e09cfa25b5d9.pdf)

stages of the rare kelp, Saccharina angustissima,

For centuries, humans have harvested different

from Casco Bay, Maine, USA, we carried out two

seaweeds for food, medicinal purposes, feed, andsets of experiments using a temperature gradientmore recently as raw materials for industrial

table. The first set of experiments combined

processes. Currently, wild harvested seaweed

temperatures between 7-18°C with irradiance ataccount for less than 5% of the total worldwide-2 -1
20, 40, and 80 μmol photons m s . The second set

supply (FAO, 2018). The majority of seaweed

combined temperatures of 3-13°C with irradianceproduction is provided via aquaculture, with 99% ofof 10, 100, and 200 μmol photons m-2 s-1. Over two

the production taking place in Asia including China,

separate 4-week trials, in 2014 and again in 2015,

Korea, Japan, Indonesia, and the Philippines, worth

we monitored gametogenesis, the early growth

US $11.7 billion annually (FAO, 2018). Althoughstages of the gametophytes, and early sporophyte

seaweed aquaculture is a fast expanding industry,development of this kelp. Gametophytes grew best

the global demand for seaweed-based products is

at temperatures of 8-13°C at the lowest irradiance of

surpassing the supply. Such demands necessitate

10-μmol photons m-2 s-1. Light had a significant effecteither domesticating new species or further

on both male and female gametophyte growth only

expanding the productivity of the existing leadingat the higher temperatures. Temperatures of 8-15°C

seaweeds. According to the FAO, only a few species

and irradiance levels of 10-100 μmol photons m-2 s-1

dominate seaweed farming, including two brown

were conditions for the highest sporophyte growth.

kelps, Saccharina japonica and Undaria pinnatifida

Sporophyte and male gametophyte growth was(Buschmann et al., 2018). To meet the present

reduced at both temperature extremes—the hottest

market requirements and to contribute in reducing

and coldest temperatures tested. S. angustissima

the over-exploitation of wild stocks, experimental

is a unique kelp species known only from a very

trials worldwide have assessed the performance

narrow geographic region along the coast of Maine,of newly farmed seaweeds as potential products,

USA. The coupling of global warming with highparticularly kelp species that have been traditionallylight intensity effects might pose stress on theharvested from wild populations.

early life-history stages of this kelp, although, as anintertidal species, it could also be better adapted to(3) Augyte S., Yarish C., and Neefus C. D., 2019:

temperature and light extremes than its subtidal

Thermal and light impacts on the early growthcounterpart, Saccharina latissima.

stages of the kelp Saccharina angustissima

水 研 機 構 研 報， 第 50 号，141 － 148， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，141－148，2021

141

Cell selection technique for establishment of low salinity tolerance
strain in Pyropia tenuipedalis†
Mahiko ABE＊1, Tomomi OHASHIRA＊1, Noboru MURASE＊1 and Masanobu KISHIOKA＊2

Abstract: Pyropia tenuipedalis is characterized by reddish thalli and direct budding from theshell substratum. The distribution of this species is limited to coastal areas in the Seto Inland Sea,
Ise Bay and Tokyo Bay in Japan. In Yamaguchi prefecture, this species has been locally used
for direct human consumption. Moreover, alanine and glutamic acid contents of P. tenuipedalis
are three times and two times higher, respectively than those of P. yezoensis “nori”. Yamaguchi
Prefectural Fisheries Research Center began development of the mariculture technique of P.
tenuipedalis in 2002, and succeeded in its commercialization in 2007. However, production has
continued to decrease since 2012. More recently, intense disappearance of thalli is observed
during January to February. One of the reasons for the production decrease is thought to be
low salinity due to recent heavy rain fall supposedly associated with global climate change. The
aim of this study was to develop low salinity tolerance strains of P. tenuipedalis through a cellselection technique. Punched out disks of thallus were cultured in a 10% seawater medium diluted
with distilled water with modified 1/2SWM-III medium for 1 to 4 months. A few cells survived
in this medium and were subsequently cultured in a 100% seawater medium with 1/2SWMIII. Surviving cells divided and regrew to thallus, and stock culture strains were establishedwith self-fertilization. A field culture trial of the established low salinity tolerance strain and theconventional strain was carried out in Koto River estuary from December 2018 to February 2019.
The low salinity tolerance strain showed better growth than the conventional strain, suggesting
the efficacy of the cell selection technique for breeding. However, some of the thalli growing on theculture plates were suddenly shortened or disappeared in early January. Time-lapse observation
with an underwater camera revealed that the disappearance of the thalli was due to predationby blackhead seabream, Acanthopagrus schlegelii. Immediate establishment of not only strains
resistant to changing environments, but also effective measures for countering predation arenecessary to increase production of P. tenuipedalis.
Key words: Pyropia tenuipedalis, breeding, cell selection, low salinity tolerance
Intoduction

has decreased due to climate change impacts, suchas seawater temperature rise and oligotrophication.

Recently, fisheries production of Pyropia yezoensis,

Many researchers are developing or seeking

commonly referred to as “nori” in Japanese language,

new strains that have tolerances to the above-

2020年12月11日受理（Accepted on December 11, 2020）＊1
National Fisheries University, Japan Fisheries Research and Education Agency, 2-7-1 Nagata-Honmachi, Shimonoseki, Yamaguchi 759-6595,
Japan＊2
Yamaguchi Prefectural Fisheries Research Center, 2861-3, Senzaki, Nagato, Yamaguchi, 759-4106, Japan
E-mail: abemahi “at” fish-u.ac.jp†
Pyropia tenuipedalis was replaced to Neopyropia tenuipedalis by Yang et al. (2020).
Yang L.-E., Deng Y.-Y., Xu G.-P., Russel S., Lu Q.-Q.,and Brodie J., 2020: Redefining Pyropia (Bangiales, Rhodophyta): four new genera,
resurrection of Porphyrella and description of Calidia pseudolobata sp. nov. from China. J. Phycol., 56(4), 862–879.

142

Mahiko ABE, Tomomi OHASHIRA, Noboru MURASE and Masanobu KISHIOKA

mentioned environmental stresses (Shimada, 2010;

approximately 2 mm in blade length release many

Sakaguchi, 2011). In Yamaguchi Prefecture, Pyropia

monospores. Monospores grow into young thalli.

tenuipedalis, which is closely related to P. yezoensis,

After release of monospores, the thalli resumes

is an indigenous species, and is a resource used to

growth (Yoshida, 1993). Therefore, P. yezoensis

promote local industries (Fig. 1). As P. tenuipedalis

can produce a large number of spores and thalli.

has reddish thalli unlike blackish P. yezoensis

However, matured thalli of P. tenuipedalis releaseand directly buds from the shell substratum, thisspecies is generally called “Aka-nori” in Yamaguchi
Prefecture, and the Japanese vernacular name is“Kaigara-ama-nori”. Aka and Kaigara means red andshell, respectively in Japanese. The distribution ofthis species is limited to coastal areas in the Seto
Inland Sea, Ise Bay, and Tokyo Bay in Japan, andthis species is designated as an endangered species.
In Yamaguchi Prefecture, this species has beenused as an important local food resource for humanconsumption (Abe et al., 2015).
The life cycle of P. tenuipedalis is quite different
from that of P. yezoensis (Fig. 2). Matured thalli of
P. yezoensis releases many carpospores. Carpospores
germinate and grow into filamentous conchocelis.
Conchosporangia are formed on conchocelis, andmany conchospores are released. Conchosporesdivide and grow into young thalli. Young thalli of

Fig. 1. Photo of Pyropia tenuipedalis collected
in January 2011.

Fig. 2. Life cycles of Pyropia yezoensis (a) and Py. tenuipedalis (b).

Cell selection techniques of Pyropia tenuipedalis

143

5000

Contents (mg 100g-1)

4500

Py. yezoensis (Nori)

4000

Py. tenuipedalis (Aka-nori)

3500
3000
2500
2000
1500
1000

Asp

Glu

Ser

Pro

Ala

Gly

Lys

Val

Met

Ile

Leu

His

Arg

2014

0

2012

500
Phe

Amino Acid
Fig. 3. Amino acid content of Pyropia yezoensis and Py. tenuipedalis.
900

many carpospores and becomes conchocelis, as in100

between P. yezoensis and P. tenuipedalis (Fig. 3).

0

Alanine and glutamic acid contents of P. tenuipedalis
are higher than those of P. yezoensis. This suggeststhat P. tenuipedalis has stronger “umami” and“sweetness” compared with P. yezoensis. Yamaguchi
Fig.4
Prefectural Fisheries Research Center focused on

P. tenuipedalis as a new mariculture species in
2002. They developed the culture plate technique

2007

Amino acid contents are completely different

2017

200

2016

thallus.

300

2015

Consequently, one spherical cell grows into one

400

2013

and monospore (Notoya and Kikuchi, 1993).

500

2011

thalli. P. tenuipedalis does not have conchospore

600

2010

divides and grows into uniseriate thalli and young

2009

instead of conchosporangia. This spherical cell

700

2008

forms a spherical cell on the tip of conchocelis

800
Production volume (kg W.W.)

the case of Fig.3
P. yezoensis. However, P. tenuipedalis

Seasons (November – March)

Fig. 4. Production volume of Pyropia tenuipedalis
mariculture from November to March during 20072017. 2007-2011: Yamaguchi Bay (blue bar), 2012:
Yamaguchi Bay and Koto River estuary (black bar),
2013-2017: Koto River estuary (red bar).

for P. tenuipedalis mariculture and succeeded in its
commercialization in 2007. This culture plate is madefrom 2007 to 2011. In 2012, it was conducted infrom calcium carbonate, and conchocelis are cultured

Yamaguchi Bay and Koto River estuary. Since 2013,

on this plate. These culture plates are set directly on

it has been conducted only in Koto River estuary.

the sea bottom in the field in November every year.

P. tenuipedalis production had the highest value inThalli growing on the culture plates are harvested

2011 with the increase in the number of the cultureby hand and dried by machine from January toplates (Fig. 4). However, production has continued

March, and then it become the final product. The

to decrease since 2012 although the number of

price for the product is 1,200 Japanese yen / 10 g in

culture plates was kept constant. More recently,

dry weight. Among seaweed products in Japan, thisintense disappearance of thalli is observed fromis the most expensive.

January to February. One of the main reasons for

Mariculture using the culture plates was

the production decrease is thought to be low salinity

previously carried out only in Yamaguchi Bay

due to recent heavy rain fall. Water discharge

144

Mahiko ABE, Tomomi OHASHIRA, Noboru MURASE and Masanobu KISHIOKA

from the Koto River Dam during the period fromwere allowed to divide and regrow to thalli, andDecember to January—the growing season for P.

stock culture strains were established with self-

tenuipedalis—clearly increased from 2008 to 2016 byfertilization of a large size of single thalli.

about 30% on average and 2 to 3 times in maximum
instantaneous flow (Water and Disaster Management

Field Culture Trial of Low Salinity Tolerant Strain

Bureau of the Ministry of Land, Infrastructure,
Transport and Tourism: Database of Dams: http://

A field culture trial of the established low salinity

mudam.nilim.go.jp/home). Therefore, we identified

tolerant strain and the conventional strain of P.

that low salinity tolerant strains were needed totenuipedalis was carried out in Koto River estuarycope with this problem.

from December 2018 to February 2019. Conchocelisof both the conventional strain and low salinity

Establishment of Low Salinity Tolerant Strain of

tolerant strain attached to the culture plates were

P. tenuipedalis

set in Koto River estuary at the end of November
in 2018. In addition, time-lapse observation at 1-hour

In this study, we used a cell selection techniqueintervals of culture plates was carried out with anfor establishment of a low salinity tolerant strain ofunderwater camera to determine the cause of the

P. tenuipedalis (Abe et al., 2019). Punched out disks

intense disappearance of the thalli.

of thalli were cultured in the modified 1/2SWM-III

The length of thalli in both strains were measured

medium prepared with 10% seawater (approximately

every 2 weeks from December 2018 to February

3 psu) diluted with distilled water for one to four2019 (Fig. 6). The thallus lengths of the conventional

months (Fig. 5). Under the low salinity conditions,and the low salinity tolerant strain were 1.0±1.0

although the majority of normal cells were bleached

mm and 1.3±1.4 mm, respectively on December

and eventually died, a few cells survived, which

10th 2018. On December 25th, the low salinity

were then cultured in the modified 1/2SWM-III

tolerant strain grew larger (13.4±9.5 mm) than themedium with 100% seawater. The surviving cells

conventional strain (7.0±5.4 mm). However, theFig. 5. Procedures of the cell selection technique used in this study.

Cell selection techniques of Pyropia tenuipedalis

145

25

Length (mm)

20
15
10

conventional strain

2019/2/24

2019/2/19

2019/2/14

2019/2/9

2019/2/4

2019/1/30

2019/1/25

2019/1/20

2019/1/15

2019/1/10

2019/1/5

2018/12/31

2018/12/26

2018/12/21

2018/12/16

2018/12/11

0

2018/12/6

5

low salinity tolerant strain

Fig. 6. The growth of thalli in the conventional and low salinity tolerant strains during thefield culture trial.

Fig.6salinity tolerant strain stopped
length of the low
growing and ranged from 12.4±11.0 mm to 16.6±
5.1 mm from January to February. The conventionalstrain grew gradually through the field culture trial
up to 15.5±6.4 mm. Thus, the low salinity tolerance
strain showed better growth than the conventionalstrain, suggesting the efficacy of the cell selection
technique for breeding.
Intense Disappearance of the ThalliSome of the thalli growing on the culture plates

Fig. 7. Photo of blackhead seabream (Acanthopagrus
schlegelii) grazing Pyropia tenuipedalis growing on
culture plates.

suddenly shortened and disappeared in early
January. Time-lapse observation with an underwater
camera revealed that the disappearance of thethalli was due to predation by blackhead seabream,
Acanthopagrus schlegelii (Fig. 7). Blackheadseabream appeared in 15% of the photos taken in
daytime. It was estimated that the population of
blackhead seabream was large in this area fromthe relationship between the interval of time-lapse
photography and the appearance of blackheadseabream in the photos. The intense disappearance
from January 4th to 5th was remarkable (Fig. 8).
Thalli on the culture plates were considerably
shortened over one night.
We unintentionally found a countermeasure forfish predation in a field trial that was conducted to
study work efficiency of the seaweed deployment.
Usually, the culture plates are placed directly on
the sea bottom. Therefore, activities for mariculture

Fig. 8. Photos of Pyropia tenuipedalis thalli on aculture plate on January 4th and January 5th 2019.

146

Mahiko ABE, Tomomi OHASHIRA, Noboru MURASE and Masanobu KISHIOKA

had better growth than the conventional strain in(a)the early culture period.
Intense disappearance of P. tenuipedalis due topredation by omnivorous fish were observed by

(b)

5cm

time-lapse observation with underwater camera
set in the field culture trial site. For P. tenuipedalis
mariculture using culture plates, it is advisable tophysically protect the seaweed from fish predation
in a cage.
Reference

5cm

Fig. 9. Specimens of Pyropia tenuipedalis collected
on January 7th 2019, including: (a) thalli on theculture plates set directly on the sea bottom, and(b) thalli on the culture plates placed within a crabbasket. Bars indicate 5cm.

g.9

Abe M., Murase N., Hatama T., Shikano Y.,
and Kanai T., 2015: New record of Pyropia
tenuipedalis from Koto river estuary, Yamaguchi
prefecture. J. Nat. Fish. Univ. 63, 244-248. (inJapanese with English abstract)
Abe M., Ohashira T., Murase N., and Kishioka M.,
2019: Influences of salinity on growth of two

management, such as cleaning sediments on the

Pyropia tenuipedalis strains selected in low

culture plates and harvesting thalli, are restrictedsalinity condition. J. Nat. Fish. Univ., 68, 11-15.

during low tide at the spring tide due to limited(in Japanese with English abstract)accessibility. In order to enable the operator to

Notoya M., and Kikuchi N., 1993: Porphyra

perform the activities regardless of the tidal height,

tenuipedalis Miura, in “An illustrated atlas of

the culture plates were placed within a crab basket

the life history of algae” (ed. by Hori T.), Vol. 2,that can be lifted up on the boat. As part of the

Uchida Rokakuho Publishing Co., LTD., Tokyo.

study, we compared the growth between thalli set

pp.214-215. (in Japanese)

directly on the sea bottom and those placed within a

Sakaguchi K., 2011: New nori strain with high water

crab basket (Fig. 9). We found that the thalli withintemperature tolerance in Mie prefecture “Mie noa crab basket grew better and that the crab basket

AKARI” (Mie ken no kousuion taisei kuro nori

was effective in physically protecting the thalli fromno shin hinshu “MIE NO AKARI”). Researchfish predation.

journal of food and agriculture, 34, 42-43. (inJapanese)
Conclusion

Shimada Y., 2010: Physiological and morphologicalcharacters of the three wild strains of Porphyra

In Yamaguchi Prefecture, P. tenuipedalis has been

yezoensis collected from Hokkaido. Aquacult.

used as a local fisheries resource, and mariculture

Sci., 58, 473-479. (in Japanese with Englishof this species was intended as a unique local

abstract)

industry. However, intense disappearance of theWater and Disaster Management Bureau of thethalli has been observed from January to February

Ministry of Land, Infrastructure, Transport andfor the past few years, which is a hindrance to the

Tourism: Database of Dams, http://mudam.

successful industrialization. One of the reasons fornilim.go.jp/home. (in Japanese) (cited 22 March,

the production decrease was thought to be low

2019)salinity due to recent heavy rainfall. We establishedYoshida T., 1993: Porphyra yezoensis Ueda, in “An

low salinity tolerant strains by developing the cellillustrated atlas of the life history of algae” (ed.

selection technique for breeding. A field culture trial

by Hori T.), Vol. 2, Uchida Rokakuho Publishingshowed the established low salinity tolerant strain

Co., LTD., Tokyo. pp.216-217. (in Japanese)Cell selection techniques of Pyropia tenuipedalis

Annotated Bibliography of Key Works147

November to March, 2010-2014. Young foliose
thallus of this species appeared in December and(1) Abe M., Murase N., Hatama T., Shikano Y., andgrew to their maximum length from January toKanai T., 2015: New record of Pyropia tenuipedalis

March. In March, mature thalli were observed.

from Koto river estuary, Yamaguchi prefecture. J.

Water temperature from November to MarchNat. Fish. Univ., 63, 244-248. (in Japanese with

was usually within the range of 6-16°C, but the

English abstract)temperature was lower in early December in 2012

We collected reddish foliose thalli from Koto River

than other years. Light reaching the growing

estuary, Yamaguchi Prefecture. In order to identify

depth of this species was 6.2±3.1% of the level at

the species, we carried out ﬁeld collections of thewater surface at high tide. The concentration offoliose thalli, morphological observation in culture

dissolved inorganic nitrogen at the sampling site

and PCR-RFLP analysis using the two regions of thewas within the range of 8.8-68.3 μmol L , which waspartial mitochondrial DNA. As for the morphologicalapproximately 10 times higher than the level that issurvey, the spherical cells were formed at the tips

assumed to cause the discoloration of P. yezoensis.

of conchocelis and developed to foliose thalli. The

Water temperature reduced at a rate of 0.33°C day-1

fragment patterns of this species in the PCR-RFLP

from November to December in 2012-2013, whichanalysis matched with Pyropia tenuipedalis. In thewas faster than the rate in other years. In this study,

present study, the foliose thalli collected at Kotoit was suggested that the growth of thalli fromRiver were identified as P. tenuipedalis, which is anspherical cells was inhibited by a long- or short-term

endangered species distributed along the coast of

rapid water temperature decrease that occurredJapan. This species was newly recorded from Koto

from November to December.

-1

River estuary, Yamaguchi Prefecture.
(4) Abe M., Murase N., Nakae M., Nakayama(2) Nakayama T., Abe M., Murase N., and Shikano,T., Nakagawa M., and Shikano Y., 2018: Water

Y., 2017: Influence of salinity on growth of red alga

temperature characteristics in growth of filamentous

Pyropia tenuipedalis and Pyropia yezoensis foliose

thallus and formations of spherical cell, uniseriate

thallus. Aquacult. Sci., 65, 321-330. (in Japaneseand foliose thallus of Pyropia tenuipedalis (Miura)with English abstract)

Kikuchi et Miyata. J. Nat. Fish. Univ., 66, 81-88. (inPyropia tenuipedalis is a new culture species inJapanese with English abstract)

Yamaguchi Prefecture, Japan. The habitat of thisWe investigated the water temperature effects

species is more brackish as compared with thaton growth of filamentous thallus and formationsof P. yezoensis, which is the common species inof spherical cell, uniseriate and foliose thallus ofJapanese nori mariculture. In this paper, the authors

Pyropia tenuipedalis with culture experiments.

investigated the relationship between salinity and

Optimal growth of filamentous thalli was observed

growth of P. tenuipedalis and P. yezoensis. It was

at 20°C. Moreover, optimal water temperatures forrevealed that P. tenuipedalis tolerates lower salinity

formations of spherical cells, uniseriate and foliose

in comparison with P. yezoensis.

thalli were 20°C, 15–20°C and 15°C, respectively.
Optimal water temperatures of each life stage were

(3) Abe M., Murase N., Hatama T., Shikano Y., anddifferent. At 10°C, the formations of uniseriate andKanai T., 2017: Environmental characteristics offoliose thalli were suppressed. At 25°C, almost all

Pyropia tenuipedalis (Miura) Kikuchi et Miyata

thalli had morphological abnormalities. Furthermore,

growing at Yamaguchi Bay, Yamaguchi Prefecture.

decreasing temperature to 15°C from 20°C enhanced

J. Nat. Fish. Univ., 65, 19-29. (in Japanese withformation of uniseriate and foliose thalli. It was

English abstract)

thought that low production in 2012 resulted from

This study surveyed environmental characteristics
of Pyropia tenuipedalis foliose thallus growing
in Yamaguchi Bay, Yamaguchi Prefecture fromsuppressed formation of uniseriate and foliose thalliat less than 10°C.

148

Mahiko ABE, Tomomi OHASHIRA, Noboru MURASE and Masanobu KISHIOKA(5) Murase N., Abe M., Fukudome K., Nakagawa M.,°C. The optimal temperature for uniseriate thalli

and Shikano Y., 2018: Influence of temperature on

developed from a spherical cell at the two-cell stage

the growth of red alga Pyropia tenuipedalis thalli.

were 15°C and 20°C. The optimal temperature

J. Nat. Fish. Univ., 66, 215-220. (in Japanese withfor foliose thalli allowed to develop from young

English abstract)

blades with a length of approximately 6 cm was 15

This study was designed to clarify the optimal°C. It was suggested that the shift in the optimal

temperatures for growth in uniseriate thalli andtemperatures from uniseriate to foliose thalli stages

foliose thalli of Pyropia tenuipedalis under laboratory

was related to the seasonal reduction of water

culture at 5°C intervals from 10°C to 25°C or 30

temperature from autumn to winter.

水 研 機 構 研 報， 第 50 号，149 － 158， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，149－158，2021

149

New diets with potential for enhancement of juvenile bivalve seedproduction and culture techniques
Yasuhiro YAMASAKI＊

Abstract: Environmental changes are now having a negative impact on a number of aquaticorganisms. In the decades after the mid-1980s, annual catch of bivalves such as the Manila clam
(Ruditapes philippinarum) continues to decrease drastically in the coastal waters of Japan, andseveral causative factors have been reported. To conserve the bivalve resources, the promotionof efficiency and stabilization of seed and juvenile bivalve production is needed in addition to thepromotion of marine ecosystem and habitat recovery. Under these circumstances, a persistentproblem is insufficient food supply for juvenile bivalves because of the difficulty in stableproduction of diet microalgae at low cost and a deficiency of microalgae that possesses all thenecessary dietary requirements. Thus, there is a compelling need for the development of dietarysupplements and/or new species of diet microalgae that contain the essential nutritional properties,
and of alternative feeds to replace live microalgal diets. In this mini-review, new diet microalgae,
a dietary-supplement, and original feeds using microencapsulation, enzymatic decomposition andfermentation technology with potential for the enhancement of juvenile bivalve seeding production
and culture techniques are introduced.
Key words: alginate hydrolysate, Eutreptiella eupharyngea, lipid-walled microcapsule, marine silage,seeding production of bivalvesIntroduction

regional and global scales since 1899, and concluded
that global phytoplankton concentration has declined

Human activities including greenhouse gas

over the past century. They also indicated that theseemissions have led to increasing global temperatures,

fluctuations are strongly correlated with basin-scale

perturbed regional weather patterns, rising sea

climate indices, whereas long-term declining trends

levels, acidifying oceans, changed nutrient loads,

are related to increasing sea surface temperatures

and altered ocean circulation (Brierley et al., 2009).

(Boyce et al., 2010).

Such a changing environment is now having a

In Japan, several studies have indicated dramatic

negative impact on a number of aquatic organisms

decreases in fisheries production and environmental(Brierley et al., 2009; Rodolfo-Metalpa et al., 2011;and ecosystem changes in coastal areas such as long-

Kroeker et al., 2013). In particular, there are growing

term decreasing trends of phytoplankton biomass

concerns about impacts of environmental change

and nutrient concentration, long-term changes inon microalgae, which are the essential primary

species composition of phytoplankton, and increasing

producers in marine and freshwater aquaticsea surface temperatures (Wanishi, 2005; Noda

ecosystems. Boyce et al. (2010) analyzed the data

and Yukihira, 2013; Abe, 2017; Yamamoto, 2019;

on available ocean transparency measurements

Nishiwaka, 2019; Kaeriyama et al., 2019). In theand in situ chlorophyll observations to estimate thedecades after the mid-1980s, annual catch of bivalves,time dependence of phytoplankton biomass at local,

especially the Manila clam (Ruditapes philippinarum),
2020年12月11日受理（Accepted on December 11, 2020）＊National Fisheries University, Japan Fisheries Research and Education Agency, 2-7-1 Nagata-Honmachi, Shimonoseki, Yamaguchi 759-6595,
JapanE-mail: yamasaky “at” fish-u.ac.jp

Yasuhiro YAMASAKI

150

in coastal waters of Japan continues to decreasejuvenile bivalves.

drastically. Though the precise cause is unclear, aAs described in the Introduction, a variety

number of factors affecting the dramatic decrease ofof studies have sought to conserve wild R.

the clam species (including overfishing) have been

philippinarum populations while providing a stablesuggested (Hamaguchi et al., 2002; Paillard, 2004;

market supply through developing clam culture.

Park et al., 2006; Tsutsumi, 2006; Matsukawa et al.,A persistent problem, however, is insufficient food2008; Toba et al., 2013; Toba, 2017). Furthermore,

supply for juvenile clams exceeding a shell length ofa wide variety of studies have aimed at conserving

1 mm (especially in late fall and spring) since clams

the clam resource (Dang et al., 2010; Paul-Pont et al.,have high food requirements, and low temperature

2010; Shigeta and Usuki, 2012; Suzuki et al., 2012;

slows the growth of the diet microalgae. Therefore,

Usuki et al., 2012; Kobayashi et al., 2012; Ikushima

the development of new species of diet microalgae

et al., 2012; Sakurai et al., 2012; Hasegawa et al.,that can grow well outdoors at low water

2012; Nakagawa et al., 2012; Sakami and Higano,temperatures and that possess the essential nutritive

2012; Houki et al., 2015; Hanyu et al., 2017; Hata etconstituents will greatly benefit juvenile clamal., 2017; Houki et al., 2018). To conserve the clam

culture. Recently, Yamasaki et al. (2019) isolated theresource, however, promotion of efficiency andmarine euglenophyte Eutreptiella eupharyngea fromstabilization of seed production and juvenile clama pond used for extensive phytoplankton cultivation

culture are needed.

at the Yamaguchi Prefectural Fisheries Research

In this mini-review, new diet microalgae, aCenter (Yamaguchi, Japan) in January 2013. The

dietary-supplement, and original feeds using

study reported from the results of both laboratory

microencapsulation, enzymatic decomposition, andand outdoor experiments that E. eupharyngea could

fermentation technology with potential for thegrow well at water temperatures ranging from 4

enhancement of juvenile bivalve seeding production

to 25℃ , but could not grow at 30℃ . Furthermore,and culture techniques are reviewed.

Yamasaki et al. (2019) demonstrated that the dietary
effect of E. eupharyngea per dry weight, on juvenile

Dietary Effect of a New Diet Microalgae andR. philippinarum of more than 1.5 mm shell length,

Mixed Algal Diets on Juvenile Bivalves

exceeded that of the diatom Chaetoceros neogracile—a known suitable diet alga for juvenile clams. These

Microalgae are used as live feeds for larvalfindings were attributed to the high nutritional value

or juvenile bivalves, crustaceans and otherof E. eupharyngea as typified by its high protein andinvertebrates in addition to the rotifer Brachionus

sugar content and high content ratio of n-3 fatty

plicatilis, which is fed to larval fish. Thus, dietacids such as eicosapentaenoic and docosahexaenoic

microalgae should contain essential nutritiveacid and n-6 fatty acids such as arachidonic acid. In

constituents, be nontoxic, be appropriately sized

addition, E. eupharyngea (Fig. 1) appears to have afor ingestion, and possess digestible cell walls tostronger dietary effect on bigger clams (e.g., moreallow the nutrients to be absorbed after ingestion

than 1.5 mm in shell length) since E. eupharyngea(Becker, 2004). In addition, it is important that thecells (cell length: 35–70 µm; cell width: 7.5–12

microalgae can be produced in large quantities atµm, Walne et al., 1986) are bigger than other diet

low cost. So far, more than 40 species of microalgae

microalgae such as C. neogracile (cell size: <10 µm).

have been isolated and analyzed to produce better

Thus, E. eupharyngea shows considerable potential

aquaculture feeds, and these species are undergoing

to become a new diet alga for the seed production

cultivation as pure strains in intensive systems

and culture of juvenile bivalves in late fall and(Becker, 2004). However, there is no microalgae

spring.

that possesses all the necessary requirements of an

A multitude of studies suggest that mixed

ideal diet alga. Therefore, the development of suchmicroalgal diets may provide a better balance of

new diet microalgae could be crucially important foressential nutrients to bivalves. Rivero-Rodríguez

the enhancement of seed production and culture of

et al. (2007) analyzed the relative fatty acidFig. 1
Dietary effect of several new diets on juvenile bivalves

(A)

151

Pavlova lutheri, which have had higher levels ofpolyunsaturated fatty acids (PUFAs), was higher
than that of other combinations of diet microalgae.
However, Geng et al. (2016) examined the effects offour different microalgae, C. calcitrans, I. galbana, N.
oculata and Diacronema viridis, on the growth ofjuvenile ark shells (Tegillarca granosa Linnaeus),and demonstrated that the best feeding effects wereobserved with the mixture of all four microalgae,
and binary algal diets were second best. Recently,
Liu et al. (2016) evaluated the nutritional value ofeight species of microalgae for larvae and early(B)post-set juveniles of the Pacific geoduck Clam
(Panopea generosa), and reported that a balanced
mixture of various dietary nutrients was important.
In particular, they suggested that the ratios
between n-3 and n-6 fatty acids, and between EPA
and DHA, are especially crucial (Liu et al., 2016).
Polyunsaturated fatty acids (PUFAs), especially then-3 fatty acids eicosapentaenoic acid (EPA, C20 : 5n-3)
and docosahexaenoic acid (DHA, C22 : 6n-3) play animportant role in bivalve growth and development(Volkman amd Brown, 2005; Martínez-Fernández

Fig. 1. A scanning electron microscope(SEM) photograph (A) and a micrograph(B) of Eutreptiella eupharyngea. The
scale bars indicate 10 µm.

et al., 2006). Thus, the fatty acid composition of dietmicroalgae might be one of the most importantfactors for bivalve growth and development.
Nevertheless, content and/or composition of thesefatty acids could be affected by microalgal growth
rates, environmental conditions, and growth phases

composition of the diatoms Chaetoceros calcitrans,(Richmond, 1986). The main environmental factors

C. muelleri, and Phaeodactylum tricornutum, asaffecting the growth and the nutritive constituentswell as the haptophyte Isochrysis galbana and theof diet microalgae are light levels, nutrients,

prasinophyte Tetraselmis suecica, and reported thattemperature, pH, and salinity (Chu et al., 1996;

these microalgae contained a high proportion ofTzovenis et al., 1997; Zhu et al., 1997). The impact ofeither EPA (C20 : 5) or DHA (C22 : 6). Furthermore,increasing water temperature and ocean acidification

they examined the dietary contribution of theseshould also be considered.

five algal species when provided as mono- or bialgal diets to juveniles of the mangrove oyster

Enhancement of Dietary Effect on Juvenile Bivalves(Crassostrea corteziensis), and concluded that C.

by the Dietary Supplements

calcitrans provided the best diet, probably due to its
high AA (C20 : 4) content (Rivero-Rodríguez et al.,Jǿrgensen (1983) observed that clams take up2007). In addition, Ronquillo et al. (2012) examined

dissolved organic matter (DOM) in seawater throughthe effect of mixed microalgal diets on the growth

epidermal tissue in the mantle and gills. Welborn

and fatty acid profile of European flat oyster

and Manahan (1990) showed that larvae of the(Ostrea edulis) juveniles. The authors indicated thatbivalve Crassostrea gigas (Pacific oyster) can take up

dietary effect of the mixture of the eustigmatophyte

dissolved glucose, maltose, cellobiose, and cellotriose,

Nannochloropsis oculata and the haptophyte

but not rhamnose or maltotriose. In addition,

Yasuhiro YAMASAKI

152

Uchida et al. (2010) reported that the growth ratecompared to other diet microalgae. Accordingly,of soft tissue in R. philippinarum was significantly

we tried to enhance the dietary effect ofpromoted by supplementing a diet of the diatom

Nannochloropsis sp. on the shell length growth of

C. calcitrans with glucose at concentrations of 10

juvenile R. philippinarum by AHs supplementation.

and 100 mg L -1. Furthermore, Taga et al. (2013)

Yamasaki et al. (2018) reported that shell length andreported the high dietary effects of the raphidophyte

total weight of clams were significantly promoted

Heterosigma akashiwo (known as a harmful algal

by supplementing a diet of Nannochloropsis sp. at

species) on juvenile R. philippinarum and suggested

4-1
the concentration of 30×10 cells mL with AHs

that the acidic sugars found in microalgal cells

at the concentration of 4 mg L-1 as compared withmay be one of the important factors determining

the groups given Nannochloropsis sp. Thus, athe growth of juvenile clams. Thus, we focused on

combination of Nannochloropsis sp. and AHs will

alginate—a known acidic sugar.

be useful to shorten the rearing period of clams at

Alginate is a natural acidic linear polysaccharide

low cost since shell length growth of the clams fed

t h a t i s c o m p o s e d o f α- L - g u l u r o n a t e a n d β

Nannochloropsis spp. added with AHs were faster- D -mannuronate (uronic acids) residues. This

than that of clams fed more costly Chaetoceros spp.

carbohydrate occurs in the cell walls of brown algae,

Particulate organic matter (POM) appears tosuch as Saccharina japonica and Undaria pinnatifida.

contribute to improvements in the quality of

Alginate and its hydrolysates are currently used in abivalves. To improve the quality of the freshwater

wide range of commercial products because of their

Clam (Corbicula japonica) by rearing in a shortsafety, low price, and bioactivities. Since alginate isperiod, Nojiri et al. (2018) examined the effect ofpoorly soluble in water, we focused on the dietaryvarious carbohydrates on the increment of glycogeneffect of alginate hydrolysates which are watercontent, and of the hyperosmotic stress on thesoluble and thus usable by clams. Results showedamino-acid uptake. As a result, Nojiri et al. (2018)that shell-length growth of juvenile R. philippinarum

showed that rice powder was effective for increasing

was significantly promoted by supplementing a dietglycogen content at the concentration of 0.1g/L,

of C. neogracile with alginate-hydrolysates (AHs) ofand suggested that insoluble carbohydrate wasat least 1 mg L-1. The most effective concentrations

suitable for the increment of glycogen content in-1of AHs were 2 to 4 mg L , but shell length growth

the freshwater clam. Furthermore, they observedin groups given AHs only without C. neogracilethat glycine was most effectively absorbed in thewas significantly inhibited (Yamasaki et al., 2015). In

freshwater clam, followed by proline, alanine andaddition, Yamasaki et al. (2016) demonstrated thatglutamic acid under hyperosmotic stress for 24

growth of adult clams (initial average shell lengthh, and concluded that the quality of C. japonica[±SD], 15.7±0.3 mm) was dramatically promoted

could be improved in a short period by feeding riceby supplementing a diet of the diatom C. neogracile

powder and rearing in palatable amino acids underwith AHs at 4 mg L-1, and metabolomics indicatedosmotic stress.

that each of the states of starvation, food satiation,and sexual maturation of R. philippinarum has

New Approaches in the Development of Originala characteristic pattern in the metabolite profile

Feeds for Bivalves(Yamasaki et al., 2016).
A few kinds of Chaetoceros spp. are widely

There is a compelling need for the developmentused for clam culture, but the cost of cultivatingof alternative feeds to replace live microalgal diet

Chaetoceros spp. is expensive. Nannochloropsis spp.

because of the difficulty in stable production of dietare used for cultivation of a various marine molluscs,

microalgae at low cost and a deficiency of microalgae

crustaceans, and zooplankton and can be produced

that possesses all the necessary requirements forin large quantities at low cost (Zhang et al., 2001).

a diet alga. In this section, several new approaches

However, the dietary effect of Nannochloropsis

in the development of original feeds using

spp. on juvenile bivalves is not always sufficient as

microencapsulation, enzymatic decomposition andDietary effect of several new diets on juvenile bivalvesfermentation technology for bivalves are introduced.
Recently, several studies have suggested that153

on Japanese pearl oyster (Pinctada fucata martensii)
spat.

microcapsules, which can easily be produced inConclusionlarge quantities, have highly customizable physicalcharacteristics and contents, and are stable forlong term storage (Aldridge et al., 2006; Costa etResearch and development of new diets suchal., 2011). Thus, microcapsules have promise as

as diet microalgae, dietary supplements, andalternative feeds to replace a live microalgal diet.

original feeds using microencapsulation, enzymatic

Willer and Aldridge (2017) demonstrated that adecomposition and fermentation technology are

new form of microencapsulated diet known as

rapidly developing. In the near future, efficiency and

BioBullets (BioBullets Ltd., Cambridge, UK) canstabilization of seed production and juvenile clam

successfully be ingested by the blue mussel (Mytilus

culture may be achieved by a combination of new

edulis). Furthermore, Willer and Aldridge (2019)

diets and live algal diets. Therefore, these techniquesdemonstrated that the use of microencapsulated

may have important implications for clam culture,

feed, which were lipid-walled microcapsulesand could contribute to the conservation of the wildcontaining 50% powdered Schizochytrium algae

clam resources and a stable market supply. Further

by weight and manufactured by BioBullets

studies are needed to develop a process for practical(BioBullets Ltd.), can lead to major improvements inutilization of these techniques for seed production

survivorship and growth in juvenile European flatand juvenile clam culture.

oysters (Ostrea edulis).
Acknowledgement

Several studies have also suggested the viabilityof marine silage (MS) and single cell detritus (SCD;
Uchida and Murata, 2002; Pérez Camacho et al.,I thank Dr. Seth Theuerkauf and Dr. Clete

2004; Uchida et al., 2004). MS and SCD can easily

Otoshi of the National Oceanic and Atmosphericbe produced in large quantities, have a suitable size

Administration, and Dr. Takuro Shibuno of thefor ingestion by filter feeder such as bivalves, andJapan Fisheries Research and Education Agency forare stable for long term storage without remarkable

the revision of the manuscript.

loss of particulate products. Thus, MS and SCD
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Annotated Bibliography of Key Works(3) Yamasaki Y., Ishii K., Taga S., and KishiokaM., 2018: Enhancement of dietary effect of(1) Yamasaki Y., Taga S., and Kishioka M., 2015:

Nannochloropsis sp. on juvenile Ruditapes

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The eustigmatophyte Nannochloropsis sp. is
widely used in the aquaculture industry because

1013-1017.
Several studies have suggested that certain types

this species can be produced on a large scale at low

of sugars are potentially a good supplement forcost. However, Nannochloropsis sp. has less dietary

growth of bivalves such as Ruditapes philippinarum.

effect on juvenile bivalves compared with other diet

We observed the dietary effects of a harmful

algae such as the diatom Chaetoceros neogracile andraphidophyte Heterosigma akashiwo on juvenile

the haptophyte Diacronema (=Pavlova) lutheri. In

clams and suggested that the acidic sugars inthis study, the use of alginate-hydrolysates (AHs)

the phytoplankton might be an important factor

to enhance the dietary effect of Nannochloropsis

determining the shell length growth of clams

sp. on juvenile Ruditapes philippinarum (average

because total sugar and acidic sugar content of H.

shell length: 1,090 µm) was attempted. As a result,akashiwo were higher than other diet microalgae.

enhancement of the dietary effect on shell-length

Therefore, we focused on alginate known as one

growth of juvenile clams was observed in theof the acidic polysaccharides, and showed thatgroups given Nannochloropsis sp. (300,000 cells

shell-length growth of juvenile clams (averagemL ) with AHs at the concentration of 4 mg L .

shell length: 432 to 507 µm) was significantly

Hence, the enhanced dietary effect of a combination

promoted by supplementing the diatom Chaetocerosof Nannochloropsis sp. and AHs will be useful to-1

neogracile (40,000 to 80,000 cells mL ) with alginatehydrolysates (AHs) of at least the concentration of 1
mg/L. In addition, the most effective concentrations-1

shorten the rearing period of R. philippinarum.

-1

158

Yasuhiro YAMASAKI(4) Yamasaki Y., Ishii K., Hikihara R., Ishimaru M.,

Eutreptiella eupharyngea, which was isolated from a

Sato F., Taga S., Kishioka M., Matsunaga S., Shikata

pond used for extensive phytoplankton cultivation

T., Abe M., Kato S., Tanaka R., and Murase N.,at the Yamaguchi Prefectural Fisheries Research

2019: Usefulness of the euglenophyte Eutreptiella

Center (Yamaguchi, Japan) in January 2013. As aeupharyngea as a new diet alga for clam culture.

result, this species grew well at water temperatures

Algal Res., 40, 101493.

of 10–25℃ , but could not grow at 30℃. Furthermore,

Microalgae are an essential feed source forthe dietary effect of E. eupharyngea per dry weight

seed production of bivalves such as Ruditapeson juvenile R. philippinarum (average shell length:

philippinarum. However, there is a deficiency

1,426 µm) exceeded that of the diatom Chaetocerosof microalgae that can provide a stable supply

neogracile. These findings are attributable toof nutrient-rich feed at low water temperatures

the high nutritional value of E. eupharyngea as

during winter and spring. To develop a new diet of

typified by its high protein and sugar content

microalga that can grow well outdoors at low waterand high content ratio of n-3 fatty acids such astemperatures and possesses the essential nutritive

eicosapentaenoic and docosahexaenoic acid and n-6

constituents, we focused on the euglenophyte

fatty acids such as arachidonic acid.

水 研 機 構 研 報， 第 50 号，159 － 160， 令 和 ３ 年
Bull. Jap. Fish. Res. Edu. Agen. No. 50，159－160，2021

159

Development of a sustainable diet for Japanese white trevally
Pseudocaranx dentex juveniles

Jonas MILLER＊1, Shuhei TANAKA＊1, Hiroki KIHARA＊1, Shinichi YAMADA＊1,
Fumiaki TAKAKUWA＊1, Keitaro KATO＊2, Amal BISWAS＊1, and Hideki TANAKA＊1

Extended Abstractof supplemental crystalline amino acids, taurine,
phytase supplementation, and palatability enhancers

The Japanese white trevally Pseudocaranx dentex,
Shimaaji in Japanese, (Bloch & Schneider, 1801) isby developing an appropriate feed formulation thewhite trevally.

a highly prized carangid fish species for sushi andIn the aquaculture industry, the flavor attractant

sashimi that is distributed in tropical and temperate

krill meal (KM) is utilized as a high-level protein

waters around the world, except in the easternsource and palatability agent for juvenile marine fish.

Pacific region. There is an increased demand for

In order to formulate a low-cost and environmentally

the white trevally due in part to the increase insustainable diet for juvenile white trevally, it isartificially hatched fingerlings which are only

necessary to find a suitable replacement for KM

cultured in Japan. Overall, published studies on thethat serves as a high-quality protein source, has anutritional requirements of juvenile white trevallywell-balanced amino acid profile, and works well as

are limited and there is ongoing research beinga flavor attractant. Furthermore, it is necessary to

conducted to determine the most suitable protein

develop a feed formulation that completely replaces

sources for developing a practical diet.

FM in practical diets. Land animal by-products

In Japan and around the world, the primary

have potential as alternative feed ingredients incommodity utilized for protein in aquaculture feed

formulated diets for marine fish. One of the mostformulations is fish meal (FM), which is produced

noteworthy animal by-products is poultry by-productfrom the mass-catch of pelagic fish species. FM

meal (PBM), which consists of rendered waste

tends to have high cost oscillations, and its highmaterial generated from poultry slaughterhouses

level of inclusion in marine fish diets tends toand processing plants. PBM, which has a good amino

have negative consequences on the environment,acid balance and high protein content has yet to be

including phosphorus pollution from the effluent

tested in in practical SBM-based diets for juvenileof fish fed FM-based diets. Soybean meal (SBM) is

white trevally as a protein source and palatability

an alternative protein source that is a promising

agent.

candidate for FM replacement in juvenile whiteThree feeding trials conducted at the Aquaculturetrevally diets. In general, marine fish species tend

Research Institute of Kindai University from 2018

to exhibit varied sensitivity to SBM due to the―2020 demonstrated that high levels of SBM

presence of phytic acid and other antinutritional

replacement by FM in combination with palatability

factors, which can contribute to issues related toenhancers KM or PBM, indispensable amino

low palatability, further leading to poor growth due

acids Lys and Met, taurine, and phytase in theto deficiency of indispensable amino acids such as

diets provided high utility when fed to juvenile

methionine and lysine. However, the amino acid,

Japanese white trevally. While SBM has been

palatability and anti-nutritional factors associatedmostly considered as an inferior protein sourcewith SBM could be overcome with the addition

to FM when included in marine fish aquaculture
2020年12月11日受理（Accepted on December 11, 2020）＊1
Uragami Station, Aquaculture Research Institute, Kindai University, 468-3 Uragami, Nachi-katsuura, Higashimuro, Wakayama 649-5145, Japan＊2
Shirahama Station, Aquaculture Research Institute, Kindai University, 1-5 Shirahama, Nishimuro, Wakayama 649-2211, Japan
E-mail: hidektana “at” gmail.com

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Jonas MILLER, Shuhei TANAKA, Hiroki KIHARA, Shinichi YAMADA, Fumiaki TAKAKUWA,
Keitaro KATO, Amal BISWAS, and Hideki TANAKA

diets, the results of the three feeding trials revealed(2) Jirsa D., Davis A., Stuart K., and Drawbridge M.,

great potential for SBM as an FM replacer. It is2011: Development of a practical soy-based diet forevident that Japanese white trevally is an excellent

California yellowtail, Seriola lalandi. Aquac. Nutr.,

candidate for alternative protein sources derived

17, e869-e874.

from plants including SBM. The diets tested

This study was carried out in an effort to develop

provided high survival and adequate growth rates

an environmentally sustainable and cost-effective

to warrant further development of practical diets

diet for California yellowtail juveniles using soy

for this species. Fish fed diets containing higher

products to replace fish meal. This research usedlevels of SBM had a lower environmental impact

a similar approach in terms of feed formulation,

than fish fed with FM-based diets, which highlights

specifically investigating the utility of soybean meal

the ecological benefits of utilizing alternative protein

and soy protein concentrate. Interestingly, fish fed

sources. It is recommended that future research

diets containing soybean meal outperformed fish fed

focuses on determining a mechanism to understandsoy protein concentrate, which further influenced

why juvenile white trevally achieve better growth

our choice of testing the utility of conventional

performance with higher levels of SBM than FM

soybean meal on white trevally juveniles. Thisin the diet. The results from the three feeding

research was conducted in the United States.

trials mentioned above will be described in future
publication.

(3) Kader Md. A., Bulbul M., Koshio S., Ishikawa
M., Yokoyama S., Nguyen B.T., and Komilus C. F.,

Annotated Bibliography of Key Works

2012: Effect of complete replacement of fishmeal
by dehulled soybean meal with crude attractants(1) Biswas A., Araki H., Sakata T., Nakamori T., Kato

supplementation in diets for red sea bream, Pagrus

K., and Takii K., 2017: Fish meal replacement by soy

major. Aquaculture, 350-353, 109-116.

protein from soymilk in the diets of red sea bream(Pagrus major). Aquac. Nutr., 23, 1379-1389.

The authors found that in red sea breamjuveniles, nutrient utilization, body composition and

The results of this study introduce the ecologicalblood parameters were improved or not significantly

benefits of replacing FM by soy-based products inaffected by replacing fish meal with soybean meal injuvenile red sea bream, such as reduced phosphorus

combination with flavor attractant supplementation.

discharge to the environment. This feeding

Growth performance results from this experimentexperiment was conducted at the Aquaculture

express a similar trend to the results presented

Research Institute of Kindai University, Japan using

in our abstract, in which soybean meal diets

the same methods that were utilized in the researchoutperformed the fish meal control diets in white

presented in our extended abstract.

trevally. This research was conducted in Japan.