46th Scientific Symposium of the UJNR
Aquaculture Panel
Marine Aquaculture in a Changing Environment
Hyatt Place Mystic
224 Greenmanville Ave
Mystic, CT
November 14th and 15th, 2018

46th Scientific Symposium of the UJNR Aquaculture Panel
Marine Aquaculture in a Changing Environment
November 14th and 15th, 2018
Hyatt Place Mystic
224 Greenmanville Ave
Mystic, CT
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 ofboth nations.
The 46th UJNR Aquaculture Symposium is the second year of the current three-year theme ofMarine Aquaculture in a Changing Environment. Environmental change impacts aquaculture inmany ways. Nutrient pollution is driving eutrophication and dead zones, ocean acidification ischanging water chemistry and climate change is already influencing our food supply, fresh water
availability, weather and way of life. Aquaculture will be impacted by, and can also impact,these environmental changes over various scales. Aquaculture of finfish, shellfish and seaweed
have different threats, benefits and opportunities related to environmental change. This year’s
presentations focus on impacts to aquaculture production due to environmental change (e.g.,
ocean acidification impacts on shellfish aquaculture) and science to mitigate these impacts(counter measures).

ProgramWednesday, November 14, 2018
Registration

13:00 - 13:30

Welcome and Aim of the Symposium
Michael Rust, US Panel Chair, NOAA Fisheries Office of Aquaculture

13:30-13:45

Production Technologies(Moderators: Simona Augyte and Masakazu Hori)
Distribution patterns of the cysts of the paralytic shellfish poisoning plankton Alexandriumtamarense and A. catenella off the Pacific coast of eastern Japan

Tomoko Sakami, National Research Institute of Aquaculture
Fisheries Research and Education Agency

13:45-14:10

Update on the progress of the scallop farming industry in Maine
Dana Morse, Maine Sea Grant College Program

14:10-14:35

Trends in aquaculture production in Japan
Satoshi Watanabe, National Research Institute of Aquaculture
Fisheries Research and Education Agency

14:35-15:00

Establishment of oyster tetraploid breeding stocks for the fast-growing oyster industry in
the Gulf region
Huiping Yang, School of Forest Resources and Conservation Institute of Food and Agricultural
Sciences, University of Florida
15:00-15:25
Development of environmentally friendly, cost-effective and nutritionally balanced
alternative protein based diets for high value marine fish culture in recirculating
aquaculture system
Md Shah Alam, Center for Marine Science, Aquaculture Program, University of North Carolina
Wilmington
15:25-15:50
Symposium Reception: One on one discussions continue
Hyatt Place Mystic

16:00-18:00

Thurssday, November 15, 2018
Temperature Resilience(Moderators: Dana Morse and Tomoko Sakami)
Seaweed aquaculture in a warming environment - building resilience with Saccharina
angustissima (Laminariales, Phaeophyceae)
Simona Augyte, Department of Ecology and Evolutionary Biology,
University of Connecticut
9:00-9:25
On the application of bio-stimulants to enhance growth and thermal resistance of sugar
kelp, Saccharina angustissima, for aquaculture production
Schery Umanzor, Department of Ecology and Evolutionary Biology,
University of Connecticut
9:25-9:50
Vibrio coralliilyticus induction of virulence toward larval oysters and corals at elevatedseawater temperatures and potential mitigation treatments

Gary Richards, Agricultural Research Service,
United States Department of Agriculture

9:50-10:15

Break

10:15-10:30
Ocean Acidification(Moderators: Schery Umanzor and Satoshi Watanabe)

Chemical changes in the environment: what does this mean to shellfish?
Shannon Meseck, Northeast Fisheries Science Center Milford Lab, NOAA

10:30-10:55

The current trends in pH and ocean acidification in an aquaculture sea area in Gokasho
Bay, Japan
Masayuki Minakawa, National Research Institute of Aquaculture,
Fisheries Research and Education Agency
10:55-11:20
Ocean acidification experimentation in seagrass-oyster ecosystems using a novel system toconduct in-situ CO2 enrichment
Masakazu Hori, National Research Institute of Aquaculture,
Fisheries Research and Education Agency
11:20-11:45
Effects of ocean acidification on the early developmental stages of the commercially
important gastropods, Ezo abalone and horned turban, in JapanRyo Kimura, Fisheries Research and Education Agency, Headquarters
11:45-12:10
Lunch Break

12:10-13:10
Responses to Environmental Impacts(Moderators: Shannon Meseck and Masayuki Minakawa)

A slow growing perspective on multi-generational responses to future change
Coleen Suckling, Animal and Veterinary Sciences, University of Rhode Island
13:10-13:35
The influence of climate and environment on the spawning, condition, and larval set of
naturalized Pacific oyster Crassostrea gigas in a US west coast estuary
Brett Dumbauld, Agricultural Research Service,
United States Department of Agriculture
13:35-14:00
The influences of environmental changes on Japanese Nori mariculture
Mahiko Abe, National Fisheries University,
Fisheries Research and Education Agency

14:00-14:25

The potential of seaweed aquaculture to reduce methane emissions in Californian livestock
Luke Gardner, University of California, San Diego,
California Sea Grant
14:25-14:50
Break

14:50-15:05
Ecological Interactions(Moderators: Brett Dumbauld and Ryo Kimura)

Condition index and fecundity of Manila (Asari) clam Ruditapes philippinarum related tohabitat environment
Natsuki Hasegawa, National Research Institute of Aquaculture
Fisheries Research and Education Agency
15:05-15:30
Ecological interactions of Horseshoe crabs and shellfish aquaculture: a case study fromNew Jersey
Daphne Munroe, Haskin Shellfish Research Laboratory, Rutgers University
15:30-15:55
Use of GoPro cameras to document fish on aquaculture gear
Gillian Phillips, Northeast Fisheries Science Center Milford Lab, NOAA

15:55-16:20

Science Symposium Closing
Fuminari Ito, Japan Panel Chair, Fisheries Research and Education Agency

16:20-16:30

List of ParticipantsAbe, MahikoAlam, Md ShahAugyte, Simona
Cowperthwaite, Hugh
Dumbauld, BrettEddy, Steve
Gardner, Luke
Hasegawa, Natsuki
Hori, Masakazu
Ito, Fuminari
Kimura, Ryo
Meseck, Shannon
Minakawa, Masayuki
Morse, Dana
Munroe, Daphne
Okumura, Takuji
Olin, Paul
Phillips, Gillian
Rexroad, Caird
Richards, Gary
Rust, Mike
Sakami, Tomoko
Takano, Masatsugu
Umanzor, Schery
Watanabe, Satoshi
Yang, Huiping
Yarish, Charles

Japan Fisheries Research and Education Agency (FRA),
National Fisheries University
North Carolina State University
University of Connecticut (UCONN)
Coastal Enterprises Incorporated
United States Department of Agriculture (USDA),
Agricultural Research Service (ARS)
University of Maine
California Sea Grant
FRA, National Research Institute of Aquaculture (NRIA)
FRA, National Research Institute of Fisheries andEnvironment of Inland Sea
Japan Panel Chair, FRA, NRIAFRA Headquarters
NOAA Northeast Fisheries Science Center Milford Lab
FRA, NRIA
Maine Sea Grant
Rutgers University
FRA, NRIA
California Sea Grant
NOAA Northeast Fisheries Science Center Milford Lab
USDA, ARS
USDA, ARSUS Panel Chair, NOAA Headquarters
FRA, NRIAFRA Headquarters
UCONN
FRA, NRIA
University of Florida
UCONN

46th Scientific Symposium of the UJNR Aquaculture Panel

Aquaculture in a Changing Environment
Symposium Abstracts
1. Distribution patterns of the cysts of the paralytic shellfish poisoning plankton Alexandriumtamarense and A. catenella off the Pacific coast of eastern Japan
Tomoko Sakami*1, Tetsuroh Ishikawa2, and Toru Udagawa3
Presenting author*
1*

National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency,
422-1 Nakatsuhamaura, Minami-ise, Mie, 516-0193, Japan, *Email: sakami at affrc.go.jp
2
Miyagi Prefectural Government, 3-8-1, Honcho, Aoba-ku, Sendai, Miyagi, 980-8570, Japan
3
National Research Institute of Fisheries Engineering, Japan Fisheries Research and Education
Agency, 7620-7, Hasaki, Kamisu, Ibaraki, 314-040, Japan
Abstract
Shellfish poisoning occurs as bivalves consume poisonous phytoplankton and the poison
accumulates in their tissues. Monitoring systems for bivalve products have improved in Japan,
and food poisoning damage has rarely occurred in recent years. However, contaminated shellfish
is detected nearly every year and presents a notable challenge to fishermen who have tovoluntarily regulate their shipments. Two dinoflagellate plankton species of the genus
Alexandrium mainly cause paralytic shellfish poisoning in Japan. Highly toxic A. tamarense isfound in low-temperature waters, whereas less toxic A. catenella is found in warmer waters.
Both species produce cysts that settle at the bottom of the sea and become the population source
for the following years. A cyst distribution survey was conducted to evaluate the risk of shellfish
poisoning. Cyst counting can be done efficiently using a fluorescent microscope; however, visual
distinction of the cysts of the two species is difficult because of their similarity in shape. A
molecular biological method has been developed to distinguish between the species. In thisstudy, we aimed to determine the difference in the distribution of the cysts between the two
species by detecting genes from the sea floor sediments using quantitative PCR. We estimatedthe gene copy numbers of A. tamarense and A. catenella in the sediment collected off the eastern
Pacific coast of Japan. Both species were detected in some ria-type inner bays in the Sanriku
region. However, only A. tamarense was detected from Sendai Bay to Kashima Nada, which
open widely to the Pacific Ocean. In addition, only A. catenella was detected south of Cape
Inubo, which is strongly influenced by the Kuroshio current. These results suggest that the
distribution patterns of the two toxic phytoplankters differ depending on topography and theocean current system.

Annotated Bibliography of Key Works
Nagai, S. 2011. Development of a multiplex PCR assay for simultaneous detection of six
Alexandrium species (Dinophyceae). J. Phycol. 47, 703–708.
In Japan, the blooming seasons of two toxic dinoflagellate species, Alexandrium catenella(Whedon et Kof.) Balech and A. tamiyavanichii Balech, sometimes overlap with those of three
nontoxic Alexandrium species, A. affine (H. Inouye et Fukuyo) Balech, A. fraterculus (Balech)
Balech, and A. pseudogoniaulax (Biecheler) T. Horig. ex Y. Kita et Fukuyo. In this study, amultiplex PCR assay was developed to enable simultaneous detection of six Alexandrium specieson the basis of differences in the lengths of the PCR products. The accuracy of the multiplex
PCR system was assessed using 101 DNA templates from the six target Alexandrium species and
27 DNA templates from 11 non-target species (128 DNA templates in total). All amplicons
obtained from the 101 DNA templates of the target species were appropriately identified,
whereas all 27 DNA templates of the non-target species were not amplified. Species-specificidentification by the multiplex PCR assay was possible from single cells of the target species.
Natsuike, M., Yokoyama, K., Nishitani, G., Yamada, Y., Yoshinaga, I., Ishikawa, A. 2017.
Germination fluctuation of toxic Alexandrium fundyense and A. pacificum cysts and therelationship with bloom occurrences in Kesennuma Bay, Japan. Harmful Algae 62, 52-59.
While cyst germination may be an important factor for the initiation of harmful/toxic algal
blooms, in situ assessments of the fluctuation in the occurrence of phytoplankton cyst
germination from the bottom sediments to the water column are rare due to lack of technology todetect germinated cells in natural bottom sediments. This study introduces a simple mesocosm
method, modeled after previous in situ methods, to measure the germination of plankton restingstage cells. Using this method, seasonal changes in germination fluxes of toxic dinoflagellate
resting cysts, specifically Alexandrium fundyense (A. tamarense species complex Group I) and A.
pacificum (A. tamarense species complex Group IV), were investigated at a fixed station in
Kesennuma Bay, northeast Japan, from April 2014 to April 2015. This investigation was
conducted in addition to the typical samplings of seawater and bottom sediments to detect thedinoflagellate vegetative cells and resting cysts. Bloom occurrences of A. fundyense wereobserved in June 2014 and February 2015 with maximum cell densities reaching 3.6×106 cells m2
and 1.4×107 cells m-2, respectively. The maximum germination fluxes of A. fundyense cysts
occurred in April 2014 and December 2014 and were 9.3×103 cells m-2day-1 and 1.4×104 cells m2
day-1, respectively. For A. pacificum, the highest cell density was 7.3×107 cells m-2 during themonth of August, and the maximum germination fluxes occurred in July and August, reaching
5.8×102 cells m-2day-1. Thus, this study revealed the seasonal dynamics of A. fundyense and A.
pacificum cyst germination and their bloom occurrences in the water column. Blooms occurred
one to two months after peak germination, which strongly suggests that both the formation of theinitial population by cyst germination and its continuous growth in the water column most likely
contribute to toxic bloom occurrences of A. fundyense and A. pacificum in the bay.
Ishikawa,T., Kusaka, K., Oshino, A., Nishitani, G., Kamiyama, T. 2015, Distribution patterns of
Alexandrium vegetative cells and resting cysts, and paralytic shellfish poisoning in Kesennuma
Bay, after the Great East Japan Earthquake. Nippon Suisan Gakkaishi 81(2), 256-266.
In 2013, paralytic shellfish poisoning (PSP) occurred in Kesennuma Bay, northern Japan, for the
first time in 24 years. From April to early May, Alexandrium tamarense increased in the inner partof the bay (up to 136,200 cells/L), expanded to the whole bay, and caused PSP. In contrast, A.

catenella increased in early September (up to 1,310 cells/L) in the center of the bay but did notcause PSP. Resting cysts of A. catenella/tamarense ranged from 30 to 6,383 cysts/cm3 anddominated the inner part of the bay. A meta-analysis of temporal trends in the density of A.
catenella/tamarense cysts in Kesennuma Bay revealed that the cysts in the surface bottom
sediment drastically increased after the Great East Japan Earthquake. High PSP toxicity wasobserved in four bivalves: Chlamys farreri (130 MU/g), Mytilus galloprovincialis (110 MU/g),
Ruditapes philippinarum (74 MU/g), and Mizuhopecten yessoensis (66 MU/g).

2. Update on the progress of the scallop farming industry in Maine
Hugh Cowperthwaite1 and Dana L. Morse*2
Presenting author*
1

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

Abstract
Climate change has profoundly impacted capture fisheries in the Gulf of Maine. Notably, thelargest fishery in the United States is in flux: landings of lobster (Homarus americanus) haveshifted northeast in recent years. These challenges - not unique to Maine and are experienced by
the Japanese fisheries - have caused great uncertainty among fishermen in Maine. Consequently,
some are turning to aquaculture, to compliment fishing incomes. Maine’s fishing andaquaculture industries have benefited from exchanges with Japan’s well-established scallop
industry, and we continue to learn directly from the Japanese.
Coastal Enterprises Inc. (CEI), Maine Sea Grant (MESG) and others are working to introduce
commercially-viable scallop aquaculture into the state of Maine, building on a 20-year-old
foundation of expertise. Maine's sister-state relationship with Aomori Prefecture is central to thisprocess, with an origin dating to the wreck of the Bath-built Chesborough, which foundered off
the coast of Aomori in 1889.
Trips to Aomori in 1999, 2010 and 2016 have focused on many aspects of scallop culture andfisheries; spat collection, nursery and growout techniques, product development, andenvironmental interactions (Beal et al 1999).
Currently, several projects are being carried out in Maine, developing the culture sector for
scallops in coastal waters. An aquaculture cooperative in Maine focused on scallops is now inplace. Optimization of spat collection has been ongoing work over several years. Biotoxin
monitoring in scallop tissues enables some producers to access the live market. A dedicatedlicense for scallop spat collection is underway. Altogether, fishermen, farmers, economic
development groups, scientists and regulators are engaged, in pursuit of the developing Maine'sscallop aquaculture industry. These collaborations - combined with expertise from Japanesecolleagues - is a powerful aggregation of expertise to solve the many problems involved.
Ex-vessel value for Maine scallops has risen for over a decade, though state and federal landings
have increased, reflecting the value that consumers place on Maine scallops specifically; and thetrend is expected to continue (Hale Group 2016). Trials by CEI and collaborators indicates that
the Japanese production machinery appears to work well, and growth rates from ongoing studies
and older works appear to fall within acceptable limits, often in excess of 25mm/year shell height(Cowperthwaite, unpublished; Morse, unpublished, Davidson et al 2014, Pottle and Hastings2001). Lastly, all indications point to a growout period from spat (seed) to commercially viable
harvest size between two and three years.
Aquaculture practitioners in Maine desire to incorporate Japanese expertise, so the industry canramp up effectively and provide new sources of protein. We seek ongoing advice with lease site
set-up and husbandry, growout techniques, biofouling and predators, equipment and machinery,handling, processing, food safety and packaging.
This innovative and unique project offers a one-of-a-kind exchange experience that is helping tofurther strengthen ties between the United States and Japan. The introduction of Japanesetechnical knowledge and equipment to our Maine scallop industry is the first of its kind in the
United States and is groundbreaking for the American aquaculture community.
Annotated Bibliography of Key Works
Beal, B.F., M. Hastings, W. Hopkins, S. Inches, D. Morse, K. Porter, T. Pottle, S. Rappaport, L.
Taylor, J. Trenholm. 1999. The culture of Japanese scallops (Patinopecten yessoensis). Reportof the Maine delegation to Aomori Prefecture, Japan, May 14-21, 1999. The Maine Aquaculture
Innovation Center, 49p.
Beal et al summarizes the delegation from Maine to Aomori in 1999, which focused principallyon spat collection. This trip was important in advancing the tech-transfer process and getting
fishermen in Maine involved in culture-related issues on scallops in state waters.
Davidson, L-A., R. Nowlan and M. Niles. 2014. Open-ocean sea scallop (Placopecten
magellanicus) culture trials in Chaleur Bay, Canada: Comparing culture gears and husbandry
practices. Can. Tech. Rep. Fish. Aq. Sci. #3121. 26p.
Davidson et al reviews culture techniques in northern regions of North America; these are allapplicable to scallop production in Maine state waters. Canadian researchers, producers andthose in the regulatory/equipment communities have all contributed substantially to theunderstanding and progress by Maine participants.
Hale Group, 2016. Farmed Shellfish Market Analysis. A report to the Gulf of Maine ResearchInstitute, Portland, Maine. 65p.
The Hale Group report is important and current information in understanding the market
possibilities for cultured scallops in Maine.
Pottle, T., and M. Hastings. 2001. Sea Scallop Demonstration Project. Final Programmatic
Report Submitted to National Fish and Wildlife Foundation. Maine Aquaculture Innovation
Center, Orono, ME. 10p.

This project run by fisherman Tom Pottle was important to establish some growth rates,
equipment use, siting and leasing issues and access to the market for whole scallops; a model
which is still in use today.
http://www.mainebiz.biz/article/20161003/NEWS01/161009997/maine-fisheries-experts-headto-japan-to-learn-scallop-practices-buy-machinery
This news piece summarizes the nationally-groundbreaking work by Coastal Enterprises Inc. inscallop production, and some of the relevant partnerships involved.

3. Trends in Aquaculture Production in Japan
Satoshi Watanabe* and Tomoko Sakami
Presenting author*
National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency,
422-1 Nakatsuhamaura, Minamiise, Mie, 516-0193, Japan, *E-mail: swat at affrc.go.jp
Abstract
A wide variety of aquatic organisms are commercially cultured in Japan. For marine species, there
are national statistical data published by the Ministry of Agriculture, Forestry and Fisheries on theaquaculture production of seven species of finfishes (Oncorhynchus kisutch, Seriola spp.,
Trachurus japonicus, Pseudocaranx dentex, Pagrus major, Paralichthys olivaceus,
Tetraodontidae spp., Thunnus orientalis), two species of bivalves (Mizuhopecten yessoensis andCrassostrea spp.), one species of prawn (Marsupenaeus japonicus), one species of tunicate(Halocynthia roretzi), and four species of aquatic plants (Saccharina spp., Undaria spp., Pyropia
spp. and Nemacystus spp.). Four freshwater species of finfishes (Oncorhynchus mykiss,
Plecoglossus altivelis, Cyprinus carpio and Anguilla japonica) are also included in the statistics.
There are also miscellaneous groups and some species have been deleted from the census due tobudgetary constraints of the related agencies. Despite the historically strong affinity of theJapanese to seafood, production of aquaculture and capture fisheries are on a long-term moderatedecreasing trend largely due to socio-economic factors in Japan. The total marine aquaculture
production decreased from 1.28 × 106 t in 1996 to 1.03 × 106 t in 2016. Bivalve aquaculture
production was relatively constant around 450 × 103 t. The production of Crassostrea spp.
(oysters) and M. yessoensis (common scallop) decreased substantially due to the Great East Japan
Earthquake in 2011, and harvest has not recovered to the level prior to the earthquake. Finfish andaquatic plant productions are on a continuous decreasing trend. Finfish production was notseverely affected by the earthquake, except for O. kisutch (Coho salmon), production of which has
quickly recovered. Although the production of some aquatic plants such as Saccharina spp, (kelps),
Undaria spp. (wakame), Pyropia spp (laver or nori) was devastated by the earthquake not only in
the Tohoku area but many other parts of Japan, the long-term decreasing trend is not attributed tothe earthquake. One of the causes for the production declines is considered to be the reduced
number of management bodies due to aging of the operators. The number of management bodies
for nori culture, for instance, decreased from 51,354 in 1963 to 3,819 in 2013. Merger of the
management bodies is one of the reasons for the decrease, but the number of farmers is also

decreasing. On the other hand, aquaculture production of finfish and bivalves is increasing in value
in recent years. The increase in finfish production is attributable to new inclusion of T. orientalis(Pacific bluefin tuna), which entered the major product species list in 2012. For bivalves,production of the common scallop leaped from JPY 25.7×109 in 2012 to JPY 62.4 ×109 in 2016.
In freshwater aquaculture, A. japonica (eel) has by far the largest production in volume and value.
The seeds for eel aquaculture are all wild caught, however, and the low availability of glass eel
has been problematic in recent years. Prompt establishment of mass production technologies foreel seeds is desired.
Annotated Bibliography of Key Workse-Stat, statistics of Japan (https://www.e-stat.go.jp/en)
The statistical data presented in this paper are available on line; however, please note that not alldata are available in English.
Demura, M. 2010. Trend in fishmeal price and its effects on aquaculture. Norinkinyu 2010.10, 4549. (in Japanese, https://www.nochuri.co.jp/report/pdf/n1010jo1.pdf)
Despite the increasing global demand on aquaculture products, aquaculture business management
is not necessarily sustainable in Japan. Some aquaculture companies producing pufferfish and red
seabream went bankrupt in 2010 due to low fish price and increasing feed price. The author
analyzed the supply and demand and price change trends of fishmeal and discussed their effects
on aquaculture business in Japan. The author urged the importance of counter measures for
insufficient fishmeal supply, such as use of unutilized fish and development of low fishmeal feed.

4. Establishment of oyster tetraploid breeding stocks for the fast-growing oyster industry in the
Gulf region
Huiping Yang
School of Forest Resources and Conservation, Institute of Food and Agricultural Sciences,
University of Florida, Gainesville, FL 32653, USA, Email: huipingyang at ufl.edu
Abstract
In the Gulf region, oyster farming is rapidly increasing in recent years to meet the seafoodmarket needs due to the decline of fishery landing since 2012. Based on the official registration
information from the Florida Department of Agriculture and Consumer Services, a total of 180
leases (about 413 acres) are now farming oysters in 2017. For oyster farming industry, triploids
are recognized as the preferred products because of their fast-growth, better meat quality, andyear-round harvest. With the fast-growing of oyster farming industry, the demands of oyster
triploid seed are growing rapidly. Seed shortage has occurred in the past several years and isbecoming major constraint to the industry. To address the demand of triploid oyster seed,establishment of tetraploid breeding stocks is needed because tetraploids are the key forcommercial all-triploid seed production. With support from the Gulf States Marine FisheriesCommission, one project focusing on establishment of tetraploid breeding stocks was initiated in2017. In Year 1, chemically-induced triploids were produced by using Florida wild populations.
In Year 2, triploid individuals were screened and identified, and the triploid females were usedfor induction of tetraploid founders by using the ever-patented technique (which is to fertilize theeggs from triploids with sperm from diploids following by inhibition the first polar bodyrelease). Currently, this project is ongoing, and data collected in 2017 and 2018 are in process ofanalysis. It is expected that establishment of tetraploid breeding stocks will be useful for triploidseed production with diversified genetic resources to support the oyster industry.
Annotated Bibliography of Key Works
Tetraploid production for all-triploid seed production
1) Guo, X. & Allen, S. 1994. Viable tetraploids in the Pacific oyster (Crassostrea gigas
Thunberg) produced by inhibiting polar body I in eggs from triploids. Molecular Marine
Biology and Biotechnology, 3, 42-50.
2) Guo, X., DeBrosse, G. A. & Allen, S. K. 1996. All-triploid Pacific oysters Crassostrea gigas(Thunberg) produced by mating tetraploids and diploids. Aquaculture, 142, 149-161.
Tetraploid production has been a challenge in shellfish due to their poor survival to spat andadult stage although there have been huge efforts in this research topic since 1980s. The
publication in 1994 by Guo and Allen reported an unique methodology for tetraploid induction in
the Pacific oyster, and the tetraploids survived to adult stage and were used for all-triploid seedproduction by crossing with normal diploids (Guo et al., 1996). The innovative method reported
for viable tetraploids spat and adult production is to use oocytes from triploid females to fertilize
with haploid sperm and followed by inhibition of the first polar body (PB 1) (Guo and Allen
1994). Later, this method has been applied to other oyster species and led the production of
viable tetraploids in the Pearl oyster Pinctada. margaritifera, eastern oyster Crassostrea
virginica, and Somonie oysters Crassostrea ariakensis (He et al. 2000, Guo et al. 2002, Allen etal. 2005). This unique methodology has been patented by Rutgers University (US5824841A) andadopted by a private company named 4Cs Breeding Technologies, Inc. (4Cs,
www.4cshellfish.com). So far, tetraploid breeding stocks have been successfully establishedmostly in oyster species (including Pacific oysters, eastern oysters, Sydney rock oysters, andSuminoe oysters) by using this method and applied for commercial triploid seed production.
Eudeline, B., Allen, S. K., & Guo, X. M. (2000). Optimization of tetraploid induction in Pacificoysters, Crassostrea gigas, using first polar body as a natural indicator. Aquaculture, 187(1-2),
73-84. doi:Doi 10.1016/S0044-8486(00)00302-1
The publication modified the method for tetraploid induction (Guo and Allen, 1994) by using
oocytes from individual females, thus the fertilized eggs would develop with a more
synchronized way to allow polar body inhibition effectively to increase tetraploid occurrence.
This is an important improvement considering the difficulty to find triploid females and the few
oocytes from triploid females.
McCombie, H., Ledu, C., Phelipot, P., Lapegue, S., Boudry, P. & Gerard, A. 2005. A
complementary method for production of tetraploid Crassostrea gigas using crosses between
diploids and tetraploids with cytochalasin B treatments. Marine Biotechnology, 7, 318-33.
Complementarily, another novel approach has been reported in the Pacific oyster by using
tetraploid founders (McCombie et al. 2005) to produce viable tetraploids by fertilizing oocytes
from diploids with sperm from tetraploids followed by inhibition of the polar body 2 (PB2). Thismethod offers an alternative way for producing more tetraploids after production of initialfounders and avoids the use of oocytes from triploid females which is always a big challenge.

This is an approach to diversify, change, and enrich the genetic background of tetraploid
populations (50% from mother in the 1st generation, and probably increase to 100% from motherin the second generation depending on the chromosome segregation in sperm formation).
Guo, X., Wang, Y., Xu, Z. & Yang, H. 2009. Chromosome Set Manipulation in Shellfish. Pages
165-194 in G. Burnell & G. Allen editors. New Technologies in Aquaculture: Improving
Production Efficiency, Quality and Environmental Management. Woodhead Publishing Limited,
Abington, Cambridge.
This book chapter reviewed systematically the shellfish polyploid breeding, including triploidand tetraploid production, concepts, mechanism, history, updates, and future application on
aquaculture industry. In this publication, two comprehensive tables are included to summarize
the triploid production, growth, and performances, and the tetraploid induction and breeding. In
addition, there are several figures included in this review to describe the chromosome
segregation mechanism for triploid, tetraploid and aneuploid production.

5. Development of environmentally friendly, cost-effective and nutritionally balanced
alternative protein based diets for high value marine fish culture in recirculating aquaculture
systemM.S. Alam*, W. O. Watanabe and P.C. Carroll
Presenting author*
University of North Carolina Wilmington, Center for Marine Science, Aquaculture Program, 601
S. College Rd., Wilmington, NC, 28403, USA, *Email: alamm at uncw.edu
Abstract
With very little domestically grown seafood, along with increasing exploitation of wild stocks,
the US seafood trade deficit now exceeds $14 billion. Economics, environmental issues and fish
feed are important factors vital to the sustainability of the aquaculture industry in both the US
and Japan. These factors increase the pressure on aquaculturists to develop eco-friendly andsustainable aquaculture practices. One of the challenging tasks remaining in the aquacultureindustry is how to prevent the pollution of water by artificial feed. Fish meal is the main protein
source used in aquafeed that made from small pelagic species of forage fish. Two nutrients infish meal that have a great impact on environment are nitrogen and phosphorus and greater
amounts of phosphorus and nitrogen result in eutrophication of farm areas. Fish meal could
contain polychlorinated biphenyls (PCBs), dioxins, and other harmful chemicals and becomes avector of contamination in farm-raised finfish. Alternate protein sources such as terrestrial
animal and plants protein source can reduce the amount of wild fish used as protein, andpotentially reduce the nutrient levels in effluent waste. One of the goals of our research is todevelop sustainable marine finfish feed with less inclusion of fish meal in recirculating
aquaculture system (RAS) that are widely considered to be “environmentally friendly”.
At the University of North Carolina Wilmington-Center for Marine Science, a series ofexperiments were conducted to test the effects of different dietary levels of soybean meal, ultralow gossypol based glandless cottonseed meal, poultry meal in the diet of southern flounder, redporgy and black sea bass. Fish (juveniles, pre adults and grow-out stages) were fed therespective test diets in aquaria, semi-pilot and commercial scale RAS systems for 8-10 weeks, 4
and 10 months, respectively. The growth performance, feed utilization, digestibility andbiochemical composition of fish tissues were evaluated.
Results suggests that about 70, 40 and 35% fish meal protein could be replaced by soybean mealand 80, 100 and 70% of fish meal could be replaced by poultry meal in the diets of black sea
bass, southern flounder and red porgy, respectively. Results also suggest that fish meal could be
replaced up to 100 and 75% by glandless cottonseed meal in black sea bass and southern
flounder diets, respectively. The digestibility, proximate composition, amino acid and fatty acidcomposition of fish tissues will be presented. These findings could be used to develop
environmentally-sound and cost-effective pre-dominantly alternative protein based diets for highvalue marine fish culture in USA and Japan.
My 8 years higher education and research on aquaculture in Japan (Masters, doctoral and JSPS
post-doctoral research in Kagoshima University 1997-2005) and my research at University ofNorth Carolina Wilmington, USA (from 2005 to date) inspired me to propose collaborative
research between Japan and USA to reduce fish meal by alternative protein sources. Professor
Dr. Yutaka Haga, Lab of Fish Nutrition, Tokyo University of Marine Science and Technology,
Japan expressed his interest for collaborative research between our institutions in this topic area.
Annotated Bibliography of Key Works
Alam, M.S., Watanabe, W.O., Carroll, P.M., Gabel, J.E., Corum, M.A., Seaton, P., Wedegaertner,
T.C., Rathore, K.S., Dowd, M.K. 2018. Evaluation of genetically-improved (glandless) andgenetically-modified low-gossypol cottonseed meal as alternative protein sources in the diet ofjuvenile southern flounder Paralichthys lethostigma reared in a recirculating aquaculture system.
Aquaculture, 489: 36–45.
One of the biggest operational expenses in marine recirculating aquaculture systems (RAS) is highquality feed. Traditionally, fishmeal (FM) is used as the main source of protein in marine fish
diets due to its high protein content, amino acid profile, digestibility, and palatability.
Unfortunately, as the aquaculture industry continues to grow it is creating a higher demand for FM
worldwide. Limited supplies along with increasing demand are causing the price of FM tocontinually increase. In an aim to search the replacement of FM by alternative protein sources insouthern flounder diets glandless cottonseed meal was tested at our University of North Carolina(UNCW)-Aquaculture Facility. Cottonseed meal (CSM) proteins from genetically-improved(glandless) low-gossypol seed (GI-CSM, 52.1% crude protein, CP), genetically-modified lowgossypol seed (GMO-CSM, 56.0% CP) and from an untreated regular (glanded) seed (R-CSM
49.9% CP) were evaluated to replace fish meal (FM) protein (59.5% CP) in juvenile
southern flounder Paralichthys lethostigma diets. The results suggest that up to 75% of fish meal
protein may be replaced by GI- or GMO-CSM protein in the diet of juvenile flounder without
adverse effects on growth performance and body composition. Cottonseed meal is a potentiallycost-effective alternative plant protein source for use in aquafeeds. These ultra-low gossypol
cottonseed flour proteins could be inexpensive protein sources for the commercial culture ofsouthern flounder and other finfish species.

Alam, M.S., Watanabe, W.O., Sullivan, K.B., Rezek, T.C., Seaton, P.J. 2012. Replacement ofmenhaden fish meal protein by solvent extracted soybean meal protein in the diet of juvenile
black sea bass Centropristis striata supplemented with or without squid meal, krill meal,
methionine and lysine. North American Journal of Aquaculture, 74:2, 251-265
Three experiments were conducted at our UNCW-Aquaculture Facility to determine the extent towhich menhaden fish meal protein (FMP) can be replaced by solvent‐extracted soybean meal
protein (SBP) in the diet of juvenile black sea bass Centropristis striata. Diets were formulated
replacing FMP by SBP at 0, 10, 20, 30, 40, 50, and 60% (experiment 1) and 0, 60, 70, 80, 90,
and 100% (experiment 2), with supplementation with squid meal, krill meal, and attractants inboth experiments. Experiment 3 was designed to replace FMP by SBP at 40, 50, 60, 70, and 80%
without supplemental squid and krill meal and at 60% and 70% with supplemental methionine
and lysine. Diets were fed twice daily to triplicate groups of fish (N = 15 per group) in 75‐L
tanks containing recirculating seawater. Fish were fed for 6, 10, and 8 weeks in experiments 1, 2,
and 3, respectively. No significant differences in body weight gain, feed efficiency, and survival
were observed among treatments in experiment 1. In experiment 2, no significant differences inpercent weight gain were observed among fish fed diets replacing FMP at 0, 60, and 70%. In
experiment 3, body weight gain was not significantly different for fish fed supplemental
methionine and lysine in 70% SBP diets compared with fish fed 0% SBP diets. No significant
differences were observed in whole‐body n‐3 polyunsaturated fatty acids among treatments inexperiment 2. Broken‐line regression of the specific growth rate data suggested that the
maximum level of FMP replacement with SBP in black sea bass diets was 67.6–68.4% with 75
g/kg squid meal and 50 g/kg krill meal in the diet and 57.2–58.0% without squid and krill meal
supplementation.
Alam, M.S., Watanabe, W.O., Dawson, M., Carroll, P. 2015. Pilot, commercial-scale testing ofpromising diets for intensive cultivation of black sea bass and southern flounder. World
Aquaculture, Dec 2015, 34-40.
In order to replace fish meal in the diet of a marine finfish, the protein source must be highenough in protein, have a similar amino acid profile, cannot harm the fish in any way, must be
palatable for the fish, needs to be cost effective, and must be accepted by the consumer. To
reduce cost in commercial fish feed industries, soybean meal is considered to be one of the mostsuitable and stable supplies of an alternative ingredient for replacing fish meal. However, there
was no published information on utilization of soybean meal as an alternative protein source inthe diets of southern flounder and black sea bass in pilot commercial scale, two high-valuemarine finfish in the Atlantic coastal region of the U.S. Therefore, commercial scale feeding
trials were conducted in three commercial aquaculture farms in North Carolina and Virginia for
about 10 months in order to investigate the replacement of fish meal (0, 50 and 30%) by soybean
meal protein in pre adult and grow-out southern flounder and black sea bass. Result suggests thata substantial amount of fish meal protein (50% for black sea bass and 30% for southern flounderdiets) could be replaced in the diets of grow-out black sea bass and southern flounder without
any adverse effect on growth and survival.
Alam, M.S., Watanabe, W.O., Myers, A.R., Rezek, T.C. Carroll, P.M., Skrabal, S.A. 2015. Effects
of dietary salt supplementation on growth, body composition, tissue electrolytes, and gill andintestinal Na+K+ATPase activities of black sea bass reared in low salinity water. Aquaculture,
446, 250-258.

Two feeding trials were conducted to investigate the effects of dietary sea salt supplementation on
growth and survival of black sea bass Centropristis striata reared under sub-optimal salinity
conditions at 15 and 10 g L− 1. Irrespective of dietary salt treatment, black sea bass juveniles
maintained good growth and survival and normal whole body electrolyte concentrations at rearing
salinities of 10 and 15 g L− 1, indicating that these fish are good osmoregulators and could be
potentially cultured under these low salinities. These findings may enable black sea bass to begrown in recirculating aquaculture systems in low-salinity brackish water. The results indicate that
dietary salt supplementation may be used to avert mortality when a producer is faced withadversely low salinity conditions due to weather and tides as may occur in coastal black sea bass
fish farms sourcing brackish water from tidal creeks.
Alam, M.S., Teshima, S., Yaniharto, D., Koshio, S.,Ishikawa, M. 2002. Influence of dietary aminoacid patterns on growth and body composition of juvenile Japanese flounder, Paralichthys
olivaceus. Aquaculture, 210:359-369.
Since essential amino acids are of vital importance, these must be found in the sources of protein
used to create finfish diets and in amounts similar to those required by the fish. If not, they mustbe supplemented to the diet in such a way that they meet the required balance for the particularspecies of fish. As per “ideal protein concept” “if the amino acid profile of the feed mimics thewhole-body amino acid profile of the animal being fed, protein utilization and growth should be
maximized.”
A feeding trial using five semi-purified diets (50% crude protein) was conducted to investigate theeffects of different dietary amino acid patterns on growth and body composition of juvenile
Japanese flounder. The control diet contained casein and gelatin as intact protein sources and fourother diets contained 30% casein–gelatin (2:1, w/w) and 20% crystalline amino acids (CAA). CAA
were added to the diets to simulate the amino acid pattern found in red sea bream egg protein(REP), Japanese flounder larvae whole body protein (FLP), Japanese flounder juvenile whole bodyprotein (FJP), and brown fish meal protein (BFP), respectively. The test diets were fed to triplicate
groups of juveniles (2.75±0.05 g) twice a day for 40 days to evaluate weight gain, survival, feed
conversion efficiency (FCE), protein efficiency ratio (PER), and apparent protein utilization(APU). The apparent retention of total dietary amino acids in the whole body and A/E ratios of thewhole body were also evaluated. The highest weight gain was observed in fish fed the dietcontaining the dietary amino acid pattern of BFP followed by fish fed the control, FJP, FLP and
the REP diets. Percent survival, FCE, PER and APU were also significantly (P<0.05) affected by
the amino acid pattern in the diets, indicating the highest value in fish fed the BFP diet. Except fora few amino acids, the amino acid composition of the whole body did not show marked differenceswith different dietary amino acid pattern. Results suggest that BFP could be more suitable as areference amino acid pattern in the diet of juvenile Japanese flounder compared to the amino acidpattern of FLP, FJP or REP.

6. Seaweed aquaculture in a warming environment – building resilience with Saccharina
angustissima (Laminariales, Phaeophyceae)
Simona Augyte

Department of Ecology and Evolutionary Biology, University of Connecticut, Stamford, CT 06901,
USA, Email: simona.augyte at uconn.edu
Abstract
Locally sourced, high quality sea vegetables, in particular kelp, are sparking consumer interestand demand in New England. The narrow-bladed sugar kelp, Saccharina angustissima, with one
narrow (1.6 ± 0.7 cm wide) and one long (average 1.8 ± 0.96 m, up to 4.4 m) blade ismorphologically and genetically distinct from Saccharina latissima, which grows much wider(20-30 cm) and is usually shorter (up to 3.5 m) in length. In the intertidal, S. angustissima is
adapted to withstand extreme hydrodynamic forces an order of magnitude higher than thesubtidal populations of sugar kelp that are usually found in more sheltered locations. It is
restricted in distribution in mid-coastal Maine and occurs in the low intertidal, exposed to highocean swells. In collaboration with kelp farmers from Maine Fresh Sea Farms and Maine Coast
Sea Vegetables, we successfully domesticated this kelp growing it to full maturity on longlines
to be used commercially for its desirable culinary traits. Based on harvest results from two
growing seasons, biomass yields of this kelp were up to 24.1 (±6.3) kg m-1 of line with a plantdensity of 400 plants m-1 of line. The phenotypic characteristics that make this kelp
exceptionally adapted to extreme sea conditions were preserved at the open-water farm sites.
Experiments with the microscopic gametophyte stages of S. angustissima show that it has thecapacity to withstand some temperature fluctuations. Overall, this domesticated kelp species has
great potential as an economically valuable sea vegetable in the Gulf of Maine. Nevertheless,because of its restricted geographic distribution, care must be taken to protect the donor
population. Although additional work needs to be done to bring this kelp to market, growing it
also offers a suite of ecosystem services including nutrient bioextraction and temporary habitat
formation for sea animals.
Annotated Bibliography of Key Works
Augyte, S., Yarish C., S.Redmond, & J.K.Kim. 2017. Cultivation of a morphologically distinct
strain of the sugar kelp, Saccharina latissima forma angustissima, from coastal Maine, USA, with
implications for ecosystem services. Journal of Applied Phycology 29: 4. 10.1007/s10811-0171102-x.
Consumer interest and demand for North Atlantic sourced sea
vegetables drives opportunities for aquaculture development in thenortheast USA. The unique morphology and desirable culinary traitsof the wild narrow-bladed kelp, Saccharina latissima forma
angustissima, were successfully translated into a cultivated crop on
two geographically distinct open-water farms in Maine, USA.
Environmental conditions, growth, and tissue analysis were
quantified. Peak meristematic growth rates for blade length occurredfrom March through April at 2.85 (±0.34) cm day−1. The kelp was
harvested from May through June with yields of up to 17 (±4.4) kg m−1
of line and plant density of 330 plants m−1 of line at the Bristol farmand yields of 13.3 (±6.2) kg wet weight m−1 line and a plant density of
400 plants m−1 of line at the Sorrento farm. Second season yields at
Sorrento were on average 24.1 (±6.3) kg m− 1 of line. Both farms grew

significantly narrower blades of f. angustissima than of the sugar
kelp, S. latissima .Common garden experiments with the twomorphotypes identified trait stability for length and width, while
blade ruffles and thickness varied with the environment.
Calculations estimating the nutrient bioextraction cap ability of thecultivated f. angustissima kelp harvested in June reveal N removal of
88.7 kg ha−1 and C removal of 1666.7 kg ha−1 (combined farm site
averages). Overall, this unique kelp form has the potential as a new
aquaculture crop for the Gulf of Maine while providing several
coastal ecosystem services.
Augyte, S., L. Lewis, S. Lin, C. D. Neefus, and C. Yarish. 2018. Speciation in the extremely
exposed intertidal: the case of Saccharina angustissima (Collins) Augyte, Yarish & Neefus comb.
nov. et stat. nov. (Laminariales, Phaeophyceae). Phycologia 57: 100-112.
Saccharina latissima is a perennial kelp with a circumboreal distribution from the North Pacificto the North Atlantic coasts. Our study clarified the taxonomy of the morphologically distinct
Saccharina latissima forma angustissima (Collins) A. Mathieson from the low intertidal zone on
exposed islands and ledges of Casco Bay, Maine, USA. To identify genetic divergence between
the two morphotypes, S. latissima and S. latissima f. angustissima, we used a multilocus
phylogenetic approach. Genetic analysis suggested low divergence between the two forms.
However, there was as much or more genetic divergence between S. latissima and S. latissima f.
angustissima as there were between other taxonomically accepted species of Saccharina. To
investigate sexual compatibility between the two forms, we made reciprocal crosses of thegametophytes and observed sporophyte formation. All crosses were successfully grown to thejuvenile sporophyte stage, suggesting that the two are reproductively compatible in vitro. It is
unknown if the two populations freely hybridize in the field. Last, we compared wave action, theecological factor most likely driving the unique morphology, at exposed sites with S. latissima f.
angustissima and protected sites with S. latissima. The mean wave force at the exposed site wasover 30 times higher in magnitude than at the protected site respectively, during the summer. The
significant differences in morphology, the lack of specimens with intermediate morphologies,
and the results of a common garden experiment suggest that the morphological differences in S.
latissima f. angustissima are heritable with a genetic basis. Therefore, on the basis of ourmolecular evidence coupled with ecological studies, we are elevating S. latissima f. angustissima(Collins) A. Mathieson to specific rank as S. angustissima (Collins) Augyte, Yarish & Neefus
comb. nov. & stat. nov
J. K. Kim, G.P. Kraemer, & C. Yarish 2015. Use of sugar kelp aquaculture in Long Island Soundand the Bronx River Estuary for nutrient extraction. Marine Ecology Progress Series 531:155166.
The present study revealed an even higher nutrient bioextraction
capacity in the cold-water species Saccharina latissima at 3 sites the mouth of the Bronx River Estuary (Bronx, NY; BRE), western Long
Island Sound (Fairfield, CT; WLIS) and central Long Island Sound(Branford, CT; CLIS), during winter and spring of the 2012−2013

growing season. These sites differ in temperature (BRE > CLIS > WIS),
salinity (BRE < WLIS = CLIS) and nutrients (BRE >> WLIS = CLIS). It wasestimated that S. latissima could remove up to 180, 67 and 38 kg N
ha−1 at BRE, WLIS and CLIS respectively, in a hypothetical kelp farmsystem with 1.5 m spacing between longlines. In the same
hypothetical kelp farm system, the estimated carbon sequestration
values are 1350 (BRE),1800 (WLIS) and 1100 (CLIS) kg C ha−1 . The
potential monetary values of N sequestration by the sugar kelp are
up to $1600 (BRE), $760 (WLIS) and $430 (CLIS) ha−1, if incorporated in
the State of Connecticut Nitrogen Credit Trading Program and acarbon-pricing scheme. The potential economic values of C
sequestration are $30−300 (BRE), $40−400 WLIS), and $24−240 (CLIS)
ha−1. These results suggest that seaweed aquaculture is a usefultechnique for nutrient bioextraction in urbanized coastal waters,such as LIS and BRE. Alternation of the warm- and cold-water species
would maximize nutrient bioextraction and augment other ecosystemservices, producing economic benefits for the region while helping tomanage non-source eutrophication.

7. On the application of bio-stimulants to enhance growth and thermal resistance of sugar
kelp, Saccharina angustissima, for aquaculture production
Schery Umanzor1*, Sookkyung Shin2, Michael Marty-Rivera1, Jang Kim2, Simona Augyte1, andCharles Yarish1
Presenting author*
1*

Department of Ecology and Evolutionary Biology, University of Connecticut, Stamford, CT
06901, USA, *Email: schery.umanzor at uconn.edu
2
Department of Marine Science, School of Natural Sciences, Incheon National University,
Incheon 22012, Korea
Abstract
Macro- and microalgae are good sources of bioactive compounds, nutrients, and bio stimulantsthat are being used in agriculture and horticulture to mitigate abiotic and biotic stresses toimprove plant productivity and food production. We tested commercially available algal extracts
to see if they can enhance growth and thermal resistance of the sugar kelp, Saccharina
angustissima under laboratory conditions. We subjected kelp juvenile sporophytes to different
temperatures, periods, and varying concentrations of extract solutions. Blade growth wasmeasured on a weekly basis for a four-week period. Preliminary results show that thecommercial extracts tested do not have a direct effect on growth but a delayed effect on thermal
resistance and blade growth. These results suggest that algal bio-stimulants could enhance theperformance of seaweed cultures on open-water farms subjected to environmental stress,
particularly in light of ocean warming and global climatic change.
Annotated Bibliography of Key Works
Craigie, J.S. 2011. Seaweed extract stimuli in plant science and agriculture. J Appl Phycol.
23:371–393
This contribution derives from the author’s presentation at the “XX International Seaweed
Symposium”. Craigie provides an overview of the utilization of seaweeds by humans, and more
relevant, a summary of seaweeds uses in agriculture. Furthermore, this paper details with
examples how the application of seaweed as bio-stimulants enhances land crops in different
ways. The author includes brand names, countries of origin, and nutritional content of various
commercially available products.
Tibubos, K.R., Hurtado, A.Q., Critchley. A.T. 2017. Direct formation of axes in new plantlets of
Kappaphycus alvarezii (Doty) Doty, as influenced by the use of AMPEP K+, spindle inhibitors,
and plant growth hormones. J Appl Phycol. 29:2 3 –245 349
This is a recent contribution describing the effect of AMPEP K+ on the formation of vegetative
shoots of Kappaphycus alvarezii. AMPEP K+ is a Canadian commercial extract of the brownseaweed Ascophyllum nodosum with enhanced levels of potassium. The authors tested thisextract in combination with colchicine or oryzalin, in addition to synthetic plant growth
regulators. They found that 5 mg L−1 of AMPEP K+ with plant growth regulators produced thelongest direct axis shoots (9.6 ± 0.33 mm), followed by 0.1 mg L−1 of AMPEP K+ combinedwith 1.0 mg L−1 oryzalin and plant growth regulators (8.7 ±0.00 mm). Their results suggest thatusing AMPEP K+ could improve shoot formation of K. alvarezii plantlets in land-sea-based
nursery cultivation.
Hurtado, A.Q. and Critchley. A.T. 2018. A review of multiple biostimulant and bioeffector
benefits of AMPEP, an extract of the brown alga Ascophyllum nodosum, as applied to theenhanced cultivation and micropropagation of the commercially important red algal
carrageenophyte Kappaphycus alvarezii and its selected cultivars. Journal of Applied Phycology
https://doi.org/10.1007/s10811-018-1407-4
The majority of publications related to using seaweed extracts on the aquaculture of otherseaweeds have assessed the effect of Ascophyllum nodosum extracts on Kappaphycus alvarezii.
This review mainly summarizes studies made on K. alvarezii exposed to different treatments of
A. nodosum extracts. A. nodosum extracts are utilized to mitigate biotic and abiotic stressors of
K. alvarezii subject to farming conditions. The authors provide examples related tomicropropagation, field cultivation, endophyte mitigation, and impacts on the resulting
carrageenan qualities of different K. alvarezii cultivars. In addition, the authors also mention
about alternative seaweed extract that has been applied to boost the growth of another cultivatedseaweed. Altogether, this review paper provides evidence of the favorable results of using
Ascophyllum extracts as biostimulants for Kappaphycus aquaculture.

8. Vibrio coralliilyticus induction of virulence toward larval oysters and corals at elevatedseawater temperatures and potential mitigation treatments

Gary P. Richards
United States Department of Agriculture, Agricultural Research Service, Delaware State
University, James Baker Center, Dover, DE, 19901, USA, Email: gary.richards at ars.usda.gov
Abstract
Recent research has advanced our understanding of the role pathogenic vibrios play in disease ofvarious aquaculture species. Vibrio coralliilyticus, formerly thought to infect just corals, is now
known to infect larval oysters, causing major losses in hatchery settings. The negative effect of V.
coralliilyticus on wild-type oyster production remains uncertain, but circumstantial evidence
suggests it may be substantial. We identified eight strains of V. coralliilyticus that infect both
Eastern oyster (Crassostrea virginica) larvae and Pacific oyster (Crassostrea gigas) larvae in U.S.
East and Pacific coast hatcheries, respectively. West Coast hatcheries have experienced major
production losses over the years, due to the presence of V. coralliilyticus. Some of the largest
hatcheries have reported losses of up to 80%, resulting in shortages in seed oysters needed forcommercial oyster production. Losses can exceed 10 billion larvae per year in the larger hatcheries.
The U.S. East Coast hatcheries are considerably smaller, but also experience losses in Easternoysters, possibly from V. coralliilyticus, but also from the known shellfish pathogen V. tubiashii.
The infection and death of corals by V. coralliilyticus is known to be enhanced atseawater temperatures ≥ 27°C. In our studies, multiple strains of V.
coralliilyticus produced high larval oyster mortalities with LD50’s ranging from 3.8 × 103 to 4 ×
104 CFU/ml of seawater, depending on the V. coralliilyticus strain and the oyster species (Eastern
vs. Pacific oysters). Studies with knockout mutations demonstrated that the transcriptional
regulator ToxR and the outer membrane protein OmpU were important in larval oyster (and coral)
infections caused by V. coralliilyticus. Gross pathological changes occur in V. coralliilyticusinfected larvae, initially in the velum and cilia and then in internal organs which become liquefied.
Infection of larval oysters is likely induced by stress and a lowering of resistance to these pathogens.
Stressors, particularly in hatcheries, likely include incorrectly adjusted seawater temperatures,
salinities, pHs, and dissolved oxygen levels or inadequate nutrition, over stocking, high overallbacterial loads, etc. To begin to mitigate vibrios in hatcheries and other venues, we identified avariety of predatory bacteria which, in nature, help to modulate Vibrio levels in seawater and/or
shellfish. They include several predatory bacteria such as Pseudoalteromonas piscicida, which
secrete digestive enzymes that inhibit and kill many kinds of bacteria including vibrios; andHalobacteriovorax, a Bdellovibrio and Like Organism (BALO) which infect Gram-negative
bacteria like vibrios and replicate within them, killing the vibrios in the process. Together thesebacteria may serve as probiotics in reducing hatchery mortalities and in killing vibrios and otherbacterial pathogens in a variety of other aquaculture settings. Bacteriophages (phages) are anothermeans to inactivate many undesirable pathogens in the environment and are increasingly being
used in aquaculture. To date, we isolated phages against V. coralliilyticus and V. tubiashii andshowed the practical application of phage therapy to reduce or eliminate mortalities from V.
coralliilyticus in larval oysters.
Annotated Bibliography of Key Works
Richards, G.P., M.A. Watson, D.S. Needleman, K.M. Chruch and C.C. Häse. 2015. Mortalities
of Eastern and Pacific oyster larvae by the pathogens Vibrio coralliilyticus and Vibrio tubiashii.
Appl. Environ. Microbiol. 81:292−297.

Vibrio tubiashii has been a well known pathogen in Eastern oyster (Crassostrea virginica) larvaeon the United States (U.S.) East Coast for many years and has also been associated with Pacificoyster (Crassostrea gigas) larvae on the U.S. West Coast. Vibrio coralliilyticus, a well-known
coral pathogen and a cause of coral bleaching, has also been associated with disease of Pacificoyster larvae, particularly in oyster hatcheries. It became evident that some of the outbreaks on
the West Coast that were thought to be from V. tubiashii were actually caused by V.
coralliilyticus. In this study, we evaluated two strains of V. tubiashii and four strains of V.
coralliilyticus in both Eastern and Pacific oyster larvae to determine their ability to cause larvalmortalities. We showed that V. tubiashii strains infected primarily the East Coast oyster larvae
while all four of the V. coralliilyticus strains infected the Eastern and Pacific oyster larvae. The
LD50 values for the V. coralliilyticus strains ranged from 1.1 × 104 to 3 × 104 CFU/ml ofseawater in Eastern oysters larvae. In Pacific oyster larvae, the LD50 range was 1.2 × 104 to 4 ×
104 CFU/ml of seawater. Together, these studies defined the host specificity of these Vibrio
strains with regard to Eastern and Pacific oyster larvae and demonstrated the ability of coralassociated pathogens to also infect and kill larval oysters. Subsequent research (Ushijima et al.,2018, see abstract below) showed similar results with other strains of V. coralliilyticus and an
up-regulation of mortalities at elevated seawater temperatures.
Ushijima, B., G.P. Richards, M.A. Watson, C.B. Schubiger and C.C. Häse. 2018. Factors
affecting infection of corals and larval oysters by Vibrio coralliilyticus. PLOS ONE.
https://doi.org/10.1371/journal.pone.0199475
Vibrio coralliilyticus is a naturally occurring marine bacterium which infects and kills corals andlarval shellfish. It is best known as the cause of coral bleaching, which has contributed to theloss or damage to coral reefs worldwide. We determined that V. coralliilyticus strains also infectand kill larval oysters and other shellfish, particularly in shellfish hatcheries, causing shortagesof seed oysters needed for commercial shellfish operations. We determined that larval oyster
mortalities were significantly higher at an elevated seawater temperature (27°C) compared to alower temperature (23°C). Thus, multiple coral pathogens were found to infect larval oysters ina temperature- and dose-dependent manner. Also identified were virulence factors that promote
the infection of both coral and oyster larvae. This work demonstrates for the first time thatelevated seawater temperatures enable V. coralliilyticus to more readily infect oyster larvae. It
also serves to warn hatchery operators to maintain seawater temperatures below 27°C.
Richards, G.P., M.A. Watson, D.S. Needleman, J. Uknalis, E.F. Boyd and J.P. Fay. 2017.
Mechanisms for Pseudoalteromonas piscicida-induced killing of vibrios and other bacterial
pathogens. Appl. Environ. Microbiol. 83:e00175-17, https://doi.org/10.1128/AEM.00175-17
Pseudoalteromonas are marine bacteria that are known to secrete antimicrobial compounds
which inhibit competing bacteria in the marine environment. We identified a second method bywhich some Pseudoalteromonas (P. piscicida) kill competing bacteria. It involves the directtransfer of digestive vesicles from the surface of the Pseudoalteromonas to the surface ofcompetitors, digestion of holes in the competitor’s cell wall by proteolytic enzymes associatedwith the vesicles, and apparent feeding of the Pseudoalteromonas off the nutrients released by
the digested bacterium in a predatory fashion. Among the P. piscicida enzymes identified wereaminopeptidase B, a trypsin-like serine protease, a chymotrypsin-like serine protease, and acysteine protease. Pseudoalteromonas piscicida inhibited and killed the pathogens Vibrio
parahaemolyticus, V. vulnificus, V. cholerae, Photobacterium damselae, Shewanella algae, andStaphylococcus aureus. Together, this data indicates that Pseudoalteromonas piscicida produce
important antibacterial compounds that have a potential role in the probiotic treatment ofaquaculture products and in reducing biofilm formation.
Richards, G.P., J.P. Fay, J. Uknalis, M. Olanya and M.A. Watson. 2016. Purification and hostspecificity of predatory Halobacteriovorax isolates from seawater. Appl. Environ.
Microbiol. 82: 922−927.
Halobacteriovorax are small predatory bacteria found in the marine environment and may serveas biocontrol agents against human, fish and shellfish pathogens. They are within a group of
predatory bacteria known as the Bdellovibrio and Like Organisms (BALO). Halobacteriovorax
were isolated from seawater and had broad specificity toward five strains of Vibrio
parahaemolyticus while two additional Halobacteriovorax strains isolated from low salinity
seawater readily infected and killed the human pathogens E. coli O157:H7 and Salmonella
Typhimurium DT104. The replication rate of Halobacteriovorax against E. coli and Salmonella
increased as salinities decreased. It is likely that Halobacteriovorax could readily be isolated
against a broad range of Gram-negative seafood pathogens. Improved methods were also
developed to purify infectious Halobacteriovorax from their host cells. Overall, the use of
Halobacteriovorax in various aquaculture applications may serve as an environmentally friendly,
non-antibiotic treatment to reduce pathogens in fish and shellfish.
Richards, G.P., J.P. Fay, K.A. Dickens, M.A. Parent, D.S. Soroka and E.F. Boyd. 2012.
Predatory bacteria as natural modulators of Vibrio parahaemolyticus and Vibrio vulnificus inseawater and shellfish. Appl. Environ. Microbiol. 78:7455−7466.
Levels of the human pathogenic Vibrio parahaemolyticus and Vibrio vulnificus are increasing inshellfish harvesting areas in many parts of the world due, in part, to elevated seawatertemperatures. A means to reduce these vibrios in shellfish is needed. When seawater andoysters were spiked with V. parahaemolyticus or V. vulnificus, the Vibrio levels rapidly
diminished over a 3-day period when natural seawater was used, but counts quickly climbed
when sterile seawater was used. In an effort to determine what was inhibiting the vibrios innatural seawater, tests showed the presence of Vibrio predatory bacteria, later classified as
Halobacteriovorax species. The Halobacteriovorax in natural seawater readily eliminatedvibrios that were added to the seawater. In contrast, oysters spiked with vibrios showed some
initial increase in Vibrio counts within the first 24 h, followed by a rapid decline to baseline
levels. Thus, it appears that Halobacteriovorax are one of nature’s tools to modulate bacterial
levels in shellfish. Harnessing this tool may provide a means to reduce or eliminate pathogens inaquaculture operations and in the seafood industry.

9. Chemical changes in the environment: what does this mean to shellfish?
Shannon Meseck*, Mark Dixon, Yaqin Li, George Sennefelder, Dylan Redman, Renee
Mercaldo-Allen, Paul Clark, Lisa Milke, and Gary H. Wikfors
Presenting author*
NOAA Fisheries, Northeast Fisheries Science Center, Milford Laboratory,

212 Rogers Avenue, Milford, CT, 06460, USA, *Email: shannon.meseck at noaa.gov
Abstract
Coastal areas, estuaries, and river deltas are experiencing an increase in carbon dioxide fromhuman activities, resulting in what is termed “coastal acidification.” Approximately 30% of
atmospheric CO2 dissolves in the oceans. As this CO2 dissolves, it reacts with water andproduces carbonic acid (H2CO3), which dissociates into bicarbonate (HCO3−), carbonate(CO32−), and hydrogen ions (H+). With increased CO2 in the water, there are multiple ways thatshellfish may be affected: (1) food availability; (2) physiological responses, and (3) sedimentacidification.
Food availability may be influenced by changes in phytoplankton community structure andnutritional value. For example, increased carbon dioxide may cause a shift in phytoplankton
biomass and community composition depending upon species’ efficiency of inorganic carbon
acquisition. Shifts in phytoplankton communities, may favor less – or more -- nutritional
phytoplankton that support production of human food.
In addition to potential changes in food supply, coastal acidification may be causing
physiological changes in marine bivalves that affect how they feed. Bivalve gills are constantly
exposed to ambient water, with the gill cilia moving water through the shell for respiration andfeeding. Depending upon environmental conditions, physiological changes to the heart, gills,
gonads, and general metabolism regulation may occur. Physiological changes could result inslower growth, higher mortality, or inability to maintain shell production, or the opposite
responses in some cases. The influence that coastal acidification has on bivalve physiological
functions is important to understand so that aquaculture may be modified to respond to achanging environment.
Finally, the same reaction that occurs in water during coastal acidification occurs at the
sediment-water interface, where bivalves settle, following bacterial decomposition of settlingorganic matter. As particles settle, aerobic and anaerobic reactions, combined with redox
processes, result in the production of CO2 in sediment porewater. Corrosive and hypoxic
sediment can impede successful settlement, recruitment, and abundance in bivalve pollutions.
Nutrient-enriched, coastal areas in the United States are already experiencing sedimentacidification, and this might be affecting bivalve recruitment at the sediment-water interface.
The research presented here will focus on the multiple ways that coastal acidification may affectmarine bivalves.
Annotated Bibliography of Key Works
Clements, J.C. and H.L. Hunt. 2018. Testing for sediment acidification effects on within-season
variability in juvenile soft-shell clam (Mya arenaria) abundance on the Northern Shore of the
Bay of Fundy. Estuaries and Coasts. 41. Pp. 471-483.
The authors conducted a field study assessing the relationship between juvenile soft-shell clamabundance and spatial, temporal, and environmental variables in the Bay of Fundy. SedimentpH, water temperature, location, and date was monitored in 2012 during the settlement season
for Mya arenaria at four different study sites along the shore. Using Akaike’s information
criterion models that incorporated environmental variables alone sediment pH and minimum air

temperature best predicted bivalve recruitment. By removing temporal effects, there was asignificant positive relationship with sediment pH and grain size. These results suggest thatcarbonate geochemistry might influence bivalve settlement.
Meseck, S.L., R. Mercaldo-Allen, C. Kuropat, P. Clark. R. Goldberg. 2018. Variability insediment-water carbonate chemistry and bivalve abundance after bivalve settlement in Long
Island Sound, Milford, Connecticut. Marine Pollution. 135. Pp. 165-175.
The authors conducted field experiments in 2013 to assess the relationship between pore water
sediment carbonate chemistry (pH, Alkalinity, dissolved inorganic carbon), grain size, andbivalve abundance at two sites in Long Island Sound (LIS), Connecticut, USA. Akaike’s linear
model was used to predict total bivalve community abundance and specific species abundance
when appropriate. Over the entire season, 29% of bivalve abundance at the sites could be
explained by grain size, salinity, and pH. When temporal effects were removed, up to 71% of
Nucula spp. abundance could be accounted for by pH, phosphate, and silica concentrations in theporewater. These results suggest that bivalve abundance in LIS may be influenced by carbonate
geochemistry and grain size.
Bermúdez, R., M. Winder, A. Stuhr, A. Almén, J. Engström-Öst, and U. Riebesell. 2016. Effectof ocean acidification on the structure and fatty acid composition of a natural plankton
community in the Baltic Sea. 2016. Biogeosciences. 13. Pp. 6625-6635.
The authors conducted mesocosm perturbation experiments in coastal waters to investigate theresponse of phytoplankton to increased carbon dioxide with respect to community composition
and fatty acid composition (nutritional statues). There results found little change in plankton
community composition from the natural condition (~347 µatm) to predicted levels for the year
2100 (~1333 µatm). The fatty acid composition was influenced by community composition,
which was driven by phosphate and silica availability. The results presented here suggest thatphytoplankton community composition is influenced more by nutrient availability than by CO2,
with little change in nutritional composition.
King, A. L., B.D. Jenkins, J.R. Wallace, Y. Liu, Y., G.H. Wikfors, L.M. Milke, and S.L.
Meseck, S. L. 2015. Effects of CO2 on growth rate, C: N: P, and fatty acid composition of sevenmarine phytoplankton species. Marine Ecology Progress Series. 537. Pp. 59-69.
The authors conducted laboratory-based experiments on 7 species of phytoplankton common incoastal estuaries of the North Atlantic where carbon dioxide was manipulated to 4 levels ranging
from the glacial minimum (<290 ppm) to geological maximum levels (>2900 ppm). Results
indicated that carbonate system-driven changes in growth rate did not result in changes toelemental composition of the species studied. Fatty acid composition was not affected byelevated CO2. The results showed little sensitivity of common coastal marine phytoplankton
species to elevated CO2 that will result in changes in nutritional status.

10. The current trends in pH and ocean acidification in an aquaculture sea area in Gokasho
Bay, Japan
Masayuki Minakawa*1, Satoshi Watanabe 2, Yuka Ishihi2, Natsuki Hasegawa2 and Toshie
Matsumoto2

Presenting author*
1

National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency,
224-1 Hiruda, Tamaki, Mie, 519-0423, Japan, *E-mail: minmas at affrc.go.jp
2
National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency,
422-1 Nakatsuhamaura, Minamiise, Mie, 516-0193, Japan
Abstract
Coastal environments are influenced by various human-induced stressors. Recently, the global
reduction of ocean pH, referred to as ocean acidification (OA), has arisen as a consequence of
oceanic uptake of anthropogenic CO2 from the atmosphere. OA has become a significant
environmental stressor to diverse marine ecosystems. OA impacts not only marine phytoplankton
production but also fisheries and seafood industries. Shellfish are farmed in many coastal and bay
areas in Japan. It is likely that shellfish aquaculture will be impacted by OA in the near future since
the shells are composed of calcium carbonate whose deposition is controlled by carbonate
chemistry and the pH of seawater. While there are many studies on pH in open ocean andcontinental shelf waters, effects of OA are not well understood in many shellfish growing areas
due to a large variation of pH associated with many biogeochemical processes. To obtain basic
information on the water quality in an aquaculture sea area in Gokasho Bay, Mie, Japan, monthlyhydrographical observations have been recorded since November 2016. Seawater samples werecollected at three depths by a 1.3 L Kitahara’s water sampler at each of 20 stations in the bay. The
samples were collected in 25 ml co-stoppered glass test tubes for measurement of pH to avoid thecontamination of CO2 from atmosphere and in 250 ml polyethylene bottles for measurements of
total alkalinity and nutrients. A CTD system (RINKO profiler, JFE Advantech) was also used forgetting hydrographical data. The pH of seawater was measured more precisely using a glass
electrode (MM-60R, DKK-TOA Co.) (±0.003 pH). The glass electrode was standardized against
the JIS buffer scale (NBS scale) and any drift in pH was corrected against the seawater buffer scale.
The total alkalinity was measured by using a one-point titration method (±0.006 meq/L). The
calcium carbonate saturation state of seawater (Ω) was calculated from the values of pH, total
alkalinity, salinity and seawater temperature. In the winter season, the value of pH was almost
constant (8.02 - 8.13) across the sampled range in the bay and was approximately the same level
as in surface water off the southern coast of Japan. In contrast, pH values varied greatly during thesummer seasons (7.75 - 8.60). The lower value was thought to be caused by the remineralizationof organic matter in the seabed, and the higher value probably resulted from high phytoplankton
productivity. The pH measurements currently recorded were generally lower than those recorded
during observations in the 1980s. The total alkalinity (TA) levels suggested that the riverine waterin the bay was mixed rapidly with the outer bay waters. The values of Ω (> 3) calculated wererelatively high across the bay. Although the mineral calcium carbonate is supersaturated in thecurrent environment of the bay, if OA advances in offshore surface water around Japan in the nearfeature, the degree of Ω in the bay water would drop with reduction of pH and could result innegative impacts on the calcareous ecosystem of the bay.
Annotated Bibliography of Key Words
Ishii, M., Koasugi, N., Sasano, D., Saito, S., Midorikawa, T., Inoue, H.Y. 2011. Ocean
acidification off the south coast of Japan: A result from time series observation of CO2 parametersfrom 1994 to 2008. J. Geophys. Res. 116, C06022.
Japan Metrological Agency. Long-term trend of pH in surface water of the Pacific Ocean.
http://www.data.jma.go.jp/kaiyou/shindan/a_3/pHpac/pH-pac.html
The authors determined that a significant trend in ocean acidification is superimposed on the large
seasonal and interannual variabilities of acidity in surface waters off the south coast of Honshu
Island, Japan, based on their observation of the carbonate system (partial pressure of CO2, total
inorganic carbon and pH). Multiple regression analysis of these parameters showed that pH andthe aragonite saturation state (Ωarago) are decreased at a rate of -0.020 ± 0.007 pH/decade, and 0.12 ± 0.05 Ωarago/decade, respectively. If future atmospheric CO2 levels keep increasing as
predicted by IPCC scenario A1FI, which postulates tentative fossil fuel use associated with very
rapid economic growth, a further reduction of -0.8 pH and -0.1 Ωarago is likely in the next 50 years.
The authors suggested that such a rapid reduction of Ωarago could have negative impacts on a varietyof calcareous organisms.
Kubota, K., Yokoyama, Y., Ishiakwa, T., Suzuki, A., Ishii, M. 2017. Rapid decline in pH of coralcalcification fluid due to incorporation of anthropogenic CO2. Nature, 7, 7694,
DOI:10.1038/s41598-017-07680-0.
Based on Boron isotopic ratio (σ11B) measurements of corals, the authors show clear evidence that
ocean acidification is affecting the pH of the calcification fluid (pHCF) in Porites corals within thewestern North Pacific Subtropical Gyre at two separate locations, Chichijima Island (Ogasawara
Archipelago) and Kikaijima Island. A comparison with the pH of the ambient seawater (pHSW)
near these islands, estimated from a large number of shipboard measurements of seawater CO2 andatmospheric CO2, indicated that pHCF is sensitive to change in pHSW. The authors suggested thatthe calcification fluid of corals will become less supersaturated with respect to aragonite by the
middle of this century (pHCF=~8.3 when pHSW=~8.0 in 2050), earlier than previously expected,
despite the pHCF-unregulating mechanism of corals.
Suga, H. P, Sakai, S., Toyofuku, T., and Ohkouchi, N., 2013. A simplified method for
determination of total alkalinity in seawater based on the small sample one-point titration method.
JAMSTEC Rep. Res. Dev., 17, 23-33.
Measurement of pH and total alkalinity is the most convenient way to describe the carbon system
in seawater samples. In this paper, the authors introduced a modified method for determination of
total alkalinity focused on reduced volume seawater samples (1 mL) and based on the one-point
titration method. The precision of this method is 0.1 - 0.2% (relative standard deviation). On theother hand, with no correction, the precision was 0.1 - 1.0%. Even though values showed asystematic error, precision was maintained through the correction based on concurrent
measurement of commercially available standards. This method allows us to describe thecarbonate system in a small amount of water with sufficient precision and accuracy to investigate
the mechanism of calcification using only a pH meter.
Fujii, T., Komai, Y., Fujiwara, T. 2001. Carbon dioxide dynamics in coastal regions of Osaka Bay.
Japan Sci. Civil Eng. Papers B2, 67, 911-915.
In coastal regions where the biological production is far larger than that in the open ocean and theshort-term change of hydrographical conditions is significant, the authors also conducted
continuous measurements of salinity, pH and dissolved oxygen (DO) at three stations in Osaka
Bay. Estimates of carbonate species in bay water were calculated by using the values of pH andtotal alkalinity. The results showed that dissolved inorganic carbon and DO fluctuatedsynchronously with high correlation (R2=0.97), and the records of DO and partial pressure of CO2(pCO2) indicated prominent diurnal variations which correspond to diurnal irradiation variations
that are due to a high productivity in the bay.

11. Ocean acidification experimentation in seagrass-oyster ecosystems using a novel system toconduct in-situ CO2 enrichment
Masakazu Hori1*, Tomohiro Kuwae2 and Masahiro Nakaoka3
Presenting author*
1

National Research Institute of Fisheries and Environment of Inland Sea, Maruishi, Hatsukaichi,
Hiroshima 739-0452, Japan, *Email: mhori at affrc.go.jp
2
Port and Airport Research Institute, Nagase, Yokosuka 239-0826, Japan3
Akkeshi Marine Station, Field Science Center for Northern Biosphere, Aikkapu, Akkeshi,
Hokkaido 088-1113, Japan
Abstract
Coastal ecosystems are under the influence of global environmental changes that threaten theirecological status and the sustainability of seafood production. Seagrass beds, which are widelydistributed along coastlines around the world, are highly important ecosystems offering various
types of ecosystem services to human beings. Especially, seagrass beds are quite important forenvironmental change mitigation and adaptation via carbon storage, acidification mitigation, andwater quality improvement, as well as providing a nursery for marine coastal organisms. Therefore,
seagrass beds are essential for harmonizing coastal fishery/aquaculture with environmental
improvement, resulting in the sustainable use of ecosystem services. Here, we developed a novel
system of in-situ manipulation of CO2 and nutrient enrichment to demonstrate seagrass mitigation
functions that combat against environmental changes. The system is called FORTES, which stands
for the Free-Ocean Real-Time Experimental System. In addition, we developed nonenvironmental risk deterrents from natural algae, specializing in arthropods and molluscs, which
can also be manipulated by FORTES. Now, we focus on the interaction between oyster aquaculture
and seagrass vegetation as a common practice to achieve better coastal management in a changing
environment. Using this system, we can demonstrate not only the mitigative effects of seagrassbeds on oyster aquaculture, but also the direct effects of environmental changes such as rising
pCO2/pH, eutrophication, and agrochemical inputs on oysters.
Annotated Bibliography of Key Works
Campbell, J.E., Fourqurean, J.W. 2013. Effects of in situ CO2 enrichment on the structural andchemical characteristics of the seagrass Thalassia testudinum. Mar. Biol. 160, 1465-1475.
Seagrasses commonly display carbon-limited photosynthetic rates. Thus, increases in atmospheric
pCO2, and consequentially oceanic CO2(aq) concentrations, may prove beneficial. Whileaddressed in mesocosms, these hypotheses have not been tested in the field with manipulative
experimentation. This study examines the effects of in situ CO2(aq) enrichment on the structuraland chemical characteristics of the tropical seagrass, Thalassia testudinum. CO2(aq) availabilitywas manipulated for six months in clear, open-top chambers within a shallow seagrass meadow in
the Florida Keys (USA), reproducing forecasts for the year 2100. Structural characteristics (leafarea, leaf growth, shoot mass, and shoot density) were unresponsive to CO2(aq) enrichment.
However, leaf nitrogen and phosphorus content declined on average by 11% and 21%, respectively.
Belowground, non-structural carbohydrates increased by 29%. These results indicate that
increased CO2(aq) availability may primarily alter the chemical composition of seagrasses,
influencing both the nutrient status and resilience of these systems.
Gattuso, J.P., Kirkwood, W., Barry, J.P., Cox, E., Gazeau, F., Hansson, L., Hendriks, I., Kline,
D.I., Mahacek, P., Martin, S., McElhany, P., Peltzer, E.T., Reeve, J., Roberts, D., Saderne, V.,
Tait, K., Widdicombe, S., Brewer, P.G. 2014. Free-ocean CO2 enrichment (FOCE) systems:present status and future developments. Biogeosciences 11, 4057-4075.
Free-ocean CO2 enrichment (FOCE) systems are designed to assess the impact of ocean
acidification on biological communities in situ for extended periods of time (weeks to months).
They overcome some of the drawbacks of laboratory experiments and field observations byenabling (1) precise control of CO2 enrichment by monitoring pH as an offset of ambient pH, (2)consideration of indirect effects such as those mediated through interspecific relationships andfood webs, and (3) relatively long experiments with intact communities. Bringing perturbation
experiments from the laboratory to the field is, however, extremely challenging. The main goalof this paper is to provide guidelines on the general design, engineering, and sensor options
required to conduct FOCE experiments. Another goal is to introduce xFOCE, a community-led
initiative to promote awareness, provide resources for in situ perturbation experiments, and build
a user community. Present and existing FOCE systems are briefly described and examples ofdata collected are presented. Future developments are also addressed as it is anticipated that thenext generation of FOCE systems will include, in addition to pH, options for oxygen and/or
temperature control. FOCE systems should become an important experimental approach for
projecting the future response of marine ecosystems to environmental change.
Kline, D.I., Teneva, L., Schneider, K., Miard, T., Chai, A., Marker, M., Headley, K., Opdyke, B.,
Nash, M., Valetich, M., Caves, J.K., Russell, B.D., Connell, S.D., Kirkwood, B.J., Brewer, P.,
Peltzer, E., Silverman, J., Caldeira, K., Dunbar, R.B., Koseff, J.R., Monismith, S.G., Mitchell,
B.G., Dove, S., Hoegh-Guldberg, O. 2012. A short-term in situ CO2 enrichment experiment on
Heron Island (GBR). Scientific Reports 2, Article number: 413.
Ocean acidification poses multiple challenges for coral reefs on molecular to ecological scales,
yet previous experimental studies of the impact of projected CO2 concentrations havemostly been done in aquarium systems with corals removed from theirnatural ecosystem and placed under artificial light and seawaterconditions. The Coral–Proto Free Ocean Carbon Enrichment System(CP-FOCE) uses a network of sensors to monitor conditions withineach flume and maintain experimental pH as an offset fromenvironmental pH using feedback control on the injection of low pH
seawater. Carbonate chemistry conditions maintained in the −0.06 and−0.22 pH offset treatments were significantly different thanenvironmental conditions. The results from this short-term
experiment suggest that the CP-FOCE is an important newexperimental system to study in situ impacts of ocean acidificationon coral reef ecosystems.

12. Effects of ocean acidification on the early developmental stages of the commercially
important gastropods, Ezo abalone and horned turban, in JapanRyo Kimura*1, Hideki Takami2, Toshihiro Onitsuka3, Tsuneo Ono4, and Yukihiro Nojiri5
Presenting author*
1

Japan Fisheries Research and Education Agency (FRA), 15F Queen’s Tower B, 2-3-3
Minatomirai, Nishi-ku, Yokohama, Kanagawa, 220-6115, Japan,*E-mail: rkim at affrc.go.jp
2
Tohoku National Fisheries Research Institute, FRA, Shiogama, 985-0001 Japan
3
Hokkaido National Fisheries Research Institute, FRA, Kushiro, 085-0802 Japan
4
National Research Institute of Far Seas Fisheries, FRA, Yokohama, 236-8648 Japan
5
Hirosaki University, Hirosaki, 036-8560 Japan
Abstract
Commercially valuable shellfish species will be affected more seriously by ocean acidification(OA) in conjunction with overfishing. We investigated the effects of OA on fertilization, larval
development, metamorphosis, and post-larval growth of valuable gastropods, Ezo abalone Haliotis
discus hannai and turban shell Turbo cornutus, using a highly accurate CO2 manipulation system,
AICAL pCO2 control system (Kimoto Electric Co., Ltd, Japan). No effects of exposure to <1000µatm pCO2 seawater were observed in fertilization, malformation, or mortality rates in both
gastropods. However, fertilization and hatching rates decreased by exposure to >1500 µatm pCO2
seawater. The malformation rates increased significantly at >1000 µatm, and the diameter of larvalshells exposed to>800 µatm pCO2 was significantly smaller than those in the control seawater(400-450 µatm pCO2) in both species. Shell growth rates of post-larval H. discus hannai weresignificantly smaller at >1500 µatm than at <1000 µatm pCO2. Scanning electron microscopic
images of post-larval shells exposed to >1000 µatm pCO2 showed abnormal development,suggesting problems with shell deposition and/or increased shell dissolution. These results indicatethat pCO2 >1000 µatm reduce developmental performance of these gastropods in early life stages.
The habitats of these gastropods are characterized by kelp beds. Photosynthesis and respiration of
the macroalgae create marked pCO2 diel fluctuations, which are larger than pCO2 change owing
to OA projected for open ocean waters by 2100. We also investigated the effects of OA in relation
with diel cycles of seawater pCO2 levels projected for the near and distant future on the early
development of Haliotis discus hannai in the laboratory. Shell length, abnormality and mortalityof larval abalone were examined under different pCO2 levels with mimicked diel cycle using the
AICAL pCO2 control system. Adult abalone with fully matured gonads were induced to spawn
and gametes were artificially fertilized at concentrations of ca. 400 (atmosphere-balanced, control
treatment), 800 or 1200 µatm pCO2., Hatched larvae 16 hours post-fertilization were exposed torunning seawater adjusted to a constant pCO2 level [ca. 400, 800 or 1200 µatm pCO2] orfluctuating pCO2 on a diel cycle [800±400 (400-1200) or 1200±400 (800-1600) µatm pCO2] in 4day experimental periods. There were no significant differences in the mortality rate, abnormality
rate and shell length of the larvae among the 400, 800, and 400-1200 µatm pCO2 treatments. Incontrast, the abnormality rate was significantly higher in the 1200 and 800-1600 µatm pCO2
treatments than in the control treatment. Larval shell length in the 800-1600 µatm pCO2 treatment
was significantly smaller than that in the other pCO2 treatments. The exposure duration exceeding
the aragonite saturation state would be a critical factor determining the negative impacts on thelarval development of abalone.
Annotated Bibliography of Key Works
Kimura, R., Takami, H., Ono, T., Onitsuka, T., Nojiri, Y. 2011. Effects of elevated pCO2 on theearly development of the commercially important gastropod, Ezo abalone, Haliotis discus hannai.
Fish. Oceanogr. 20, 357-366.
This is one of the first papers investigating the effect of ocean acidification on commercially
important gastropod species in the world. This is also a very early paper using an AICAL systemthat precisely controls the pCO2 concentration during the experiment.
Kurihara, H. 2008. Effects of CO2-driven ocean acidification on the early developmental stages of
invertebrates. Mar. Ecol. Prog. Ser. 373, 275-284.
This review paper describes the effects of ocean acidification on the early development of many
marine invertebrates, including bivalves (oyster and mussel). It provides an overview of the effectsof carbon dioxide concentration on the development of marine invertebrates.
Onitsuka, T., Takami, H., Muraoka, D., Matsumoto, Y., Nakatsubo, A., Kimura, R., Ono, T., Nojiri,
Y. 2018. Effects of ocean acidification with pCO2 diurnal fluctuations on survival and larval shellformation of Ezo abalone, Haliotis discus hannai. Mar. Env. Res. 134, 28-36.
This paper assessed the effects of constant and diurnally fluctuating pCO2, mimicking the rhythmof actual ocean conditions on development and shell formation of larval abalone, using an AICAL
system which can precisely control pCO2. It is suggested that the effects of ocean acidification on
development and shell formation of larval abalone can be determined by intensity and exposure
duration to pCO2 over the threshold associated with aragonite saturation state (Ω-aragonite).

13. A slow growing perspective on multi-generational responses to future change
Coleen Suckling
Animal and Veterinary Sciences, University of Rhode Island, Kingston, Rhode Island, USA,02882, Email: coleensuckling@uri.edu
Abstract
Our oceans are changing, becoming increasingly undersaturated with respect to carbonates asatmospheric CO2 concentrations increase. Predicting how organisms will respond to these changes
has become a major area of research, particularly for those species needing to maintain homeostaticand biomineralizing processes. In recent years there has been a shift in focus from shorter (hoursweeks) towards assessing responses over longer term exposure periods (months-years) advancing
our understanding in this field. Given that predicted changes span across years and decades, andthat organisms will be producing offspring, a greater consideration of multi-generational response
is needed to better understand how organisms will respond under future climates. For many species,this is still in its infancy with the majority of focus on organisms with rapid life cycles. In an
experimental context this is convenient as numerous generations can be achieved withinweeks/months. However, they cannot be a substitute for higher trophic level organisms that havemuch more complex life cycles, developmental and physiological processes. High trophic levels
typically comprise of slower growing organisms with longer life cycles and deferred maturity (i.e.
years). With these, rearing multiple generations becomes time-consuming, difficult and almost
unfundable within normal grant time-scales (i.e. 3 years). This presentation will address thisknowledge gap by discussing the physiological, energetic, somatic and reproductive responses ofa slow growing benthic invertebrate, the European Green Sea Urchin (Psammechinus miliaris),bred across several generations under IPCC predicted CO2 conditions. This sea urchin species
currently has a low level of commercial interest within Europe, but our results indicate that theseanimals will become much more marketable under future CO2 conditions.
Annotated Bibliography of Key Work
Suckling, C.C., Clark, M.S., Peck, L.S., Cook., E. 2014. Experimental influence of pH on theearly life-stages of sea urchins I: different rates of introduction give rise to different
responses. Invertebrate Reproduction and Development, 58 (2): 148-159.
The authors identified that focusing on only the early life stages within an organism’s life-cycle todetermine marine invertebrate responses to future climate change is not a suitable approach. When
this focus is taken, often the larvae are derived from present-day ambient conditions and thendirectly transferred to low pH or high CO2 (~1000 ppm). We showed that this elicits a grossnegative response which will consequently skew our predictions. It was identified that this focus
instead provides important information on present day phenotypic flexibility which can provide
insights into natural selective pathways. The authors also identified that the focus on early life
stages is too narrow because contrasting responses were found between planktotrophic larvalstages and juveniles.
Suckling, C.C., Clark, M.S., Beveridge, C., Brunner, L., Hughes, A., Cook, E., Davies, A.J., Peck,
L.S. 2014. Experimental influence of pH on the early life-stages of sea urchins II: increasing
parental exposure times gives rise to different responses. Invertebrate Reproduction andDevelopment. 58 (3): 161-175.
Suckling, C.C., Clark, M.S., Richard, J., Morley, S.A., Thorne, M.A.S., Harper, E.M., Peck, L.S.
2015. Adult acclimation to combined temperature and pH stressors significantly enhances
reproductive outcomes compared to short-term exposures. Journal of Animal Ecology, 84 (3):773-784.
These studies (2 and 3) further challenge the suitability of directly transferring present day ambient
sourced early life stages to future conditions (see study 1 above). We widened the focus across theentire life cycle by pre-exposing parents prior to assessing offspring responses to future climatechange conditions. The authors for the first time present temporal models to identify when marine
invertebrates are able to acclimate to future ocean acidification conditions. It was shown that it
takes at least 42 days exposure to for temperate European Green Sea Urchins (Psammechinus
miliaris) adults and 8 months for the Antarctic Sea urchin (Sterechinus neumayeri) to acclimate to
IPCC predicted climate change conditions. These studies are important because they highlight theneed to utilise longer exposure times in predictive experimental designs – by needing toincorporate at least one full gametogenic cycle within experimental designs for predicting

organismal responses to future changes to determine whether acclimation is possible. This timeframe will differ depending on the latitudinal source of your study animals.
Kelly, M.S., Carboni, S., Cook, Hughes, A. 2015. Sea urchin aquaculture in Scotland. In:
Echinoderm Aquaculture (Eds. Brow, N. Eddy, S.). Wiley Blackwell, New Jersey. pp. 211-224.
This chapter outlines the echinoderm aquaculture effort in Scotland (my previous work place). It
highlights that the model species (Psammechinus miliaris – the European Green Sea Urchin) I
have utilised in my climate change studies (e.g. see studies 1-3 above) has been identified as agood candidate for aquaculture. It provides an overview of examples to support its use inaquaculture – i.e. gonad deposition and colour can be positively influenced via pigmented finishingdiets.

14. The Influence of Climate and Environment on the Spawning, Condition, and Larval Set of
Naturalized Pacific Oysters Crassostrea gigas in a US West Coast Estuary
Brett R. Dubauld1*, Alan J. Trimble2, Jennifer L. Ruesink2, and Zachary Forster3
Presenting author*
1

USDA-ARS, Hatfield Marine Science Center, Newport, OR 97365, USA, *Email:brett.dumbauld at ars.usda.gov
2
Department of Biology, University of Washington, Seattle, WA 98195, USA
3
Washington Dept. of Fish and Wildlife, Ocean Park, WA. 98640, USA
Abstract
The Pacific oyster Crassostrea gigas was introduced to the US west coast in the early 1900’s
where it replaced the native oyster Ostrea lurida and has become the mainstay for the shellfish
aquaculture industry. Pacific oysters only regularly spawned and became “naturalized” inseveral discrete estuarine locations where conditions allowed for both adult oyster conditioning
and spawning and larval survival, retention and settlement. The shellfish industry relied on“natural” set at these locations and/or continued to import seed from Japan until the advent of
hatchery technology in the late 1970’s. Since that time the industry has gradually shifted tohatchery production of oyster larvae with large operators establishing their own in-househatcheries and smaller companies incorporating remote setting facilities and purchasing productfrom hatcheries that specialize in producing late stage eyed larvae. Most operators continued totake advantage of “wild” set until a prolonged set of failures in larval production termed the seedcrisis occurred in both hatcheries and in these estuaries like Willapa Bay, Washington where
wild set was and continues to be a feature of management on state established oyster reserves.
The failures at hatcheries have been clearly linked to water chemistry and ocean acidification
where aragonite saturation state is reduced and larval oysters are unable to deposit shell, butevidence for direct effects of carbonate chemistry in estuaries like Willapa Bay is more equivocal
due to complex interactions with other factors. Variables like temperature, salinity, andphytoplankton as food as well as other sources of larval mortality are still related to theproximity of the ocean and cold upwelled water, but have been less studied in this estuary andsome of these factors also influence gametogenesis and spawning in adult oysters. Wesummarize historical records for spawning and setting in this naturalized Pacific oyster
population including a long term record of oyster condition. We propose a new effort to evaluate
some of these additional factors and compare these records with data from locations in Japan
where these oysters were originally sourced and other locations with similar climate records
where Pacific oysters have been introduced in order to understand and potentially mitigate forthe effects of anthropogenic changes to these systems and an uncertain future climate.
Annotated Bibliography of Key Works
Barton, A., G.G. Waldbusser, R.A. Feely, S.B. Weisberg, J.A. Newton, B. Hales, S. Cudd, B.
Eudeline, C.J. Langdon, I. Jefferds, T. King, A. Suhrbier, and K. McLaughlin. 2015. Impacts of
Coastal Acidification on the Pacific Northwest Shellfish Industry and Adaptation Strategies
Implemented in Response. Oceanography 28: 146-159. This is the most recent review of thehistory and science underpinning the effects of changing seawater chemistry on bivalve shellfish
larvae and the impacts that have already taken place to the commercial shellfish aquacultureindustry on the US West Coast. Multiple authors contributed to this review which addresses a
broad audience but covers the leading research on direct effects to bivalve larvae as well as
monitoring seawater conditions and adapting to these changes.
Dumbauld, B.R., J.L. Ruesink, A.C. Trimble, and B.E. Kauffman. 2011. The Willapa Bay oyster
reserves in Washington State: Fishery collapse, creating a sustainable replacement, and thepotential for habitat conservation and restoration. Journal of Shellfish Research 30: 71-83. The
authors review the history of the state oyster reserves in Willapa Bay, Washington where Pacificoysters were introduced in 1928 and one of a few locations on the US West Coast where anaturalized population became established. They review the record of larval set in this estuary
and attempt to link this to broad scale oceanographic conditions (but see Hales et al 2017 andRuesink et al. 2018 below for a more detailed discussion of water chemistry within this estuary).
Hales, B., A. Suhrbier, G.G. Waldbusser, R.A. Feely, and J.A. Newton. 2017. The carbonate
chemistry of the "Fattening Line," Willapa Bay, 2011-2014. Estuaries and Coasts 40: 173-186.
The authors present detailed data on seawater chemistry (especially PC02 and aragonite
saturation state) for Willapa Bay, Washington where Pacific oysters have been the mainstay ofthe oyster aquaculture industry for almost 100 years and there is a long term record of spawningand setting. They reconstruct this record for a longer historical period and their data suggest thatrecent conditions provide a smaller window of optimal conditions (low aragonite saturation state
and warm enough temperatures for oyster spawning) than occurred historically. While they did
not sample larvae (see Ruesink et al 2018 below) and therefore can’t confirm effects, they
substantiate the complexity of measuring these effects and attributing them to a single cause inan estuary where conditions are variable.
Ruesink, J.L., A. Sarich, and A.C. Trimble. 2018. Similar oyster reproduction across estuarine
regions differing in carbonate chemistry. Ices Journal of Marine Science 75: 340-350. Theseauthors measured seawater chemistry in Willapa Bay,Washington, but unlike Hales et al (2017) ,they also present simultaneously collected data on four cohorts of Pacific oyster larvae that werecollected over three summers. The southern end of Willapa Bay has two arms which create
distinctly different characteristics because one is much more affected by riverine conditions thatcause reduced aragonite saturation relative to the other. Oyster settlement differed greatlybetween cohorts, but they did not find differences they could attribute to this different waterchemistry and instead found thermal conditions were perhaps more important.
Weisberg, S.B., N. Bednarsek, R.A. Feely, F. Chan, A.B. Boehm, M. Sutula, J.L. Ruesink, B.
Hales, J.L. Largier, and J.A. Newton. 2016. Water quality criteria for an acidifying ocean:
Challenges and opportunities for improvement. Ocean & Coastal Management 126: 31-41. This
review represents a call for developing ecologically relevant water quality criteria foracidification and augmenting coastal water quality monitoring efforts to characterize this at
appropriate temporal and spatial scales. Historical data for oyster larval recruitment in Willapa
Bay is one of two case examples presented that demonstrate the challenges involved in relating
biological endpoints to appropriate water chemistry criteria in estuaries where conditions arevariable.

15. The influences of environmental changes on Japanese Nori mariculture
Mahiko Abe*1, Noboru Murase1, Masahiro Kobayashi2, Eiji Fujiyoshi3 and Motoya Tamaki3
Presenting author*
1

National Fisheries University, FRA, Shimonoseki, Yamaguchi 759-6595, Japan, *Email:abemahi at fish-u.ac.jp
2
National Research Institute of Fisheries Science, FRA, Yokohama, Kanagawa 236-8648, Japan
3
Seikai National Fisheries Research Institute, FRA, Nagasaki, Nagasaki 851-2213, Japan
Abstract
Nori mariculture has a long history of over one hundred years and is one of the most importantfisheries industries in Japan. Recently, nori production has decreased due to environmental
changes, such as seawater temperature rise and low nutrient levels. Seawater temperature risedelays the start of nori mariculture because water temperature stays above 23ºC in autumn andspores begin to be released, thereby shortening the season. Low nutrient levels in the ambient
waters cause bleaching of the thalli, reducing the quality and price of nori. Now, we are trying tofind new culture species and new strains of nori around the coasts of Japan. The thallus has asimple structure, and cells within the thalli sometimes have mutations. Mutant cells may havepotential for tolerance of high water temperatures and low nutrient levels, as well as other favorable
characteristics. More recently, shortening of the thalli is observed frequently. One of the causes ispredation by herbivorous fishes (i.e. Acanthopagrus schlegelii, Mugil cephalus cephalus ) andducks (i.e. Anas penelope, Anas acuta). In Japan, it has been reported that herbivorous fishes cause
disappearance of marine forests, which is referred to as “isoyake”. The duration of isoyake causedby herbivorous fish has become longer than before because of water temperature rise. The isoyake
caused by activities of the fish is reduced below 17ºC. On the other hand, the herbivorous fishes
eating nori are still active at low water temperature. Even if some measures are taken to control
herbivorous fish, birds would alternatively eat nori. It is necessary to develop effective measures
to control these herbivores immediately.
Annotated Bibliography of Key WorksAbe, M., Kobayashi, M., Fujiyoshi, E., Tamaki, M.., Kikuchi, N., Murase, N. 2013. Use of PCRRFLP for discrimination of Japanese Porphyra and Pyropia species (Bangiales, Rhodophyta). J.
Appl. Phycol. 25, 225-232. DOI. 10.1007/s10811-012-9856-7
In this paper, the authors developed a PCR-RFLP method useful for discrimination of 16
Japanese and two non-Japanese Porphyra and Pyropia species. These species have simple
morphological features, making morphological species identification difficult. The two primer
sets used in this study were able to amplify with PCR single fragments on the mitochondrial
DNA (partial mitochondrial DNA related to ATP6 gene and trnC – rns). All 18 species weresuccessfully distinguished using a combination of five restriction enzymes (TaqI, SspI, AciI,
Cfr13I and AluI). It was concluded that PCR-RFLP analysis is a useful tool for discrimination ofwild strains of Porphyra and Pyropia species for potential use in mariculture.
Nakayama, T., Abe, M., Murase, N., Shikano, Y. 2017. Influence of salinity on growth of red
alga Pyropia tenuipedalis and Pyropia yezoensis foliose thallus. Aquacult. Sci. 65, 321-330.
Pyropia tenuipedalis is a new culture species in Yamaguchi Prefecture, Japan. The habitat of thisspecies is more brackish as compared with P. yezoensis, which is the common species inJapanese nori mariculture. In this paper, the authors investigated the relationship between salinity
and growth of P. tenuipedalis and P. yezoensis. It was revealed that P. tenuipedalis tolerates
lower salinity in comparison with P. yezoensis.
Abe, M., Fujita, Y., Kobayashi, M., Fujiyoshi, E., Tamaki, M., Fukui, Y., Satomi, M., Murase,
N. 2015. Effects of antibiotics on survivals and growth for Pyropia yezoensis protoplasts.
Aquacult. Sci. 63, 1-8.
The nori thallus sometimes has cell mutations. For the breeding of Pyropia species, it wasnecessary to develop an axenic cell culture method. In this paper, we investigated a way of
isolating axenic protoplast of P. yezoensis and the effects of twelve antibiotics on the survivaland growth of the protoplasts. The axenic protoplasts could be isolated by a series of treatmentswith sodium citrate seawater. The growth rates of the protoplasts treated with FRM and ABPC
were 1.1–2.7 times higher than that of the control, while PCG and SM suppressed growth. The
authors succeeded in developing an axenic culture method of Pyropia species, but the survival,
growth and morphogenesis of the cells in the axenic culture varied. Improvement of themethodological stability is necessary.

16. Potential of seaweed aquaculture to reduce methane emissions in Californian livestock
Luke Gardner1,2,*, Michael Graham2, Scott Hamilton2, Kenneth Kalscheur3, Stacey Gunter4
Presenting author*
1

California Sea Grant, University of California, San Diego, Scripps Institution of Oceanography,
9500 Gilman Drive, Dept. 0232, La Jolla, California 92093, USA, *Email: lgardner at ucsd.edu
2
Moss Landing Marine Laboratories, 8272 Moss Landing Rd., Moss Landing, California 95039.
3
United States Department of Agriculture - Agricultural Research Service, U.S. Dairy Forage
Research Center, 1925 Linden Drive, Madison, Wisconsin 53706.

4

United States Department of Agriculture - Agricultural Research Service, Southern Plains
Range Research Station, 2000 18th Street, Woodward, Oklahoma 73801.
Abstract
Release of greenhouse gases into the atmosphere is a major environmental, social, and economic
threat worldwide. Methane is a particularly potent greenhouse gas that is 25 times more efficientat trapping heat in the atmosphere than carbon dioxide. It is well documented that ruminant
livestock contribute significantly to the global release of methane via digestion. The UnitedStates is the fourth largest cattle producer in the world and cattle contribute 26% of the total U.S.
methane emission. Dairy cattle are the largest methane producers among livestock. California isthe dairy industry’s most productive state and has 1.4 million dairy cows that account for the
majority of the enteric methane emission. Consequently, California recently enacted legislationin 2016 mandating a 40% reduction from present day methane emissions by 2030 (2016).
Several methane reductions are expected to come through existing technologies associated withlandfills and energy producers. However, the state’s largest contributor to methane production iscattle and methods to reduce gases produced from cattle have largely been unexplored in
California. Elsewhere, recent research has shown that some species of sub-tropical seaweed can
reduce methane production in cattle by up to 99% when used as a feed additive at 2% inclusion
rates of organic matter. The mode by which seaweed reduces methane production is still unclear,but most recent research attributes the reductions to the bioaccumulation of halogenated methane
analogues produced as algal secondary metabolites. The advantages of seaweed over otherdietary manipulations is that seaweed has shown the most significant methane reductions withminimal declines in digestibility, and seaweed production does not compete with terrestrial foodproduction systems. Temperate species have not yet been assessed for methane reducing
properties, but many of the species present in the California Current Ecosystem are closely
related to these sub-tropical species. The nutrient-rich waters off the Californian coast are ideal
for the development of seaweed aquaculture, offering an excellent opportunity to address theunderdevelopment of aquaculture in California and the reduction in methane emissions.
Annotated Bibliography of Key Works
Machado, L., Magnusson, M., Paul, N. A., de Nys, R., Tomkins, N., 2014. Effects of Marine andFreshwater Macroalgae on In Vitro Total Gas and Methane Production. PLOS ONE. 9, e85289.
The authors for the first time assess an array of different sub-tropical seaweeds for methane
reducing properties in cattle using in vitro techniques. They find that a red seaweed species
called Asparagopsis taxiformis is the most effective seaweed tested reducing methane production
by up to 99%.
Maia, M. R., Fonseca, A. J., Oliveira, H. M., Mendonca, C., Cabrita, A. R., 2016. The Potential
Role of Seaweeds in the Natural Manipulation of Rumen Fermentation and Methane Production.
Sci Rep. 6, 32321.
This research is important in that it assesses the effect of the basal feed source (substrate) on
methane reduction for a number of different seaweed species. The authors find that there is asignificant effect on methane reduction based on whether the basal feed is either grass or cornhighlighting the need to further examine the seaweed interaction with different cattle feeds.

Li, X., Norman, H. C., Kinley, R. D., Laurence, M., Wilmot, M., Bender, H., de Nys, R.,
Tomkins, N., 2018. Asparagopsis taxiformis decreases enteric methane production from sheep.
Animal Production Science. 58, 681-688.
The authors for the first time assess the effect of seaweed on methane reduction using a wholeanimal experimental design. The article reports that sheep feed a 3% diet of seaweed
Asparagopsis taxiformis reduce their methane emissions by 81% without any decrease in weightgain compared to the control treatment.

17. Condition index and fecundity of Manila (asari) clam Ruditapes philippinarum related tohabitat environment
Natsuki Hasegawa*1, Toshie Matsumoto1, Satoshi Watanabe1, Kazuhiro Hanyu2, Dai Miyawaki3,
Ryogen Nanbu4, and Masami Hamaguchi5
Presenting author*
1*

National Research Institute of Aquaculture, Japan Fisheries Research and Education Agency,
422-1 Nakatsuhamaura, Minamiise, Mie, 516-0193, Japan, *Email: hasena at fra.affrc.go.jp
2
Suzuka Branch, Mie Prefecture Fisheries Research Institute, Suzuka, Mie, 510-0243, Japan
3
Aichi Fisheries Research Institute, Gamagoori, Aichi, 443-0021, Japan
4
National Research Institute of Fisheries Engineering, Japan Fisheries Research and Education
Agency, 7620-7, Hasaki, Kamisu, Ibaraki, 314-0408, Japan
5
National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research
Agency, 2-17-5, Maruishi, Hatsukaichi, Hiroshima, 739-0452, Japan
Abstract
Reproductive traits are important information for maintenance or recovery of Manila (asari) clam
Ruditapes philippinarum fisheries production, which has dropped considerably in Japan over the
past half century. Relationships between condition index and the number of eggs (fecundity) of
the asari clam were assessed at several clam habitats in Ise Bay, Japan, during spring and autumn
spawning seasons in 2015. Condition index was calculated from the shell size and soft body wetweight. Fecundity was quantified using an indirect enzyme-linked immunosorbent assay (ELISA)
with monoclonal antibody specific to an asari clam yolk protein. Land elevation (i.e. subtidal orintertidal), association with river input (river mouth or not), closedness of the shore andchlorophyll a concentration in the water column were the habitat parameters analyzed in this study.
A correspondence analysis revealed that the condition index in both spawning seasons was
positively corelated (r = +0.48 to +0.80) with the 1st and 2nd correspondence analysis scores on
various habitat parameters. On the other hand, the fecundity, which is summed for each spawningseason (three sampling days) and normalized to 30 mm shell length, was negatively correlatedwith the 2nd habitat score (spring: r = -0.64, autumn: r = -0.80). These results show that thenutritional condition as determined by the condition index is not the sole factor affecting fecundityof the clam. Actually, the fecundity was high in the intertidal zones and enclosed river mouth areas,
whereas the condition index was higher in the subtidal zones and open non-river mouth areas. The
information obtained about asari clam reproduction would be useful for stock enhancement byimproving reproduction in the populations. This study was conducted as part of a research projectfor recovery of asari populations by Fisheries Agency of Japan.
Annotated Bibliography of Key Works
Toba, M., and Y. Miyama 1994. Relationship of size to gonadal maturation and spawning in
artificially conditioned Manila clams. Nippon Suisan Gakkaishi, 60 (2), 173-178.
In this study, the authors observed sexual maturation and measured
the number of eggs released per spawned female subjected to thermal
spawning stimuli in breeding experiments for evaluating the size
dependent batch fecundity of asari clam. Smaller clams at 0 yr old(groups A and B) produced as many eggs per soft tissue weight aslarger clams at ≥ 1 yr (groups D and E). No significant differences in
egg diameter, percent fertilization, and larval survival fromstraight-hinge to pediveliger stage were observed among the clam
size groups.
Kakino, J. 1996. Growth and survival of Japanese littleneck clam Ruditapes philippinarum incage test related to the sharpness index. Nippon Suisan Gakkaishi, 62 (3), 376-383.
The author evaluated the variation in asari clam growth in a tidal flat in Tokyo Bay, Japan, byusing the “sharpness index” defined as width / length of shell. The author concluded thatjuveniles of the clam offshore grew faster than those that were shoreside, but the juvenile
survival rate in the intermediate zone of the flat was higher than those in the offshore andshoresides. Juveniles having a high sharpness index tended to show a high survival rate. At thesame age, the juveniles with a high sharpness index grew faster than or equal to those with a low
sharpness index.

18. Ecological interactions of horseshoe crabs and shellfish aquaculture: a case study fromNew Jersey
Daphne Munroe
Haskin Shellfish Research Laboratory, Rutgers University
6959 Miller Ave., Port Norris, NJ, 08349, USA, Email: dmunroe at hsrl.rutgers.edu
Abstract
Globally, shellfish aquaculture is growing rapidly. For the first time in history, global seafood
supply from aquaculture exceeded that of wild fisheries. Although shellfish culture is viewed as
an ecologically sound industry, as farm production expands it faces key challenges assuringecological and social sustainability. Understanding the ways shellfish farms interact with mobile
coastal wildlife, particularly species of special concern, is among those challenges (Barrett et al.,2018; Collier et al., 2018).
Farms for Eastern oyster (Crassostrea virginica) are commonly located in shallow coastal areas
along the US east coast, use a range of equipment, and involve regular access to care for andharvest livestock. In some cases, these farms are located in areas used by American HorseshoeCrabs (Limulus polyphemus) as they come ashore during spring to spawn along sandy Atlantic
Coast beaches. Horseshoe crabs are economically and ecologically important; their blood is highly
valuable in the medical industry and migratory shorebirds including the red knot (Calidris canutusrufa), a threatened migratory shorebird, feed on crab eggs when stopping in Delaware Bay.
Limited studies have been done to examine the interactions among horseshoe crabs and intertidal
oyster farms (Kwan et al., 2018 and Munroe et al., 2017), and recently concern has been raised
about horseshoe crab ability to traverse oyster farms to reach spawning habitat. A case study
examining potential farm interactions with Horseshoe Crabs was done in Delaware Bay, New
Jersey, during the 2018 crab spawning season. Experiments included a range of controlled
experiments and surveys during high and low tide using sonar and walking respectively, to observe
crab behavior at farm and non-farm sites. In all cases, results indicate that crabs can successfully
traverse farms and reach spawning beaches, and crabs do not differentially use farm versus nonfarm areas of the Delaware Bay flats. These results provide important context for developing
frameworks for managing ecological interactions among farms and species of concern.
Annotated Bibliography of Key Works
Barrett, Luke T., Stephen E. Swearer, and Tim Dempster. 2018. Impacts of marine andfreshwater aquaculture on wildlife: a global meta‐analysis. Reviews in Aquaculture. 1-23. doi:
10.1111/raq.12277
In this review, authors from the University of Melbourne in Australia demonstrate that evidencein support of higher wildlife biomass and diversity is found around aquaculture farms. They
argue that to properly understand the possible impacts of farms on wildlife, studies have thus far
failed to address issues of impacts on behavior, reproduction or fitness. If animals that tend toaggregate around farms suffer poor fitness relative to those occupying non-farm habitats, thefarm becomes an ‘ecological trap’.
Callier, M.D., Byron, C.J., Bengtson, D.A., Cranford, P.J., Cross, S.F., Focken, U., Jansen, H.M.,
Kamermans, P., Kiessling, A., Landry, T. and O'beirn, F., 2017. Attraction and repulsion ofmobile wild organisms to finfish and shellfish aquaculture: a review. Reviews in Aquaculture.
The ways in which finfish and shellfish farms act as attractant or repulsive areas for mobile
marine species is reviewed. At finfish farms, food inputs and physical structure of the farm tend
to drive aggregating effects. Likewise, at shellfish farms, the farm structure (both farm gear andshellfish themselves) and increased feeding opportunities via biodeposition, the shellfish crop or
fouling organisms tend to attract wildlife to farms. In general, the authors note that a great dealof variability in attractiveness or repulsiveness exists when considering the suites of habitats,farms, and mobile fauna that have been studied thus far.
Kwan, Billy KY, Hoi Kin Chan, and Siu Gin Cheung. 2018. Habitat use of globally threatened
juvenile Chinese horseshoe crab, Tachypleus tridentatus under the influence of simulated
intertidal oyster culture structures in Hong Kong. Aquatic Conservation: Marine and Freshwater
Ecosystems 28: 124-132.
The authors conducted this study to test if juvenile Chinese horseshoe crabs differ in abundance
and if their feeding trails differ at oyster farm spat collection locations. They performed their test
at a shallow mudflat in Hong Kong which is home to a large population of juvenile horseshoecrabs. Addition of oyster spat collectors (bricks on the mudflat) lead to a significant reduction in
the number of juvenile horseshoe crabs, and a reduction in the length of feeding trails.
Munroe, D., Bushek, D., Woodruff, P., Calvo, L. 2017. Intertidal rack-and-bag oyster farms
have limited interaction with Horseshoe Crab activity in New Jersey, USA. Aquaculture
Environment Interactions, 9: 205-211. https://doi.org/10.3354/aei00227
In this study, the authors sought to assess whether intertidal oyster farms altered the distributionof mature horseshoe crabs as they came ashore to spawn in the lower Delaware Bay. In total,they used a combination of field surveys of crabs on mudflats and stranded along the upper
shore, and controlled tests to determine if crabs can move past farm gear. Results show noevidence that horseshoe crab distribution within this area is altered by the presence of oyster
farms.

19. Use of GoPro Cameras to Document Fish on Aquaculture Gear
Gillian Phillips*1, 2, Renee Mercaldo-Allen1, Peter Auster3, Paul Clark1, Erick Estela1,
Yuan Liu1,2, Lisa Milke1, Dylan Redman1, Julie Rose1
Presenting author*
1

NOAA Fisheries, Northeast Fisheries Science Center, Milford Laboratory,
212 Rogers Avenue, Milford, CT, 06460, USA, *Email: gillian.phillips at noaa.gov
2
Integrated Statistics, 16 Sumner Street, Woods Hole, Massachusetts, 02543, USA
3
University of Connecticut, Department of Marine Sciences & Mystic Aquarium,
1080 Shennecossett Road, Groton, CT, 06340, USA
Abstract
Oyster cages are an increasingly common style of aquaculture gear in Long Island Sound. Cages
are used to contain and protect shellfish during grow-out but also provide three-dimensional
structure for the local wild fish community. Understanding the ecosystem services provided byaquaculture gear may inform regulatory decisions and increase public understanding of shellfish
farming. Point-of-view (GoPro) cameras attached to oyster cages were used to visually assessfish activity near a high-density cage farm as well as adjacent single cages. To compare fishinteractions with cages to natural structured rock-reef habitat, t-platform stands were fabricatedto mount cameras adjacent to boulders while minimizing additional structure. Seawater samples
were collected for environmental DNA (eDNA) analysis to detect fish species that may not be
observed in video. Video collected by cameras attached to oyster cages and near boulders
facilitated visualization and quantification of the wild fish communities along with behavioral
interactions between animals and these physical elements of habitat. Preliminary results suggestthat aquaculture cages support a higher abundance and greater diversity of fish species and life
stages than did the natural rock-reef habitat. Analysis of eDNA detected differences in fish
community composition between sites.
*The Federal Government does not endorse the use of GoPro™ cameras

Annotated Bibliography of Key Works
Watson D.L., Harvey E.S., Anderson M.J., and Kendrick G.A. 2005. A comparison of temperate
reef fish assemblages recorded by three underwater stereo-video techniques. Marine Biology.
Vol 144. Pp 415-425.
The authors deployed underwater stereo-video cameras to sample the relative density and species
richness of temperate reef fishes in Southwest Australia. This study compared diver deploy, unbaited remote and baited remote cameras. Their definition of MaxN “Species presence and the
maximum number of individuals belonging to each species in the field of view at one time
(MaxN).” This metric avoids repeated counting of fish within a given time frame and gives aconservative estimate of relative density relating to area of survey.
Dealteris J.T., Kilpatrick B.D., and Rheault R.B. 2004. A comparative evaluation of the habitat
value of shellfish aquaculture gear, submerged aquatic vegetation and a non-vegetated seabed.
Journal of Shellfish Research. Vol 23:3. Pp 867-874.
The authors set out to evaluate habitat value of shellfish aquaculture gear (SAG) submerged
aquatic vegetation (SAV) and shallow non-vegetated seabed (NVSB) over one year in Port
Judith, RI, USA. To identify the community around oyster cages lift were diver deployed underoyster cages and left to soak for two weeks. Submerged aquatic vegetation and non-vegetatedseabed sites were sampled with quadrats, drop nets, and venture-driven suction dredge. Sessile
invertebrate growth was documented on eelgrasses and on oysters and oyster cages. All fish
>5mm were collected. The authors documented that the physical habitat of shellfish aquaculture
gear has greater surface area compared with the other two habitat types as well as had asignificantly higher abundance and species richness of organisms per meter squared throughout
the year. The authors concluded that shellfish aquaculture gear has greater habitat value
compared to their control sites.
Tallman J.C. & Forrester G.E. 2007. Oyster grow-out cages function as artificial reefs fortemperate fishes. Transactions of the American Fisheries Society. Vol 136:3. Pp 790-799.
The authors compared fish habitats within Narragansett Bay, RI, USA using trap surveys. They
compared three oyster grow-out sites, six natural rock reefs and one artificial reef built for fish
habitat looking to identify patterns in fish density, growth and disappearance rates. Their traps
were encompassing juvenile and adult fish in the summer and fall. The study showed that oyster
cages provide habitat for fish associated with hard bottom habitats including scup, tautog andblack sea bass.
Dumbauld, B.R., J.L. Ruesink, and S.S. Rumill. 2009. The ecological role of bivalve shellfish
aquaculture in the estuarine environment: A review with application to oyster and clam culture inWest Coast (USA) estuaries. Aquaculture Vol 290. Pp 196-223.
This review article focuses on bivalve shellfish aquaculture on the US West Coast. Much likenatural rock reefs, cages become colonized by epibenthic, emergent, and encrusting organisms
and may provide sheltering fish with camouflage, refuge from predation, respite from highcurrent flow, and a source of food. Multi-tiered off-bottom cages, an increasingly popularmethod for growing oysters on a smaller footprint, offer vertical three-dimensional structure andsurface area similar to that afforded by natural reefs. In traditional on-bottom shellfish culture,
where live oysters and shell cultch are distributed on sediments for grow-out and spat collection,oysters act as ecosystem engineers creating hard bottom irregular substrate on otherwise

featureless seafloor. Evaluation of habitat services provided by oyster aquaculture can bechallenging. Shellfish farms vary in spatial size and bottom area covered, variety and quantity of
aquaculture gear, and have a seasonally variable footprint as gear is relocated or harvested.
Aquaculture of oysters, whether in cages or grown on the seafloor, may represent valuable
estuarine habitat.

Closing remark of 46th scientific symposium of UJNR
Dr. Fuminari Ito, Japan chair

Good afternoon, everyone.
Now, we come to close the 46th Scientific Symposium of the UJNR Aquaculture Panel, “Marine
Aquaculture in a Changing Environment, impacts to aquaculture production due toenvironmental change and science to mitigate these impacts”.
First of all, I would like to congratulate all of you for the successful conduct of this ScientificSymposium. I am quite sure that we would be able to have another good result of cooperative
activities among the Aquaculture Panel, one of the most active UJNR panels.
The 10th Three-Year Plan is “Marine Aquaculture in a Changing Environment". The Special
Report on Global Warming of 1.5°C (SR15) was accepted during the 48th session of theIntergovernmental Panel on Climate Change (IPCC 48) held in Korea this October. There has
been an increasing interest in risks, adoptability and sustainability of varied systems which could
possibly be influenced by global climate change. In the field of aquaculture, it is also importantto further conduct research and to develop technologies to tackle issues associated withenvironmental change.
Last year, the first scientific symposium was held under the theme “potential of aquaculture to
mitigate impacts of environmental change” in Hiroshima, Japan. We exchanged information anddiscussion on potentials for sustainable seafood production through aquaculture of a variety of
species in different environments. Today we discussed in detail the effects of ocean acidification
on aquaculture organisms, and so on.（当⽇の感想を挿⼊）
Next year will be the final year of the three-year plan, and the theme for the symposium, which isplanned to be held in Japan, will be “Application of Aquaculture Technology to Provide
Sustainable Seafood and Reduce Impacts of Environmental Change.”In the next year's symposium, we will be able to share scientific knowledge about the latest
research results for the future of aquaculture.
Through these three-year symposium presentations and discussion, I believe that we will
successfully deepen the issue of counter measures for global environmental change, which are
expected to contribute in the development of the sustainable aquaculture industry in not only the
US and Japan, but also the rest of the world.
Finally, I would like to extend my deep appreciation to Dr. Rust, Dr. Olin, Mr. Otoshi, who
could not unfortunately join the symposium, and all the staff concerned for their great
contributions, which led to the success of this symposium.
Again, I would like to express my heartfelt congratulations and declare this symposium closed.
Thank you very much for your cooperation, and good afternoon.