Aquaculture and Stock
Enhancement of FinﬁshProceedings of the Thirty-fourth
U.S.-Japan Aquaculture Panel
Symposium
San Diego, California
November 7–9, 2005
Robert Stickney, Robert Iwamoto, and Michael Rust, editors© 2002 Natalie Fobes, all rights reserved

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

National Marine Fisheries Service
NOAA Technical Memorandum NMFS-F/SPO-85

Aquaculture and Stock Enhancementof Finﬁsh

Proceedings of the Thirty-fourth
U.S.-Japan Aquaculture Panel
Symposium
San Diego, California
November 7–9, 2005
Robert Stickney, Robert Iwamoto, and Michael Rust, editors

NOAA Technical Memorandum NMFS-F/SPO-85
October 2007

U.S. Department of Commerce
Carlos M. Gutierrez, Secretary
National Oceanic and Atmospheric Administration
Vice Admiral Conrad C. Lautenbacher, Jr., USN (Ret.)
Under Secretary for Oceans and Atmosphere
National Marine Fisheries Service
William T. Hogarth, Assistant Administrator for Fisheries

Suggested citation:
Stickney, R., R. Iwamoto, and M. Rust (editors). 2007. Aquaculture and Stock
Enhancement of Finﬁsh: Proceedings of the Thirty-fourth U.S.-Japan Aquaculture
Panel Symposium, San Diego, California, November 7–9, 2005. U.S. Dept.
Commerce, NOAA Tech. Memo. NMFS-F/SPO-85, 76 p.
A copy of this report may be obtained from:
Northwest Fisheries Science Center
2725 Montlake Boulevard East
Seattle, Washington 98112
Or online at:
http://spo.nmfs.noaa.gov/tm/
Reference throughout this document to trade names does not imply endorsement by
the National Marine Fisheries Service, NOAA.

ii

Table ofContents

Introduction
Species Reports
SeriolaCurrent Situation of Technical Developments in Seed
Production of Yellowtail (Seriola quinqueradiata) in Japan1

Keiichi Mushiake, Hideki Yamazaki, and Hiroshi Fujimoto

Sciaenids

Culture of Spotted Seatrout (Cynoscion nebulosus) in a Closed,
Recirculating System

5Reginald B. Blaylock, Angelos Apeitos, Jason T. Lemus, andJeffrey M. Lotz

Disease Surveillance in Wild and Cultured Stocks of White
Seabass (Atractoscion nobilis)

11Mark S. Okihiro

Sebastes

Juvenile Mass Production of Black Rockﬁsh (Sebastes
schlegeli) in FRA Miyako Station: Present Status and Problems

21

Masahiro Nakagawa, Junichi Adachi, Ken Shimizu, andSatoshi Shiozawa

Paralichthys

Construction of a BAC Library from XY Japanese Flounder
Using Frozen Sperm Genomic DNA

27

Hiroyuki Okamoto, Hiroyuki Nagoya, Eiichi Yamamoto, Takashi
Sakamoto, Kanako Fuji, Nobuaki Okamoto, Kazuo Araki, andIchiro Nakayama

Scombrids
Status of Paciﬁc Blueﬁn Tuna (Thunnus orientalis) Seed
Production in Amami Station of NCSE FRA

31

Hideki Nikaido, Takayuki Takebe, Kentaro Ide, Hitoshi Imaizumi,
Nobuhiro Tezuka, and Syukei Masuma

Genetics and Reproduction

Visualization Tools to Probe Early Stage Fish Abnormalities

37

Susumu Uji and Tadahide Kurokawa

Special Topics

Airlift-PolyGeyser Combination Facilitates Decentralized
Water Treatment in Recirculating Marine Hatchery Systems

43

Ronald F. Malone and Sripavani Gudipati

Preparation of Marine Silage and its Potential for Industrial
Use

51

Motoharu Uchidaiii

Special Topics (continued)

Techniques for Live Capture of Deepwater Fishes with Special
Emphasis on the Design and Application of a Low-costHyperbaric Chamber

57

Jeffrey E. Smiley and Mark A. Drawbridge

A Preliminary Report on the Use of Porphyra Protoplast as a
Food Substitute for Culturing Aquatic Animals

63

TakaoYoshimatsu, Alok Kalla, Toshiyoshi Araki, Dong-Ming Zhang,and Shuichi Sakamoto

Aquaculture and Stock Enhancement Technologies Based on
Recently Discovered Calcium-sensing Receptors in Finﬁsh

69

Steve Jury, H. William Harris, Timothy Linley, David Russell,
Marlies Betka, Susan Anderson, Shinji Harakawa, and Akikuni Hara

Biological Studies on the Digestive Mechanism in Abalone
Kentaro Niwa, Hideaki Aono, and Tomoo Sawabe

iv

75

Introduction

The U.S.-Japan Cooperative Program in Natural Resources (UJNR) was establishedon January 29, 1964. Since its creation, the UJNR has evolved to become one ofthe oldest and most effective cooperative agreements between Japan and the UnitedStates. In 1969, the UJNR Aquaculture Panel was created as a vehicle for scientistsof both countries to meet and discuss aquaculture research accomplishments, needs,
and priorities, as well as provide opportunities for cooperative research and scientiﬁcexchange.
Under the auspices of the UJNR Aquaculture Panel, U.S.-Japan scientists havemet annually since 1971 without interruption. The venue for these meetings hasalternated between the two countries. In November 2005 the meeting (comprisedof a business meeting, scientiﬁc symposium, and several ﬁeld trips) was held at the
Hubbs-SeaWorld Research Institute, San Diego, California, and nearby environs. The
scientiﬁc symposium was coordinated by Mark Drawbridge of Hubbs Sea-World and
Dr. Kenneth Leber of Mote Marine Laboratory, Sarasota, Florida. By prior agreementof the U.S.-Japan panels, contributors to the proceedings were requested to submit
manuscripts in the form of extended abstracts.
Yasuji Sakai, Japan
Robert Iwamoto, United Statesv

vi

Current Situation of Technical Developments in Seed Production of Yellowtail(Seriola quinqueradiata) in Japan
Keiichi Mushiake
Headquarters, Fisheries Research Agency
15th Floor Queen’s Tower B
2-3-3 Minatomirai, Nishi-ku, Yokohama
Kanagawa, Japan, 220-6115
mushiake@fra.affrc.go.jp
Hideki Yamazaki
Yashima Station of National Center for Stock Enhancement
FRA, Takamatsu
Kagawa, Japan, 761-0111
Hiroshi Fujimoto
Obama Station of NCSE
FRA, Obama
Fukui, Japan, 917-0117
Keywords: Yellowtail, Seriola quinqueradiata, seed production, broodstock management
Abstract
The National Center for Stock Enhancement (NCSE, formerly Japan Sea-Farming Association), of theFisheries Research Agency, introduced the stock enhancement program for yellowtail (Seriola
quinqueradiata) in 1977. Technical developments in induced spawning as well as larval and juvenile rearing
techniques have increased the production of this species to 1 million juveniles per year at NCSE. This
project faced three major drawbacks: high mortality of larvae, cannibalism, and the smaller size of released
juveniles in comparison with their wild counterparts. The high mortality of larvae was overcome by utilizing
strong aeration during the early larval stages, while cannibalism was controlled by grading juveniles by size
selection. The two-month delay in the spawning season of reared broodstock (the usual spawning season islate April to early May), which caused the smaller size of released juveniles, was solved by developments inadvanced spawning techniques. Photoperiod and water temperature manipulations were used to produce
eggs in February, thus producing yellowtail juveniles that can be released into the wild at a size similar tothat of the wild stock.

Introduction
Yellowtail (Seriola quinqueradiata), which is alarge migratory species that moves north and south inwaters adjacent to the Japanese archipelago, is an
important species of the fishery stock in westernJapan and the coastal areas of the Sea of Japan. In
2002, the annual catch was 50,000 tons and theaquaculture industry produced 140,000 tons.
Because there is apprehension that the yellowtail
stock may be reduced by capture of wild juveniles(called mojako) for aquaculture, yellowtail juveniles
were chosen as a target species for sea ranching in
1977. Komame and Goto Stations of the National
Center for Stock Enhancement (NCSE, formerly
Japan Sea-Farming Association), of the FisheriesMushiake

Research Agency (FRA), started to develop thetechniques for broodstock management of yellowtail
in 1977. In 1978 Yashima and Kamiura Stations of
NCSE started seed production of the species,
followed by Goto Station of NCSE in 1981.
Yellowtail is a unique species distributed only
near the coast of Japan. The major spawning groundis the south-central area of the East China Sea where
the spawning season starts around February. In
Shikoku and the northern part of Kyushu, it starts
around April or May, so the season becomes later as
one goes further north (Hamada and Mushiake,2006).

1

Brief history of seed production in yellowtail
Collection of eggs and culture of larvae andjuveniles were started in order to obtain basic
information in 1960. A marked decrease of catch
quantity in 1970s led NCSE to begin the technicaldevelopment for broodstock management in 1977
and seed production in 1978.
In NCSE, the brief history of the technicaldevelopments for yellowtail culture was as follows(Shiozaw and Yamasaki 2006):
1. 1977–1989. NCSE was engaged in seedproduction in large quantities, thereby
enabling the production of one million
juveniles in just one station by 1986.
2. 1990–1995. The technical developments
focused on the quality of seed production,rather than quantity, through the introductionof formulated diets and developments in the
improvement of survival rates during theearly stages.
3. 1996–2005. Since 1996 advanced egg
collection and early seed production
techniques have been implemented.
In the present paper, some technical developments inseed production since 1990 are presented, as well as
the details of both improvement techniques forsurvival in seed production and advanced spawningof broodstock.

Improvement techniques for survival in seedproduction of yellowtail
The drawback of seed production for yellowtail
was the high incidence of larval mortality that
occurred during the early larval stages. Moreover,cannibalism was observed from when they reached atotal length of about 15 mm until they grew tojuvenile size. Those were the two major causes ofmortality, and some measures have been taken tocounteract the problems.
One approach was aeration which led to a highsurvival rate during the early larval stages. In thepast, the sinking of hatched larvae, seen before andafter the mouth opening stage, was a major problem
leading to a high mortality (Shiozawa and Yamasaki.
2006). With the conventional method, slight aeration
was used during the early stages of rearing. The
water in the tank was slightly up-welled vertically,
though only locally in the tanks, leading to highmortalities due to the sinking of larvae. Since 1995,
the problem has been eliminated by employing
stronger aeration using air-block pipes set in eachcorner at the bottom of the tanks. This method
enabled the tank water to have a stronger upwelling2

current that prevented the larvae from accumulating
at the bottom of the tanks. The method markedly
increased the survival rate up to 10 days after
hatching. Compared with the previous method, thesurvival rate at the early stage was increased from
21.7% to 58.1% in 1995 and from 14.6% to 57.4% in
1997.
The other problem was cannibalism. The best
way to prevent cannibalism is to grade the juveniles(Shiozawa and Yamasaki 2006). When yellowtail
reach 10 mm total length (TL), they go up to thesurface at night, so grading is carried out during asingle night when the fish reach around 15 mm TL,
as that is the size when cannibalism is known to start.
The floating, sleeping juveniles are siphoned out of
the rearing tank and transferred to a net pen of 3 mmmesh size in another tank. Only individuals that
couldn’t pass through the mesh remain within the net
thereby separating the fish into two size groups.
Figure 1 shows the total length distribution in thelarge and small sized fish that were selected by thismethod. The fish were clearly divided into twogroups at around 18 mm and the positive effects of
selection are shown by the survival rate. This size
selection at night has clearly doubled the survivalrate, when compared to the nongraded fish. That is,according to size selection at 15 mm TL, the survival
rate of 15–30 mm TL was improved from 33.8% to78.9% in 1994 and from 24.4% to 52.1% in 1995. At
present, survival rates of around 15% at 30 mm in TL
are obtained. In exceptional cases, the survival rate
can even reach up to 30%.

Figure 1. Size frequency (TL) of yellowtail larvae gradedwith a 3 mm mesh net at night when the fish averaged 15 mm
TL.

MushiakeAdvanced spawning of yellowtail broodstock
Broodstock of wild origin were reared in net
cages and used for egg collection. Egg collection
was mainly carried out from late April to early May
until 1995 (Mushiake 1994). The collection was
carried out after the broodstock were maintained at
19°C. When the oocyte diameter reached over 700
micrometers, the broodstock were injected with 600
IU/kg body weight with human chorionicgonadotropin (HCG). The procedure enables egg
collection 48 hours after the hormone injection. The
hormone should be injected when broodstock
maturity is in the last phase of yolk deposition.
About one million eggs were obtained. However, thesize of hatchery-reared juveniles was much smaller
than that of their wild counterparts that were caught
during the same period because of a two month delayin the spawning season of reared broodstock.
Therefore in 1996, in addition to the previous rearing
methods for captive broodstock, a rearing
environment that had short and long photoperiods
and manipulated water temperatures was provided inorder to induce earlier maturation. These
manipulations were followed by HCG injection andspawning was successfully induced between
February and March (Mushiake et al. 1994, 1998).
Now eggs are available for seed production in thehatchery at the same time as the ones that are
spawned naturally in the wild.
As shown in Figure 2, the growth of the hatcheryreared juveniles originated from the advanced
spawning on February, as represented by closed
circles, clearly showed that the juveniles had asimilar size and almost the same growth as their wild
counterparts, as indicated by open circles. In fact, thehatchery-reared juveniles were released with a brandmark into the Seto Inland Sea and collected at a fish
market in December 1999. The body weight of thewild and the released fish was 1.2 kg and both types
traded for the same price in the fish market. The seedproduction at present is capable of producing fish of asimilar size to the wild stock by utilizing eggs
obtained from advanced spawning in February(Fujimoto et al. 2006).
Thus far, the following technical developments
were shown in the present study.
1. The methods of egg collection and seedproduction techniques were the optimal ones
and solve the various problems faced in thepast, so the techniques have been alreadyestablished for yellowtail.
2. With regard to the advanced egg collection
method during February and March, theMushiakehatchery-reared juveniles that were produced
by advanced spawning showed growth rates
to their wild counterparts. These developed
techniques were effective for the promotionof stock enhancement.
3. At present, yellowtail aquaculture depends
on captured wild juveniles. However, there
is a concern that a decrease in wild juveniles
may have some adverse effect on thefundamental management of yellowtail
aquaculture. According to our studies, there
was no difference between yellowtail
produced in advanced spawning and thosehatched in the wild. The next challenge will
be to utilize the hatchery seed of yellowtail
for aquaculture purposes.

Figure 2. Growth of hatchery-reared juvenile yellowtail
originating from advanced spawning in February (closed
circles) compared with wild spawned yellowtail (open circles).

3

References
Fujimoto, H., H. Yamasaki, and A. Suda. 2006. Releasefor stock enhancement. In M. Matsuyama, Y. Hiyama, K.
Mushiake, and H. Yoshiaki (eds.), Biology, stockenhancement and aquaculture of buri [Japanese yellowtail(Seriola quinqueradiata)].
Hamada, K., and K. Mushiake. 2006. Advanced
spawning of yellowtail Seriola quinqueradiata as early as
December by manipulations of both photoperiod and watertemperature. Nippon Suisan Gakkaishi, 72(2):186-192.
Mushiake K. 1994. Studies on technical development ofbroodstock management in striped jack and yellowtail.
Doctoral thesis. Hiroshima University, Higashi-hiroshima.

4

Mushiake, K., K. Kawano, W. Sakamoto, and I.
Hasegawa. 1994. Effect of extended daylength on ovarian
maturation and HCG-induced spawning in yellowtail fed
moist pellets. Fish. Sci. 60:647–651.
Mushiake, K., K. Kawano, K. Kobayashi, and T.
Yamasaki. 1998. Advanced spawning in yellowtail,
Seriola quinqueradiata, by manipulations of thephotoperiod and water temperature. Fish. Sci. 64:727–731.
Shiozawa, S., and H. Yamasaki. 2006. Seed production ofjuveniles. In M. Matsuyama, Y. Hiyama, K. Mushiake,and H. Yoshiaki (eds.), Biology, stock enhancement andaquaculture of buri [Japanese yellowtail (Seriola
quinqueradiata)].

MushiakeCulture of Spotted, Seatrout (Cynoscion nebulosus) in a Closed,
Recirculating System
Reginald B. Blaylock, Angelos Apeitos, Jason T. Lemus, and Jeffrey M. Lotz
Gulf Coast Research Laboratory
The University of Southern Mississippi
P.O. Box 7000
Ocean Springs, Mississippi 39566-7000
reg.blaylock@usm.edu
Keywords: Spotted seatrout, intensive culture, recirculation, Cynoscion nebulosus, larviculture
Abstract
The spotted seatrout (Cynoscion nebulosus) is the most popular sport fish among anglers in the estuarine
and nearshore waters of Mississippi. In partnership with the Mississippi Department of Marine Resources,
we have begun to evaluate the feasibility of using cultured spotted seatrout to supplement the natural stocks
in Mississippi waters. Presently, culture of seatrout consists of captive spawning of broodstock and rearingof larvae in earthen ponds containing estuarine water and mixed wild zooplankton. We have designed arecirculating system in which to rear spotted seatrout larvae and juveniles intensively. As a test of theintensive culture system, approximately 80,000 spotted seatrout (2 day posthatch [PH]) larvae were obtained
from Sea Center Texas in Lake Jackson. Larvae were stocked in eight, 1,000 L tanks at a density of 10
larvae/liter. Feed consisted of ss-rotifers (range 70-110 µm), Artemia nauplii, and dry food. Mean survival
from day 2 PH through day 25 PH was 25% and the larvae had a mean total length of 16.64 mm ±0.79 mm
and a mean weight of 0.04 g ±0.01 g. Cannibalism commenced at day 7 PH. At day 24 PH the juveniles weretransferred to a nursery system. At day 38 PH the fish were transferred to a larger 15×3.5×1 m, 55,000 L
raceway. Survival during the nursery phase averaged 88.84% ±5.80%. Larvae averaged 45.01 mm ±1.93mm and 0.79 g ±0.10 g.

Introduction
Worldwide, marine fish populations are
declining. Current capture fisheries, while relatively
stable over the last 15 years, may be unsustainablepast the year 2040 (New 1997). Of the fish
populations that have been assessed, more than 30%
are currently overfished (Pew Oceans Commission
2003). In response to the growing problem, theMagnuson-Stevens and Sustainable Fisheries acts
required that plans for management and restoration of
overfished species be put in place. Typically, theplans include catch/size limitations or habitat
restoration, but additional techniques to assist
management agencies are needed.
Stock enhancement—the release of cultured fish
into the wild—actually served as the technique of
choice in marine fisheries management throughout
most of the nineteenth and twentieth centuries.
However, the lack of evidence of effectivenesscombined with concerns over maladaptive behaviors,
artificial genetic selection, and disease problems incultured fish created skepticism about the desirabilityof the approach. The advent of new hatchery andtagging technologies, the continued decline ofBlaylockmanaged stocks, and an accumulation of data fromstudies in Japan (Tsukamoto et al. 1989, Tsukamoto
1993), Norway (Svasand et al. 1990), and Hawaii(Leber 1995) inspired Blankenship and Leber (1995)
to revive the idea that successful marine stockenhancement is possible through carefully planned
research that incorporates principles of fish culture,
ecology, health, genetics, and assessment.
The spotted seatrout (Cynoscion nebulosus) is anestuarine and nearshore species ranging from New
York to Texas (Robins and Ray 1986). In thesoutheastern U.S. and in the Gulf of Mexico, thespotted seatrout is widely esteemed among
recreational anglers. In Mississippi waters, thespotted seatrout is the premier sport fish. However,increasing human population places pressure on
marine fisheries resources through habitat destruction
and demand for food and recreation. The popularity
of seatrout, combined with its dependence on
threatened inshore habitats, make it potentiallyvulnerable to depletion. It is from this perspectivethat the Gulf Coast Research Laboratory incooperation with the Mississippi Department of5

Marine Resources and recreational angler
Organizations founded SPEC, the Seatrout
Population Enhancement Cooperative, a program todetermine whether stock enhancement is a feasible
option for management of spotted seatrout in
Mississippi.
In this paper, we present the framework of the
program we developed for the intensive rearing of
spotted seatrout for stock enhancement in Mississippi
waters.

Seatrout culture
The culture of seatrout is not new. Wild seatrout
juveniles were stocked into brackish ponds with
mixed results as early as the 1940s (Colura et al.
1992). Colura (1974) first described induced
spawning of spotted seatrout. Arnold et al. (1976)
and Taniguchi (1981) described methods forspawning and rearing spotted seatrout in thelaboratory. Colura et al. (1976) and Tucker (1988)
described experimental methods of fingerling
production for spotted seatrout. Although Arnold’s
original production system included early larvalrearing in which he achieved as high as 30% survival
through 30 days, his system evolved into theextensive pond culture approach described in Colura
et al. (1992) whereby prefeeding larvae are
introduced into brackish water ponds containing wild,
mixed zooplankton. Part of the reason fortransitioning to pond rearing was inconsistent andlow yields in laboratory experiments. Colura’s
extensive system produced an average survival of
28.7% over 20–30 days of rearing (Colura et al.
1992). The Texas Department of Parks and Wildlife
currently uses a variation of this method to produce
approximately 3 million 30-day-old larvae annually.
Since 1993 Texas has released approximately 35
million 30-day-old juvenile seatrout (David Abrego,
personal communication).
In coastal Mississippi, which is estuarine, pond
culture is not feasible for a variety of reasons,
including the highly variable salinities, the lack ofavailable space, and the shallow water table. Thus
our program has focused on intensive tank culture.
As a result, the process is more complex and labor
intensive than pond rearing; however, preliminary
results suggest that the process produces a result at
least as good as extensive culture.

Mississippi program
Our program focuses on five primary objectives:
1) broodstock development—the development oftechnology for capturing, transporting, quarantining

6

and maintaining broodstock; 2) Spawning—thedevelopment of methods for conditioning healthy
broodstock for natural spawning in captivity; 3)culture—the development of methods to maximize
production of fish that can be tagged for release inintensive, indoor recirculating systems; 4) tag andrelease—the development of technology to efficiently
and reliably tag and release juveniles; and 5)
postrelease assessment—the development of methods
to monitor juveniles after release and analyze theinformation to assess the effectiveness of the
program.
Broodstock development. Broodstock development
is patterned directly after the Texas Parks andWildlife program (David Abrego, personalcommunication). Wild animals are caught locally on
hook-and-line using barbless hooks. Seatrout aredelicate animals and must be handled with great care.
Soft catch-and-release nets or standard nets linedwith plastic are required for landing the fish. Fish aretransferred from the hook into holding pens that serve
as a reservoir to contain all fish caught during acollecting event. Upon completion of the collecting
event, fish are transferred using the plastic-lined nets
into hauling tanks for transport to the lab. Stress Coatand supplemental oxygen are added to the haulingtank. It is important to note that at no point during
this process are the fish removed from the water. The
population structure of seatrout in Mississippi is not
yet known, so until we evaluate the population
structure, we assume animals from the Pascagoula
River, the Biloxi Bay area, and Bay St. Louis are
distinct populations.
Upon arrival at the laboratory, fish are placed ina staging area that allows acclimatization andrecovery from the stress of capture and transport.
After a brief acclimatization period, fish enter thequarantine area. Because our goal is to maintain fishin optimal condition for long-term holding andbecause all wild fish have parasites, we initiallyquarantine the fish to eliminate diseased fish andparasites that are likely to cause problems in captivity(i.e., parasites with direct life cycles). We practicestandard animal care facility sanitation practices suchas access control, hand/foot/equipment disinfection,and barrier protection after the quarantine period.
Quarantine. Quarantine consists of three stages.
First, fish enter pretreatment to remove ectoparasites.
Pretreatment consists of two steps, a 5 minutefreshwater dip that detaches many of theectoparasites, particularly Amyloodinium ocellatum,

Blaylockfollowed by a 24 hour treatment with Praziquantel(2.5–5.0 mg/l) to rid the fish of dangerous
monogeneans. Following pretreatment, fish enter
primary quarantine in a 12,000 L raceway for aminimum of 10 days, during which they are observedfor health problems (and treated if necessary) andtransitioned to frozen food. Seatrout are slow to
begin feeding in captivity, so after a few days of
isolation we slowly introduce live, laboratory-reared
shrimp. Once the fish begin to eat the live shrimp,
frozen shrimp are added. Eventually, frozen squid
are added. Ten to 14 days after entry into primary
quarantine, the fish are treated with formalin (175
mg/L for 60–90 min) to remove any residual
ectoparasites. After formalin treatment, the fish enter
the third stage—secondary quarantine—where they
are observed and retreated as necessary for an
additional 20 days.
Maturation. After quarantine, fish enter thematuration facility. During the move, the fish areanesthetized, weighed, measured, sexed, tagged
according to sex, and fin clipped for DNA analysis.
Fish are stocked into 12,000 L circular tanks fittedwith egg collectors in a temperature and photoperiod
controlled environment. Water quality is maintainedusing a suspended trickle bioball biofilter, sand filter,
coral filter, protein skimmer, and ozonation unit.
Each tank is stocked with 20 fish, ideally in a 1:1 sex
ratio. The fish are fed a combination of shrimp,
squid, and cigar minnows at 3% of body weight threetimes per week using the Texas Parks and Wildlife
protocol (David Abrego, personal communication).
The ratio of shrimp, squid, and cigar minnows varies
depending on season. During spawning, animals arefed shrimp (≈36%), squid (≈45%) and cigar minnows(≈18%). Postspawning animals are fed at the samerate a diet consisting of approximately 16% shrimp,
42% squid, and 42% cigar minnows. The
temperature and photoperiod cycle mimics thenatural cycle, but is condensed to 150 days based on
the Texas Parks and Wildlife protocol to provide
quick turnaround or off-season spawning.
Larviculture. Our larviculture system is a
modification of the combined experiences reported in
Arnold et al. (1976), Taniguchi (1981) and Tucker(1988) as well as the literature on the culture of othermarine fishes including the red snapper (Ogle andLotz 2005). Seatrout spawn at night. The eggs arebuoyant and are carried with the current out of thetank into an egg collector where they are retained.
From the egg collector they are placed in a conicalincubator and agitated with air. Viable eggs float,

Blaylockand upon hatching (≈48 hours) the larvae are placed
at a density of 10/L in 1,000 L larval rearing tanks
previously stocked with 50,000 cells/ml of live
Tahitian strain Isochrysis galbana. Beginning on day
t2 posthatch (PH), enriched ss-rotifers at 10/ml andcultured copepod nauplii at 0.5-1.0/ml are added. On
day 7 PH enriched brine shrimp nauplii are offered at
1/ml. Copepods are discontinued on day 8 PH androtifers are discontinued on day 9 PH. Brine shrimp
are increased to 2/ml on day 10 and to 4/ml on day 13
PH. Sprinkles of dry, size-00 mash are offered
beginning on day 15 PH and the amount is slowly
increased over time. Brine shrimp are decreased to
2/ml on day 18 PH. Dry, size-0 crumble concurrent
with 00-mash begins to be offered on day 20 PH andsize-1 crumble is added to the mixture on day 23 PH.
Harvest to the nursery occurs on days 24 and 25 PH.
Of the larvae stocked into the system, we achieved
approximately 25% survival through day 24 PH.
Nursery phase. Trout are typically released into
ponds after the initial culture period; thus, protocols
for seatrout nursery operations are largely
experimental, particularly for large-scale production.
Our system is based on that used in our red snapper
culture operation (Ogle and Lotz 2005) and consistsof fourteen, 1,000 L tanks equipped with adjustable
spray bars. Cannibalism began on day 7 of the study.
By day 24, cannibalism was a significant source ofmortality. We did two things in the nursery to try andminimize the problem. First, we used the adjustable
spray bars and pumps to create currents sufficient tokeep the juveniles active. Second, we manipulated
the density. Not all species respond to changes indensity in the same manner (e.g., Kestemont et al.
2003) and the response of seatrout was unknown.
Thus we initially stocked at three different densities(four tanks at 0.8 fish/L, four at 1/L, four at 1.5/L,
and two at 3.5/L). Initially, size-1 crumble wasslowly sprinkled into the tanks to acclimatize the fish,
but food level was increased to 3% body weight per
day based on the average weight of a sample ofjuveniles going into the nursery. Originally, thenursery phase was intended to last for at least 14
days, however concerns over our ability to maintainwater quality in the high density tanks led toharvesting at day 10 of the study (~35 days PH). We
achieved an average of 88.8% survival (range of
79.8–95.7%), and although the average survival was
lower in the high density tanks (82.4%), thevariability was such that we can not summarily
exclude a high density nursery as an option for futurebatches of juveniles. Although fish stocked at thehighest density were smaller than those stocked at7other densities, major size discrepancies within tanks
were not observed.
Growout. Growout for this batch was accomplishedin a 15×3.5×1 m rectangular raceway (approximately
50,000 L capacity), equipped with suspended trickle
bioblock biological filters, sand filters and a protein
skimmer. Fish were fed progressively larger pellets
at approximately 3% body weight based on periodic
samples. All fish died at approximately day 60 PH
during hurricane Katrina. The juveniles wereapproximately 80–100 mm, and we estimatedapproximately 75% survival to day 60. There wasmore size variability than in the nursery, perhaps due
to unequal access to food created by poor circulationin the raceway.
Tagging and release. At the time of the hurricane,plans for experimental tagging with coded-wire andelastomer tags to assess handling and insertion
procedures, tag placement, and tag retention werebeing developed. Acoustic tags will be explored in
the future. Growth rates and feed conversion ratios
were being monitored. We anticipate that releaseprocedures will involve a fish pumping device likethose widely used in the aquaculture industry forstocking and harvesting netpens.
Seatrout are known to vary genetically among
localities (Gold et al. 2003, Wiley and Chapman2003), thus, the test group of juveniles from Texas
could not have been released in Mississippi. A
project to assess genetic differences among fish fromdifferent localities in Mississippi is in development toprovide information critical to the development of asound release strategy. Until that information isavailable, we restrict ourselves to releasing juveniles
only in the area from which the broodstock wereacquired. Initial issues addressed by the release will
include survival after release, percent recovery, andmovement. More complicated issues such as theeffect of size-at-release and time of release will be
addressed in the future.

References
Arnold, C. R., J. L. Lasswell, W. H. Bailey, T. D. Williams
and W. A. Fable, Jr. 1976. Methods and techniques forspawning and rearing of spotted seatrout in the laboratory.
Proc. Annu. Conf. Southeast Assoc. Fish Wildl. Agencies
30:166–178.
Blankenship, H. L., and K. M. Leber. 1995. A responsible
approach to marine stock enhancement. Am. Fish. Soc.
Symp. 15:167–175.
Colura, R. L. 1974. Induced spawning of the spotted
seatrout, Cynoscion nebulosus (Cuvier). Proc. World
Maricul. Soc. 5:319–330.
Colura, R. L., B. T. Hysmith, and R. E. Stevens. 1976.
Fingerling production of striped bass (Morone saxatilis),
spotted seatrout (Cynoscion nebulosus), and red drum
(Sciaenops ocellatus) in saltwater ponds. Proc. World
Maricul. Soc. 7:79–92.
Colura, R. L., T. L. King, J. D. Gray and B. W.
Bumguardner. 1992. Analyses of six years of spotted
seatrout (Cynoscion nebulosus) pond culture trials.
Aquaculture 107:313–332.
Gold, J. R., L. B. Stewart, and R. Ward. 2003. Population
structure of the spotted seatrout (Cynoscion nebulosus)
along the Texas gulf coast, as revealed by genetic analysis.
In S. A. Bortone (ed.), Biology of the spotted seatrout, p.
17–29. CRC Press, Boca Raton, Florida.
Kestemont, P., S. Jourdan, M. Houbart, C. Melard, M.
Paspatis, P. Fontaine, A. Cuvier, M. Kentouri, and E.
Baras. 2003. Size heterogeneity, cannibalism, andcompetition in cultured predatory fish larvae: Biotic andabiotic influences. Aquaculture 227:333–356.
Leber, K. M. 1995. Significance of fish size-at-release on
enhancement of striped mullet fisheries in Hawaii. J.
World Aquacult. Soc. 26:143–153.
New, M. B. 1997. Aquaculture and the capture fisheries:
Balancing the scales. World Aquacult. 28(2):11–30.
Ogle, J. T., and J. M. Lotz. 2006. Characterization of anexperimental indoor larval production system for red
snapper. N. Am. J. Aquacult. 68:86–91.
Pew Oceans Commission. 2003. America’s living oceans:
Charting a course for sea change, recommendations for a
new ocean policy, May 2003.
Robins, C. R., and G.C. Ray. 1986. A field guide toAtlantic Coast fishes of North America. Houghton
Mifflin, Boston, Massachusetts.

8BlaylockSvasand, T., K. E. Jorstad, and T. S. Kristiansen. 1990.
Enhancement studies of coastal cod in western Norway,
part 1, recruitment of wild and reared cod to a local
spawning stock. J. Cons. Int. Explor. Mer. 47:5–12.

Tsukamoto, K., H. Kuwada, J. Hirokawa, M. Oya, S.
Sekiya, H. Fujimoto, and K. Imaizumi. 1989. Sizedependent mortality of red sea bream, Pagrus major,
juveniles released with fluorescent otolith-tags in News
Bay, Japan. J. Fish Biol. 35(Supplement A):59–69.

Taniguchi, A. K. 1981. Survival and growth of larvalspotted seatrout (Cynoscion nebulosus) in relation totemperature, prey abundance, and stocking densities.
Rapp. P-V Reun. J. Cons. Int. Explor. Mer. 178:507–508.

Tucker, J. W., Jr. 1988. Growth of juvenile spotted
seatrout on dry feeds. Prog. Fish-Cult. 50:39–41.

Tsukamoto, K. 1993. Marine fisheries enhancement inJapan and the quality of fish for release. Eur. Aquacult.
Soc. Spec. Publ. 19:556.

Wiley, B. A., and R. W. Chapman. 2003. Population
structure of the spotted seatrout, Cynoscion nebulosus,
along the Atlantic coast of the United States. In S. A.
Bortone (ed.), Biology of the spotted seatrout, p. 31–40.
CRC Press, Boca Raton, Florida.

Blaylock9

Disease Surveillance in Wild and Cultured Stocks of White Seabass(Atractoscion nobilis)
Mark S. Okihiro
California Department of Fish and Game
4065 Oceanside Blvd., Suite G
Oceanside, California 92056
ms.okihiro@att.net
Keywords: white seabass, Atractoscion nobilis, fish, infectious, disease, survey, aquaculture
Abstract
Success of the Ocean Resources Enhancement and Hatchery Program (OREHP) in California has beendue, in part, to a comprehensive disease surveillance program geared toward rapid detection of a wide range
of infectious and parasitic diseases afflicting both wild and cultured stocks of white seabass (Atractoscion
nobilis). Isolation and identification of white seabass (WSB) pathogens is accomplished via a number ofproven diagnostic methodologies including necropsy, cytology, histology, electron microscopy, microbiology,
serology, and polymerase chain reaction assays. For WSB, major pathogens of concern are: viral nervous
necrosis virus (VNNV), Piscirickettsia salmonis, and an as yet uncharacterized herpes virus. Use of aneffective disease surveillance program has helped ensure that hatchery epizootics are detected in a timely
fashion, and that treatment is appropriate and efficacious. Since OREHP is a stock enhancement program,
there is an additional goal of minimizing disease transfer from cultured to wild fish.
Disease outbreaks are inevitable with intensive culture; the key is determining which pathogens pose
acceptable risks to wild stocks. The most dangerous pathogens are lethal and highly contagious; the worst
are novel pathogens to which wild WSB have no immunity. Avoiding introduction of novel pathogens
requires determining which diseases occur naturally. A broad sampling of wild populations can determine
normal pathogen load and exposure level. The most efficient method of wild fish evaluation is to determine
serum antibody levels to specific pathogens using enzyme-linked immunosorbent assays (ELISAs). ELISAs
have allowed OREHP to make informed decisions regarding enhancement efforts in California. ELISA
results have demonstrated that exposure to VNNV is widespread among wild WSB. Those data give
OREHP the option of releasing VNNV-exposed, but healthy, WSB without fear of unleashing a new plague
into native stocks. In contrast, exposure to P. salmonis has not been conclusively demonstrated among wild
fish, and that novel disease requires a strict discover-and-euthanize approach when found in cultured fish.

Introduction
White seabass (Atractoscion nobilis) belong to
the family Scianidae and are one of the largest
croaker species found in the Pacific Ocean. The
primary host range is from Point Conception, inCentral California, south through Baja, Mexico.
Historically, the fish has been found as far north as
Alaska (Eschmeyer and Herald 1983). White seabass(WSB) have a maximum size of approximately 40 kg
and have an estimated lifespan of more than 30 years.
WSB are a popular commercial and recreational fish
species, with peak catches of 50,000 to 65,000 fish
per year from sport boats (commercial passenger
fishing vessels) alone in the late 1940s. Beginning in
the early 1950s, catches from both recreational andcommercial fishermen started to decline dramatically.
The fishery was essentially nonexistent by the late1970s.

OkihiroIn response to the collapse of wild stocks, theOcean Resources Enhancement and Hatchery
Program (OREHP) was formed in 1983. OREHP is acooperative effort between the California Departmentof Fish and Game (CADFG), Hubbs-SeaWorld
Research Institute (HSWRI), several California
public utility companies, and a number of volunteer
organizations. The primary goal of OREHP is tohelp restore marine fish populations in California
waters. OREHP works actively to culture several
marine finfish species, but the major focus ofremediation efforts is on WSB; WSB are currently
the only cultured marine fish being released into
California waters. As part of the OREHP program, adedicated marine fish hatchery was constructed in
1995. The Leon Raymond Hubbard Jr. Marine Fish
Hatchery (also known as Carlsbad Hatchery), located11

in Carlsbad, California, has produced more than
100,000 juvenile WSB for release into the Pacific ineach of the past five years. The record year was 2004
when the hatchery released more than 270,000 fish,
either directly from the hatchery, or from one of 14
growout/netpen facilities located throughout
Southern California.
The release number for 2005 was expected to be
only half that of 2004. The primary reason for the
reduced number is that 2005 was one of the worst
years for diseases since the hatchery was constructed.
Flexibacter maritimus and disseminated Vibrio
epizootics occurred at the hatchery and four netpensites. Piscirickettsiosis, which is a lethal rickettsial
disease caused by viral nervous necrosis virus(VNNV) (Piscirickettsia salmonis), was confirmed at
one netpen site and was investigated at another. Gillflukes (Anchoromicrocotyle guaymensis) plagued
four netpen sites. As of November 2005, there was asevere herpes virus outbreak at the Carlsbad Hatcheryinvolving more than 50,000 WSB. There is no single
reason for the increased number and severity of
disease outbreaks that year, but the 2004–2005 winterwas one of the wettest on record for California, andwater quality early in 2005 was marginal at best withwide fluctuations in temperature, salinity, andparticulate loads. Although no specific testing wasdone, contaminant loads (i.e., xenobiotics andbacteria) from rainwater runoff were assumed to behigh throughout Southern California.
Disease outbreaks are an unfortunate, andsometime inevitable, consequence of almost all highdensity marine aquaculture and finfish enhancement
programs. To maximize mitigation efforts, while atthe same time minimizing disease impacts to both
cultured and wild fish, OREHP uses a three pronged
approach: 1) a comprehensive biosecurity program,
2) health inspections of cultured fish, and 3) wild fish
disease surveillance. Biosecurity protocols are
geared toward disease prevention and limiting the
spread and impact of diseases when they occur.
Biosecurity is a key component of any aquaculture
operation, but is not discussed here.
Health inspections are the second component of
OREHP’s overall disease prevention progam; allcultured fish are thoroughly evaluated prior torelease. At a minimum, cultured WSB are inspected
twice. The initial health check is done at the hatcheryprior to transport to net pen facilities; the second
check is done at the net pens just prior to fish being
released into the Pacific. Additional healthassessments are done as needed if fish are behaving
abnormally or if there is an increase in daily
mortality.

12

The third arm of OREHP’s health assessment
and disease prevention program is an ongoing effort
to survey wild stocks of marine fish species—withspecial emphasis on those species targeted for
mitigation or aquaculture. The goal of this diseaseassessment survey is to determine which pathogens
and diseases are naturally occurring among wild
marine fish stocks. This baseline information is
critical if we are to avoid releasing lethal pathogens
into potentially naïve populations of wild marine fish.

Finding a Competent Fish Pathologist
One of the most important steps in any
aquaculture venture or finfish enhancement program
is locating a qualified and experienced fish
pathologist. Fish pathologists can have either aveterinary or fisheries background, but should be
qualified in the diverse array of subjects required for
disease prevention, detection, control, andmanagement. Some of the required fields ofexpertise (or at least familiarity) include: general
biology, marine biology, fish diseases, generalpathology, fish pathology, cytology, hematology,
serology, histology, histopathology, general anatomy,
fish anatomy, and molecular biology. Somebackground in toxicology, microbiology, fish
physiology, surgery, and genetics is also helpful.
While finding a fish pathologist with acomprehensive and diverse educational backgroundis important, equally important is finding one withextensive practical experience with a variety of fish
diseases (infectious and noninfectious) in a variety offish species. Ideally, experience should be with large
groups of fish (i.e., herd health) and with managing
problems associated with outbreaks of highly
contagious diseases among high-density populations.
The ideal fish pathologist would be one who isalready familiar with the particular fish species inuse. However, because many remediation efforts
involve marine fish species being cultured for the
first time, this is usually not possible. The next best
approach is to hire a person with sufficient flexibility
that he or she can adapt his or her training andexperience to the species at hand and to the uniquedisease problems associated with that species.
Eventually, given sufficient time and examination ofa sufficient number (i.e., hundreds to thousands) of
diseased fish, almost any qualified fish disease
specialist can become experienced with a given fish
species.
Once the appropriate fish disease expert islocated and gains experience with the particular fish
species being cultured, the next major hurdle is toOkihiroensure that this disease knowledge base (often
painstakingly acquired over a period of years) is not
lost when the fish pathologist leaves, retires, or getseaten by a shark while diving. Knowledge transfer tothe next pathologist can be simplified by 1) having acomprehensive and detailed file of pathology reports,
2) having detailed written protocols for most (if notall) of the tasks involved with disease diagnostics,
prevention, and control, and 3) having an extensive
photographic record for all of the major diseases
encountered in that particular species. Photos should
include gross photos of necropsy findings; wet mountcytology photos, light micrographs of histologic
lesions, as well as transmission electron micrographsof ultrastructural lesions and pathogens. A
comprehensive and well-organized photographic
record of diseases, for a given fish species, will go along way in bridging the knowledge gap for the next
fish pathologist.

Diagnostic Approach
The diagnostic approach used by OREHP
includes five major components: 1) history andbackground information, 2) clinical assessment oflive fish, 3) necropsy and gross examination, 4)
sample selection, storage and transport, and 5)
sample analyses using a variety of diagnostic assays.
History and background information. Obtaining athorough history is the first step in obtaining a
reliable diagnosis. Background information should
include specifics on the species, age, size, andnumber of fish involved. If the fish are at a growout
facility, it is important to determine from what part of
the hatchery the fish originated and how they weretransported. The number of transport trips that wereneeded, as well as any problems encountered duringtransport, are also important pieces of information.
Daily mortality (both raw numbers and percentage ofthe population) and mortality patterns over thepreceding two weeks are important indicators ofhealth, as are feeding rates and patterns. Some
OREHP pen operators feed by hand, which gives
them a good opportunity to observe their fish, assess
feeding rates, and evaluate overall condition andhealth. Information with respect to tank or pen
maintenance is also helpful. Breakdown of
mechanical equipment can obviously impact water
quality and fish health. Consistent vacuuming oftank bottoms and cleaning of end screens are theprimary maintenance items to look for. Inconsistentcare—because of personnel shortages or sickness—should also be looked for and noted. The majority ofOkihirogrowout facilities have logbooks, which can beaccessed to determine if pen maintenance wasconsistent and if there were problems with either
mechanical systems or volunteers.
Water quality is the final item to check. Major
water quality parameters that can affect fish healthand influence disease outbreaks are: 1) temperature,2) dissolved oxygen, 3) xenobiotic contaminant
spills, and 4) red tide events (dinoflagellate plankton
blooms). Marked temperature fluctuations (those>3°C) that occur over a short period of time (hours todays) often precede outbreaks of infectious disease.
Clinical assessment. Knowledge of normal behavior
for the fish species, age group, and enclosure isimportant if deviations from the norm are to be
detected. With WSB, it is important to know how thedifferent age groups (larvae, juvenile, subadult, andadult) act when they are in round versus rectangulartanks, concrete raceways, fiberglass raceways, or
netpens. Do fish school? At what age do theyschool? In what types of enclosures do they school?
Are fish normally up near the surface, in the middleof the water column, or near the bottom? Answers to
all of these questions are important to define what isnormal behavior for a given species, age group, andtank setup. This knowledge can only be acquired overtime, so it is always important to take the time toobserve living fish in both sickness and health. Somegeneral clinical signs to look for in unhealthy fish
are: 1) weight loss (head out of proportion to thebody), 2) anorexia (sick fish rarely feed), 3) lethargy(sick fish swim slowly) and absent or reduced escapereflex, 4) weakness (sick fish are pushed towards end
screens or pulled towards drains), and 5) abnormal
pigmentation (sick fish are usually darker thannormal).
In addition to these nonspecific signs of disease,
there are other more specific clinical indicators of ill
health. With WSB, even heavily parasitized fishrarely flash (i.e., attempt to scratch themselves on
fixed objects). Still, when flashing is observed, it iswise to check for external parasites. Terminal
spinning or spiraling in juvenile WSB is usuallyassociated with herpes virus gastroenteritis. A
variety of cutaneous and ocular lesions can also be
observed among a group of fish and will help indicate
which fish should be selected for necropsy and which
diseases are most likely to be encountered. For
example, with WSB, mottled skin pigmentation isoften indicative of Ichthyobodo infestation, while
focal white areas of depigmentation are characteristicof P. salmonis. There is never 100% uniformity withrespect to the clinical presentation, but with careful13

observation, patterns will usually emerge and will
help indicate from what the majority of fish are
suffering.
Necropsy. A thorough necropsy is the mostimportant step in determining why fish are dying. A
good necropsy, performed by an experienced
pathologist, will often quickly reveal what disease isinvolved and what the specific etiologic agent is. A
well-executed necropsy is also the cheapest, most
cost efficient means of making the right diagnosis.
Of course, necropsy results are only as good as
the fish that are examined, so proper selection of theappropriate fish is critical. Live moribund fish arealways preferable to dead fish, fresh dead is preferred
to a fish that has been dead for several hours, and adead fish several hours old is preferable to one thathas been frozen. A good pathologist will always
select his or her own fish to examine because for theuninitiated, all sick fish generally look alike. A good
pathologist will select fish for necropsy based on
specific clinical findings after observing fish in situ.
If obvious moribund fish are not available, thenfish should be caught from areas where sick fishcongregate. In concrete raceways, this means toward
the back, near endscreens. With fiberglass raceways,
this means checking the corners, and with circular
tanks with center standpipes and drains, it means
checking the middle of the tank. The number of fish
to necropsy is variable, but the OREHP standard is tosample six fish per group. The number of fishsampled may be as few as four, if the cause of
disease is obvious, or as many as 400, if there is needfor a large number of different diagnostic assays.
Moribund WSB are euthanized with MS222(tricaine methane sulfonate). Total length (TL) andstandard length (SL) are taken in centimeters;
determining wet body weight (in grams) is optional.
If a blood sample is required, it is taken prior to or
immediately following euthanasia. The grossexternal exam is the next step and involves carefulassessment of the skin, fins, gills, pseudobranch, oralcavity, tongue, and eyes. The skin and fins arechecked for irregular pigmentation, erosion, andulcers. The gills and pseudobranch are assessed forcolor, filament damage, and parasites. The oralcavity and tongue are checked for parasites, lesions,and deformities. Deformities of the maxilla andmandible are relatively common among some groupsof WSB. The eyes are evaluated primarily forevidence of emphysema associated with exposure togas supersaturation. Following the gross external
exam, scrapings are taken from the gills and skin for
cytological assessment of parasite and bacterial loads.

14

For the gross internal exam, the fish is placed inright lateral recumbency (i.e., right side down, headto the left). The entire left abdominal body wall isremoved with blunt/blunt tipped scissors to avoid
puncturing the swim bladder and to avoid lacerating
gonad or liver. Once the abdomen is open, the gonad
is examined to determine sex. Almost all WSB >12cm TL can be sexed grossly. After sex is determined,
the heart, left liver lobe, pyloric cecae, stomach, andswim bladder are examined. The left liver lobe andstomach are then reflected ventrally to expose thespleen, intestinal tract, right liver lobe, gall bladder,and endocrine pancreas. The last step is to pull theswim bladder away from the spine and to examine
the head, trunk, and tail kidney.
For the majority of cultured WSB, the brain andotoliths are not examined during routine healthchecks. Brains are dissected and sampled only when
hatchery fish exhibit neurologic signs, have sufferedcranial trauma, or when viral nervous necrosis(VNN) is suspected. For wild WSB, otoliths areroutinely sampled for age assessment, and brains are
frequently removed for VNN evaluation. The
dissection is made by using a hacksaw with a metal
cutting blade to make a frontal section above the eyes
to access the brain dorsally. If the initial cut is notdeep enough, the cranial vault can be further opened
with a scalpel or razor blade. The brain is removed
by using fine scissors to sever the olfactory lobes,
cranial nerves, and then the spinal cord. Otoliths arelocated in the semicircular canals located below thebrainstem.

Sample Collection, Storage, Transport, andAnalyses
OREHP employs a diverse array of diagnostic
methodologies including cytology, histology,
electron microscopy (EM), microbiology, polymerase
chain reaction (PCR) assays, and enzyme-linked
immunosorbent assays (ELISAs). Proper sample
collection and handling, prior to analyses, ensuresthat the maximum amount of information will beobtained.
Cytology. Wet mount cytology is used for the
diagnosis of many common bacterial and parasitic
infections in WSB. Cytological samples include gilland skin scrapings and occasional squash
preparations used to assess nodular masses. Gill andskin scrapings are collected using a dull scalpel tominimize cutting into deeper tissues; the goal is to
scrape parasites and bacteria off target surfaces. For
gills, samples of filament tissue are collected from

Okihirothe first or second gill arch. With skin samples, thebase of the left pectoral fin is initially scraped,
followed by a pass along the skin below the dorsal
fin. Small (1–2 mm3) samples of skin and gill aretransferred onto glass slides and spread into a thinlayer by applying a glass coverslip. Coverslippedsamples are examined immediately via dark fieldlight microscopy for parasites and bacteria.
The usefulness of the exam is dependent on how
long the fish has been dead, when the sample wastaken, and the thickness (between the slide andcoverslip) of the sample. Ideally, samples are taken
as soon as possible after the fish has died andexamined as soon as possible after the slide has been
prepared. If a fish has been dead for too long, andthe fish remains in water, the offending parasites may
leave the body. If a fish has been dead for too long,and the fish remains out of water, the offending
parasites may be obscured by excess skin or gill
mucus, or may have died and be difficult to locate.
Squash preparations are typically used to
examine focal nodular lesions in parenchymal organs
such as liver or kidney. Squash preps are also used todetermine etiologies of ocular infections. Squash
preps are made by excising a small (1 mm3) piece ofthe mass, with a scalpel and crushing it between a
glass slide and coverslip. For dry lesions, a small
drop of clean seawater can be included prior toadding the coverslip.
Both squash preps and skin scrapings are
examined via light microscopy using a standard
binocular microscope (Nikon Labophot) and dark
field/phase contrast condenser. For WSB, the dark
field condenser is almost exclusively used for wet
mount preparations. The best wet mount
preparations are thin (<30 µm between the slide andcoverslip). Thin preparations allow clear
visualization of bacteria and smaller protozoa.
Thicker preparations (e.g., those with large scales orparticulates) are suitable for large parasites (e.g.,
flukes), but make identifying bacteria difficult or
impossible. The majority of marine finfish pathogens
are motile, so examining slides quickly will allow
location and identification of parasites by focusing
the search on movement.
Histology. Histology is employed when necropsy
and or cytology fails to provide a diagnosis, or when
confirmation of a gross diagnosis is required ordesired. Histology allows the extent of tissue damage
to be determined and results in a permanent record
for a disease or pathogen. Histology has good
application for larval fish as almost all major organs,for multiple fish, can be examined on a single slide.

OkihiroHistology is also a faster and cheaper diagnostic
alternative for some viral infections (e.g., VNN) andrickettsial pathogens (e.g., P. salmonis).
For histology to be effective, a key factor is toobtain samples as quickly as possible after euthanasia
or natural death. Autolysis, from bacterial or hostenzymes, and bile, will rapidly degrade organs andtissues, making interpretation difficult or impossible.
For WSB, 10% formalin is routinely used as a
fixative. Samples are handled as carefully as possible
to minimize crushing artifacts and to preserve normalarchitecture. Samples from larger parenchymalorgans are cut with Teflon-coated razor blades to a
thickness of 1 cm (or thinner) to ensure rapid andthorough fixation. A 10:1 ratio of formalin to tissue
is used. Larval fish <1.5 cm TL are fixed whole. For
larval and small juvenile fish >1.5 cm TL, theabdominal cavity is opened prior to fixing the entire
fish. For large juvenile and subadult WSB, the gall
bladder is carefully removed and either discarded or
fixed in a separate container.
Formalin-fixed tissues are trimmed with a razor
blade, placed in 4.0 cm by 2.8 cm plastic cassettes,
and submitted to a commercial laboratory (Central
Histology Facility in Sacramento, California) for
routine paraffin processing. Tissue sponges are usedwith smaller larvae to prevent fish from slipping
through slots in cassettes and being lost during
processing. Embedded tissues are sectioned at 5–7µm; sections are mounted on glass slides and stainedwith hematoxylin and eosin (HE). All HE slides areevaluated in house by the OREHP fish pathologist.
Slides are assessed to determine which organs areaffected, number and types of lesions, lesion severity,and presence of microbial pathogens. If etiologic
agents are not readily apparent, special stains may beordered and HE slides may be sent out for review byother fish pathologists.
Electron microscopy. EM is used to confirm theidentity of known WSB viral and rickettsial
pathogens, and to help identify and classify new
microbial pathogens. For transmission electron
microscopy (TEM), samples are taken from freshly
euthanized fish. TEM samples are trimmed to 1 mm3
cubes with Teflon-coated razor blades, andimmersion fixed in chilled Karnovsky’s solution.
Fixed samples are stored at 4°C prior to processing.
In cases where samples were not fixed in
Karnovsky’s, TEM samples can be obtained fromeither formalin-fixed material or from paraffin
histology blocks. Formalin-fixed material istransferred to Karnovsky’s, while paraffin-processedmaterial is excised from blocks using scalpel blades,

15and deparaffinized prior to processing. Karnovsky’sfixed tissues are processed and embedded in epoxy
resin, sectioned at 1 µm, and stained with toluidine
blue. Following initial screening of toluidine blue
stained sections, ultrathin (800–900Å) sections are
cut with a diamond knife, mounted on copper grids,
and stained with 2% uranyl acetate and Reynolds’
lead citrate.
Some TEM assessment is done by the OREHP
pathologist, but the majority of scope work, and all of
the processing and sectioning, is done by the
California Animal Health and Food Safety (CAHFS)
laboratory in Davis, California. Mounted ultrathin
sections are viewed with either a Zeiss or Phillips
transmission electron microscope. Cells from target
organs are examined for subcellular lesions and thepresence of viral particles in the nucleus orcytoplasm. For nonviral pathogens, lesions arescanned to locate, identify, and characterize
rickettsial or protozoan parasites.
Samples for direct EM examination are used to
identify gastrointestinal (GI) parasites that are shedinto the lumen of the GI tract. Direct EM is
specifically used by OREHP to try and identify asuspect herpes viral pathogen. Direct EM samples
are taken from pyloric cecae and intestine; thestomach is avoided to prevent sample degradationfrom gastric acids. The gall bladder (closely
associated with the intestine in WSB) is similarly
avoided to prevent damage from bile salts. Contentsof pyloric cecae or intestine are emptied into plastic
cryovials using forceps, and are frozen and stored at–70º to –80ºC. Direct EM samples are shipped on
dry ice to the CAHFS laboratory. Samples are rinsed
and filtered prior to EM examination for viralparticles.
Microbiology. Microbiology includes the fields ofmycology, bacteriology, and virology. All are geared
towards isolation and growth of microorganisms on
artificial media or in cell culture. Fungal infections
in cultured WSB are rare. Diagnosis is usually madevia cytology, but occasionally, fungal colonies will
be isolated on Sabouraud-dextrose agar. Major
bacterial pathogens of WSB are Vibrio spp. andFlexibacter maritimus. Although infections are also
routinely diagnosed using wet mount cytology,
culture on artificial media is necessary for bacterial
identification and to determine antibiotic sensitivity.
Skin is the primary target organ for both Vibrio(usually as a secondary invader) and Flexibacter(usually as a primary pathogen). Vibrio also occurs
as a disseminated infection, attacking kidney andbrain.

16

Samples are ideally taken from freshly
euthanized fish, but can be taken from euthanized ordead fish that have been stored at 4°C (or on ice) forup to 12 hours. Sterile technique, employing flamesterilized metal loops or prepackaged sterile plastic
loops, is used to take initial samples. Flexibacter iscultured on specialized cytophaga agar, while Vibrio
species are cultured on blood agar. Colonies take 1–
3 days to appear. The hatchery manager, Paul Curtis,
routinely processes fungal and bacterial samples at
the Carlsbad Hatchery. Some Vibrio samples havebeen sent to the University of California at Davis(UC Davis) for speciation.
The major rickettsial pathogen of concern for
WSB is P. salmonis. The primary target organ for P.
salmonis is the liver and lesions appear as multifocal,
pale tan to yellow granulomas. P. salmonis requires
culture on a chinook salmon (Oncorhynchus
tshawytscha) embryo cell line (CHSE-214) and allliver samples from suspect fish are shipped on ice
overnight to UC Davis.
There are three viral pathogens of concern for
cultured WSB: viral nervous necrosis virus (VNNV),
viral hemorrhagic septicemia virus (VHSV) and asuspect herpes virus that has only been identified
using TEM. Samples for virus isolation (VI) assays
include: eye and brain (for VNNV), spleen andkidney (for VHSV) and pyloric cecae and intestine(for herpes virus). The liver has also occasionallybeen sampled for VI. With liver, the left lobe iscollected (the right lobe and associated gall bladder
are not sampled). Splenic samples are taken byfreeing the organ from the gastrosplenic ligament(mesentery), being careful not to incise the gall
bladder. Trunk and tail kidney are sampled by sharp
dissection, using either a scalpel or razor blade to free
the organ from the attached spine. Eyes are dissectedby grasping conjuctival tissues with forceps, applying
traction and using scissors to cut periocularconnective tissue, extraocular muscles, and the optic
nerve. The dissection and removal of the brain,pyloric cecae, and intestine are covered above.
VI samples are stored in chilled (4°C) minimal
essential media and shipped overnight to UC Davison ice. VNNV is cultured on a snakehead fish cellline (SNN-1), while VHSV is cultured on a carp cellline (EPC). Both VNNV and VHSV havecharacteristic cytopathic effect when grown on theappropriate cells lines. Positive cultures are
confirmed with either PCR or TEM. Attempts havebeen made to culture the herpes virus using several
fish cell lines, but have to date been unsuccessful.

OkihiroEnzyme-linked immunosorbent assay. ELISAs
have been developed by Dr. Ronald Hedrick’slaboratory at UC Davis to assess pathogen exposurein WSB to three major pathogens: VNNV, VHSV,and P. salmonis. ELISAs work by detecting
antibodies specific for the three pathogens incirculating blood. Although ELISAs detect exposureand not infection, the main advantage is that a fish
could have been exposed months or possibly years
prior to sampling and still have detectable antibody
levels in peripheral blood.
For WSB <15 cm TL, a 1 ml syringe with a 2.5
cm, 26 gauge needle is used to draw blood. The
needle is inserted on the ventral midline, adjacent tothe anal fin, to access the large vertebral vein locatedventral to the spinal column. Approximately 0.3–1.0
ml of blood can be obtained from a single fish.
Blood from 2–6 small fish is often pooled for a single
serum sample. For larger juvenile WSB, a 2.5 or
3.75 cm 18 gauge Becton Dickinson Vacutainer
needle (and associated Vacutainer sleeve) is used to
draw blood. One to 10 ml of blood can be collectedfrom a larger juvenile or subadult fish. Blood
samples are collected in Vacutainer SST serum
separation tubes. Blood is allowed to clot at room
temperature for 20–30 minutes and then stored at 4°C
for up to 48 hours. Clotted blood is centrifuged for
6–10 minutes at 1,000–1,300 RCF (g) in a swing
bucket centrifuge. Serum is pipetted off and stored
as 0.5–1.0 ml aliquots in 2 ml plastic cryovials.
Serum is frozen and stored at –70º to –80°C. Frozen
serum samples are shipped on dry ice to UC Davisfor final ELISA assessment.
ELISAs are layered assays conducted in
multiwell plastic plates. The initial antigen layer isderived from viral or rickettsial pathogens grown incell culture. The second layer is serum from wild or
cultured WSB that has varying concentrations ofantibodies specific to the pathogen antigens. The
third layer is a biotinylated-rabbit anti-WSB
polyclonal antibody. The rabbit antibodies bind
specifically to the WSB immunoglobulin. The fourthlayer is a horseradish peroxidase (HRP) streptavidin,
which binds selectively to biotin on the rabbit
antibodies. The final layer is tetramethylbenzidine, asubstrate specific for the HRP enzyme. Positive
samples change color from yellow to blue. Degree of
optical density (OD) is determined by an automatedplate reader. Degree of OD is dependent on theamount of pathogen-specific WSB antibody present
in the serum sample; the greater the OD, the higher
the antibody titer. Positive samples are those with
OD higher than that of positive-control samples.
Positive-control serum is obtained from either

Okihirocontrolled laboratory exposures or from confirmed
disease outbreaks among hatchery fish.
Polymerase chain reaction. PCR assays have beendeveloped by Dr. Ronald Hedrick’s laboratory at UC
Davis to detect VNNV, VHSV, and P. salmonis
infection in WSB. PCR is a sensitive, highly
accurate molecular assay designed to detect pathogen
genetic material (RNA or DNA) via use of specificprimers followed by exponential gene amplification.
The primary drawbacks are: 1) the assays cannot
determine level of infection, 2) the assays cannot
determine degree of host damage, and 3) a relatively
slow turnaround time.

Diseases and Pathogens of Cultured White
Seabass
Diseases of cultured WSB can be divided into
noninfectious, infectious, and parasitic.
Noninfectious diseases are not covered here.
Infectious diseases include viral, bacterial, rickettsial,and fungal diseases. There are three major viraldiseases of concern for cultured WSB: VNN,
VHSV, and herpes virus gastroenteritis (HVGE).
VNN is a lethal, highly contagious disease of larval
WSB; HVGE is a lethal, highly contagious disease ofjuvenile WSB. VHSV has not been confirmed in
WSB, but is of concern because it has been isolated
from several baitfish species caught in Southern
California waters (Hedrick et al. 2003). Bacterial
diseases include cutaneous infections by F. maritimus
and various Vibrio species. In addition, at least one
Vibrio species (tentatively identified as V.
alginolyticus) is the causative agent of a lethal
disseminated infection involving kidney and brain.
Two rickettsial diseases afflict WSB: one is atransient, benign infection of the gills by an
Epitheliocystis sp.; the other, piscirickettsiosis, is alethal systemic infection caused by P. salmonis.
Fungal infections, as stated above, are rare.
Parasitic diseases of cultured WSB include
metazoan, protozoan, and crustacean parasites. No
cestodes have been recovered from cultured WSB,
but three trematode species have been found in gillsof netpen fish: Anchoromicrocotyle guaymensis(previously identified as Cynoscionicola
psuedoheterocantha), Gyrodactylus sp., and an
unidentified fluke species. Protozoan parasites
include: three ciliates (Uronema marinum,
Trichodina sp., and at least one unidentified ciliate),four flagellates (Icthyobodo sp., Cryptobia sp.,
Hexamita sp., and one unidentified flagellated
protozoan), and one sporozoan (a probable17

myxosporidian parasite commonly found inmesonephric ducts of WSB in growout pens).
Parasitic isopods have occasionally been observed
attached to fins of WSB at one netpen site in
Huntington Beach.
The three most important diseases of cultured
WSB are VNN, HVGE, and piscirickettsiosis. All
three are lethal, highly contagious, and, for the mostpart, untreatable. As such, these three diseases posethe greatest challenge to OREHP, both from ahatchery production standpoint and from a diseaserisk standpoint to wild fish stocks.
Viral nervous necrosis. VNN is a lethal nodaviraldisease of larval WSB (Curtis et al. 2001). Prior to
the spring of 2003, VNN epizootics were a regularoccurrence at the Carlsbad Hatchery. Since
installation of an ozone water treatment system at thehatchery in 2003, there have been no outbreaks. The
etiologic agent is a single-stranded RNA virus. VNV
primarily infects larval WSB 20–40 days posthatch;target organs are retina of the eye, brain, and spinal
cord. Clinically, affected larval fish present as
paralyzed fish, floating on their sides at the surface of
the water. Histologically, target organs havecharacteristic necrotizing and cystic lesions.
Presumptive diagnosis is made via observation oflarval fish with classic clinical signs. Confirmationof VNN is made with histology, TEM, culture on
SNN-1 cells, and PCR. There is no treatment.
Herpes virus gastroenteritis. HVGE is a lethal,highly contagious viral disease of juvenile WSB. It
primarily affects smaller (<15 cm TL), younger (<4months) hatchery seabass exposed to ambient lagoon
waters during fall and winter months. There havebeen two confirmed outbreaks and both occurred at
the Carlsbad Hatchery: one in the fall of 2002, thesecond in the fall of 2005. Mortality can exceed
3,000/day if HVGE occurs simultaneous with
Flexibacter. Typical clinical signs (whenuncomplicated with Flexibacter) are: 1) highmortality among fish with no cutaneous lesions, 2)yellow-white stringy fecal casts in the water, and 3)
terminal spiraling prior to death. Target organs arestomach, pyloric cecae, and intestine. Gross lesions
include moderate to massive dilation of the entire GI
tract, excessive amounts of clear fluid in the GI tract,occasional gastric intussusception, variable ascites,and dilated urinary bladders. Histologically, there ismultifocal necrosis of mucosal epithelium of pyloric
cecae and intestine, with minimal inflammation.
Some epithelial cells may have marginated chromatin18

and an absence of nucleoli. The diagnosis isconfirmed with TEM. There is no PCR assay
available and the virus has not been successfullygrown in cell culture. There is no treatment.
Piscirickettsiosis. Piscirickettsiosis is the third
major disease of concern for OREHP (Arkush et al.
2005, Chen et al. 2000). The causative agent is a
Piscirickettsia salmonis. P. salmonis epizootics haveoccurred twice since the hatchery opened. The firstoutbreak occurred in 1998 and involved multiple
hatchery systems as well as several netpen sites. The
second outbreak occurred in April 2005 and involved
a single netpen site at King Harbor in Redondo
Beach. Clinically, P. salmonis infected WSB are
slow moving, moribund fish with scattered, small,white foci of depigmentation over the flanks.
Characteristic gross lesions include: linear streaking
in gill filaments, a fibrinous necrotic exudate over theheart, and multifocal, pale tan to yellow, nodular
masses in the liver. The diagnosis can be confirmedwith histology, culture on CHSE-214 cells, PCR, or
TEM. Although treatment with tetracycline
antibiotics is theoretically possible, treatment has
thus far not been attempted.

Management of Virulent Disease
The mortality rate among even the most virulent
infectious disease in cultured WSB is never 100%, so
the question becomes: what should OREHP do withthe thousands of fish that have survived exposure to
VNNV, HVGE, and P. salmonis? Disease epizootics
that occur at netpen sites can involve up to 10,000
cultured WSB; outbreaks in the hatchery’s raceway
system can impact up to 60,000 older (3–6 month)juvenile fish. Survivors are often fish into which
OREHP has invested a significant amount of time,effort, and money, and which represent a highpercentage of total release numbers. If this were an
aquaculture operation, the answer would be simple:
as long as there was no danger of pathogen transfer tohumans, surviving stocks would either be sold or
eaten. But since this is an enhancement program,
OREHP has to consider the relative risk of diseasetransfer to the wild fish stocks they are seeking tohelp.

Disease Surveillance of Wild WSB
To assess the risk to wild seabass from lethal,highly contagious diseases occurring in cultured
WSB, OREHP initiated a comprehensive disease
surveillance program in 2002. The goal is to survey
wild marine fish populations in order to determine

Okihirowhich lethal infectious diseases are naturally
occurring or endemic to wild stocks. The rationale isthat if a disease is endemic among wild fish, then therelative risk from exposed or infected hatchery fish is
relatively small.
Although WSB were the primary focus of wild
fish surveys, other native California marine fish
species in which OREHP is interested were also
sampled. Some of these other species include:
California sheephead (Semicossyphus pulcher),
California halibut (Paralichthys californicus),
California yellowtail (Seriola lalandi), lingcod(Ophiodon elongatus), vermillion rockfish (Sebastes
miniatus), bocaccio rockfish (S. paucispinis), andcowcod rockfish (S. levis). OREHP’s plan of action
was simple: 1) sample a large number of wild fish, 2)
assess pathogen exposure and infection rates, and 3)
use the data to make informed decisions regarding
release of infected or exposed hatchery fish.
Total catch numbers after 3.5 years of sampling
were: 272 wild WSB, 124 bocaccio rockfish, 43vermillion rockfish, 6 cowcod rockfish, 146
California halibut, 83 sheephead, and 17 assorted
other fish species. Sample numbers for just wild
WSB were: 195 ELISA serum samples, 100+ brain
samples for VNNV VI and PCR, 100+ eye samples
for VNNV VI and PCR, 80+ spleens for VHSV VI
and PCR, 80+ kidneys for VHSV VI and PCR, 80+liver samples for P. salmonis isolation and PCR and100+ intestinal content samples for direct EM
assessment of herpes virus. All wild fish samples
were sent to UC Davis and sample analyses are
ongoing.
Preliminary ELISA results indicate that VNNV
exposure is widespread among wild WSB. Among
wild juvenile or subadult WSB (i.e., those <72 cm
TL), 18% (14/78) of serum samples were ELISApositive for VNNV exposure. Among wild adult
WSB, 53% (9/17) of serum samples were VNNVpositive with ELISA. In contrast, none of the 94
serum samples analyzed thus far via ELISA havebeen positive for P. salmonis exposure. ELISA
results for VHSV are pending, and currently there is
no ELISA assay specific for the herpes virus that
occurs in WSB.
All attempts to isolate virus or rickettsia fromwild WSB have failed. A small number of PCR
samples have been assessed and these have also
turned up negative for virulent pathogens known tooccur in cultured WSB. Results from the majority of
PCR samples, however, are still pending. Of themore than 100 intestinal content samples taken, 50
have been analyzed via direct EM for herpes virus. A
single direct EM sample had suspect hexagonal

Okihiroprofiles, but did not contain nucleocapsid material
and was classified as negative for herpes virus.

Wild Fish Disease Survey Conclusions
Preliminary results from OREHP’s wild fishsurvey lead to three conclusions. First, VNNV isprobably a naturally occurring disease among wild
WSB. Second, it is likely that P. salmonis is an
exotic disease. Third, the disease status of wild WSB
for VHSV and herpes virus is still unknown.

Impact on OREHP Policies and HatcheryOperations
The impact wild fish disease survey data have on
hatchery operations is that OREHP can now makeinformed decisions regarding what to do whenvirulent disease outbreaks occur. With VNNV
epizootics, the hatchery can reasonably decide tosalvage survivors and release them at a later datewhen they are healthy. In contrast, with P. salmonis,
the assumption has to be that this disease does notoccur among wild WSB, and that any cultured
seabass infected or exposed to this pathogen shouldbe euthanized. With herpes and VHSV, there still is
not enough information to determine whether or notthese two diseases are endemic to wild fish stocks.
We believe that herpes virus is probably a naturally
occurring disease among wild WSB, but until there isdefinitive proof, OREHP policy will be to operate asif it too were an exotic disease.

References
Arkush, K. D., A. M. McBride, H. L. Mendonca, M. S.
Okihiro, K. B. Andree, S. Marshall, V. Henriquez, and R.
P. Hedrick. 2000. Genetic characterization andexperimental pathogenesis of Piscirickettsia salmonis
isolated from white seabass Atractoscion nobilis. Dis.
Aquat. Organ. 63:139–149.
Chen, M. F., S. Yun, G. D. Marty, T. S. McDowell, M. L.
House, J. A. Appersen, T. A. Guenther, K. D. Arkush, and
R. P. Hedrick. 2000. Piscirickettsia salmonis-likebacterium associated with mortality of white seabass
Atractoscion nobilis. Dis. Aquat. Organ. 43:117–126.
Curtis, P. A., M. Drawbridge, T. Iwamoto, T. Nakai, R. P.
Hedrick, and A. P. Gendron. 2001. Nodavirus infection ofjuvenile white seabass, Atractoscion nobilis,cultured insouthern California: First record of viral nervous necrosis(VNN) in North America. J. Fish Dis. 24:263–271.
Eschmeyer, W. N., and E. S. Herald. 1983. Croakers:
Family Sciaenidae. In R. T. Peterson (ed.). A field guideto Pacific Coast fishes of North America, p. 218–222.
Houghton Mifflin Company, Boston, Massachusetts.

19

Hedrick, R. P., W. N. Batts, S. Yun, G. S. Traxler, J.
Kaufman, and J. R. Winton. 2003. Host and geographic
range extensions of the North American strain of viralhemorrhagic septicemia virus. Dis. Aquat. Organ. 55:221–
220.

20

OkihiroJuvenile Mass Production of the Black Rockfish (Sebastes schlegeli)in FRA Miyako Station: Present Status and Problems
Masahiro Nakagawa
Miyako Station
National Center for Stock Enhancement
Fisheries Research Agency
4-9-1, Sakiyama, Miyako
Iwate 027-0097, Japanmnakagaw@affrc.go.jp
Junichi Adachi
Shibushi Station
National Center for Stock Enhancement
Fisheries Research Agency
Ken Shimizu
Fisheries Stock Enhancement Department
Fisheries Research Agency
Satoshi Shiozawa
Amami Station
National Center for Stock Enhancement
Fisheries Research Agency
Keywords: Sebastes schlegeli, black rockfish, mass production, stock enhancement
Abstract
We reviewed the results of black rockfish (Sebastes schlegeli) mass production at FRA Miyako Station,
Japan, between 1982 and 2004. Miyako Station began testing juvenile production of the species in 1980.
Initially, live food was mainly used for the tests, but cultivation and securing that food required a large
amount of labor. Two types of food, Artemia nauplii and formulated feed, were used for juvenile production
beginning in 1999; thus, a juvenile production system that was free from the difficulties involved with thecultivation of most live foods was established. A juvenile black rockfish with a total length of 30 mm
currently costs 8.5 yen. The survival rate of juveniles now averages approximately 50%, but it used tofluctuate widely from 19.6% to 81.4%. Early mortality of juveniles; in particular those up to 10 days after
birth, was higher and more variable. Even now, early mortality is an obstacle to juvenile production. To
stabilize the technique for juvenile production of this species, it is essential to develop a method to curb earlymortality.
Introduction
Juveniles of six rockfish species (Sebastes
schlegeli, S. inermis, S.s pachycephalus, S.s vulpes, S.
thompsoni and S. oblongus) are currently produced inJapan. Of those species, the black rockfish, grows
fastest (Nagasawa 2001) and moves in a limited
range after being released (Nakagawa et al. 2004).
For those reasons, in 2002, 3.5 million juvenile black
rockfish—70% of all rockfish produced that year
were reared for stock enhancement and mariculture

Nakagawa(Fisheries Agency & Fisheries Research Agency
2004). The species is also important to mariculture
in Korea, as is the Japanese flounder (Paralichthys
olivaceus) (Lim et al. 2004).
A study of the production of juvenile black
rockfish was undertaken by the fisheries
experimental station of Aomori Prefecture in the
1970s. That was followed by production of juvenilesof the species in Hokkaido, Akita, Yamagata, Miyagi,
Hiroshima, Fukushima, Niigata and Ehime

21

Prefectures (Kusakari 1995). The Japan Sea-Farming
Association (JASFA, predecessor of the Miyako
Station of the National Center for Stock
Enhancement, Fisheries Research Agency) began
testing mass production of juveniles in 1980. The
technique for juvenile production is now considered
to be almost established, but the scale of rearing
tanks varies among research agencies. Developmentof the juvenile production technique at Miyako
Station in recent years has therefore been aimed at
reducing the unit cost of juvenile production. On theother hand, the survival rate of juveniles with total
lengths up to 30 mm has now reached approximately
50%, but it has varied greatly from 19.6% to 81.4%(Shiozawa 1985, 1986, 1988; Shimizu 1989, 1990,
1991, 1992; Adachi 1993, 1994, 1995, 1996, 1997;
Nakagawa 1998, 1999a, 2000, 2001, 2002, 2003).
This cannot be described as a stable survival rate.
This report reviews results of the examination ofjuvenile production at Miyako Station.
Subsequently, we describe the tasks that remain to beaccomplished.

Changes in Feed Composition
Table 1 shows changes in the types of feed given
to larvae and early juvenile black rockfish.
Table 1. Changes in feed composition on mass production of
black rockfish, Sebastes schlegeli.

Second period (1994–1998). An automatic
feeding machine was developed that was capable ofdispensing a formula feed consisting of minuteparticles (400 µm), making it possible to employ it
early in life the fish. Rearing during the period began
by providing a combination of three types of feed:rotifers, Artemia nauplii and formula feed. The
production system, which had been centered on live
feed, was changed to one based on formula feed,
thereby saving labor.

Third period (1999–present). It was thoughtpossible to start using Artemia nauplii as an early live
food because the average total length of newborn
larvae of the black rockfish is 7 mm. Rotifers andArtemia nauplii had been used previously in
combination as live food for juvenile production, but
it was suggested that juvenile production was
possible by feeding Artemia nauplii alone (Nakagawa
2000). To test the hypothesis black rockfish larvae
were fed either a combination of live rotifers andArtemia nauplii (Group I) or Artemia nauplii alone(Group II). Larvae were reared for 15 days in 500 L
tanks. The average total length ±SD after 15 days ofrearing was 12.4 mm ±1.1 mm in Group I larvae and12.6 mm ±0.7 mm in Group II. No significant
difference (p > 0.05) was found between the twogroups. The average survival rates were 65.1% inGroup I and 64.9% in Group II. Therefore, two typesof feed, Artemia nauplii and formula feed, were used
for mass production of black rockfish beginning in
1999. A juvenile production system that does not
require rotifer cultivation was thus established.

Current Rearing Conditions Used for Mass
Production

First period (1982–1993). Juveniles were
produced by giving live foods such as rotifers(Brachionus plicatilis), Artemia nauplii, cultured
Altemia, eggs of pollock (Theragra chalcogramma)
and Japanese flounder, frozen larvae of black
rockfish, frozen water fleas and frozen mysids.
Dependence on live food was high and a large
amount of labor was required to cultivate and secure
feed during the period. The spread of disease anddeterioration of rearing water were feared because
fish eggs were used as food.

22

For juvenile production, it is most important toensure that few individual larvae are deformed orswim with marked instability. The larvae were
quantified, and healthy larvae were transferred using
a 50 mm diameter siphon hose to 50,000 L squareconcrete tanks (8×4.8×1.6 m) at a density of 10
larvae/1 L. For production of juvenile black rockfish,
we used a titanium and steel heater that used heatfrom a boiler, a ventilation device with a blower, anautomatic bottom vacuum cleaner and an automatic
feeder for formula feed. Artemia nauplii at 5–300
million per tank were provided in two or three
feedings each day. Incubation of Artemia eggs wascontrolled at a tank temperature of 22°C andeggshells and Artemia nauplii were separated 42 hafter hatching. Artemia nauplii were enriched for 24
h at 20°C by addition of nutrients (Plus-Aqualan,

Nakagawa

BASF Japan Ltd.) at a density of 50 g/1,000 L. One
liter of freshwater chlorella concentrate (Chlorellasp., Fresh Green 600, Nisshin Science Co., Ltd.) was
added to the rearing water in two or three portions a
day for up to 30 days after birth (DAB). Formula
feed was given from about 10 DAB in amounts of
50–5,000 g per tank in 8–16 portions daily. The
water temperature was increased by 1ºC/day from
13°C at the beginning of rearing period to the
optimum rearing temperature of 18ºC, after which it
was maintained at that temperature. The daily waterexchange rate was set at 50–100% of the total from
0–10 DAB, 100–300% from 10–20 DAB, 300–800%
from 20-30 DAB, and 800–1,200% after 30 DAB.
Sand-filtered seawater was used for rearing
larvae and early juveniles, and for incubation andnutrient enrichment of Artemia nauplii. The tankfloors were cleaned using an automatic bottom
vacuum cleaner every day and dead larvae werecounted at that time. The total length of the larvae orjuveniles was measured every five DAB, andspecimens (fixed in 10% formalin) were collected todetermine their developmental stages.

certification method, but no significant differenceswere found among periods (p > 0.05). Therefore, it
was concluded that changes in feed composition did
not cause a decline in survival rate. Labor savings injuvenile production and improvement in production
efficiency became possible through a reduction in thevariety and type of feed required for juvenile
production.
Table 2. Results of mass production breeding experiments inthe black rockfish, Sebastes schlegeli.

Survival Rate and Unit Number of Juveniles
Produced
Table 2 presents the results of juvenile
production examinations that were conducted using
50,000 L tanks. The number of juveniles produced
represents the sum of the number of juveniles foreach 1,000 L of rearing tank (hereafter called the unit
production number) because the number of tanks
used varied among years. As indicated in theclassification above, 1982–1993 was defined as thefirst period, 1994–1998 constituted the second
period, and 1999 and after was the third period. The
unit production numbers of juveniles, rearing times,
and the total lengths of juveniles at the end of
production covering each of three periods werecompared using Scheffé’s F certification method.
Results showed no significant differences among theperiods in the unit production numbers of juveniles(p > 0.05), but the total lengths of juveniles at the endof production and the rearing times differedsignificantly between the first and third periods(p < 0.05). An increase in mortality rates as a resultof handling at harvest was observed when the total
length of juveniles averaged 25 mm at the end of
production. The significantly prolonged rearing
times were attributable to the increased size of theharvest, which was intended to raise the survival rate
after harvesting. Survival rates were converted into
variables and compared using Scheffé’s F

Nakagawa

Growth and Development
Figure 1 shows the growth of larvae andjuveniles reared in 50,000 L tanks. The equation Y=
5.6609e0.184x (r2 = 0.9967) represents the relationshipbetween the total length and DAB. Variations
became greater as rearing progressed.
Table 3 shows results of observations of thedevelopmental stages of larvae and juveniles rearedin 50,000 L tanks. Larval development wasclassified into seven stages according to the methodof Nagasawa (2001): A preflexion larva, B flexion
larva I, C flexion larva II, D postflexion larva,
E transforming larva, F pelagic juvenile I and G
pelagic juvenile II.
Larvae that reached stage B immediately after
birth were observed in 2001 and 2003. Tanks inwhich the larvae were born had only small capacities(1,000 L). Therefore, the possibility of larvae born
the previous day or earlier remaining in the tank wasconsidered extremely low. Therefore, it was assumed
that the larvae that reached stage B immediately after
birth had overgrown in the mother’s body. This
seems to have resulted from a time gap between thegrowth of the larvae and their delivery, but its

23mechanism and causes remain unknown. It took
about 5 days for larvae to move between stages from
A to D, but 10 days were required from stage D to E
and more than 15 days from stage E to F. The later
the stage, the more days were required to develop to
the next stage. In all years except 1999, the larvae
reached developmental stage G 40 DAB.

Figure 1. Growth of larvae and early juveniles in black
rockfish, Sebastes schlegeli. Closed circles and vertical lines
indicate mean total length and one unit of standard deviationof total length.
Table 3. Developmental-stage composition (%) in larval andearly juvenile Sebastes schlegeli.

Mortality Patterns
The survival rate of black rockfish at Miyako
Station reached nearly 50%, but it remained unstable,
fluctuating widely from 19.6% to 81.4% (Table 1).
Daily changes in the mortality rate during the rearing
time (Figure 2) and in the shapes of individuals wereobserved during juvenile production from 1998–2003
to accurately determine mortality patterns in early
juveniles. Although minor differences were visible,
depending on the rearing lot, increases in themortality rate were observed in larvae and juveniles
from 0–10 DAB, 18–22 DAB and 30–35 DAB, apattern that was common to all years. It isnoteworthy that a mortality rate amounting to 10% of
the total in one day was observed in early juveniles
from 3–4 DAB in 2000. Normal larvae at that time
had reached developmental stages B–C, and theaverage total length was 7.7 mm ±0.3 mm. However,dead fish were numerous in developmental stages A–
B and their average total length was 6.8 mm ±0.2mm, or smaller than at birth. Those dead fish were
presumed to have died of starvation because they had
deformed mouths and vertebrae. Furthermore, every
fish in that population had an empty stomach.
Between 18 and 22 DAB, both surviving and deadfish reached developmental stage D or E, with nosignificant difference in average total length.
Numerous food organisms were found in thedigestive tracts of fish that died at 18–22 DAB,
which differed greatly from the scenario in dead fish
found 0–10 DAB. One fish was seen swimming
belly-up on the surface of the rearing water the day
before the increase in mortality rate. When dissected,that individual was found to have an air bladder thatwas far more inflated than those of normal fish,
implying death from gas-balance disorder. Aerating
the seawater immediately before it is poured into therearing tanks minimizes mortality. Mortalities oflarvae from 30 to 35 DAB occurred from cannibalismthat was attributed to differences in growth rates.

Unit Cost of Juveniles
Based on rearing data for 2003, we calculated theunit cost of juvenile black rockfish reared to a total
length of 30 mm. Computed expenses included
wages; electricity and heating bills; costs of feed,materials, and consumables; as well as rental costs,
but they did not include depreciation costs of the
facilities. Unit costs of juveniles were calculated by
dividing the costs of the two production processes ofbroodstock rearing and juvenile production by the
number of fish produced. That result was 8.5 yen.
Itemization revealed that wages accounted for 39.6%

24

Nakagawa

Figure 2. Changes in the mortality of black rockfish Sebastes
schlegeli larvae and early juveniles during 1998–2003. Closed
circles indicate mortality.

of the total, followed by electricity and heating bills
at 25.3% and the cost of feed at 15.8%. Those three
items added up to more than 80% of the total
expenses. Unit costs of juveniles by size were
calculated tentatively, including expenses forintermediate rearing as indicated by Nakagawa andOkouchi (2003a). Results were: 8.5 yen for 30 mm
fish, 10.4 yen for 40 mm, 13.5 yen for 60 mm, 15.9
yen for 80 mm, and 18.3 yen for 100 mm.

Future Tasks
For efficient juvenile production, it is necessary
to develop a technique to secure quality newborn
larvae intensively and in massive quantities. Eachday, more than 1 million newborn larvae of multiplespawning fish such as Japanese flounder are now
obtainable on a stable basis, thanks to advances in thetechniques used for broodstock rearing (Thuzaki1994). However, the black rockfish, which isviviparous, spawns only once a year (Kusakari 1995).
In addition, the larvae produced per broodstock are as
few as 200,000 (Nakagawa and Okouchi in press).
The fish do not synchronize their spawning in groups(Nakagawa and Hirose 2004). Therefore, it has beendifficult for most juvenile production agencies toobtain larvae of the species in large quantities at one
time. Using a cannulation method, Yamada andKusakari (1991) developed a technique to estimate
the birth dates of larvae from examination ofembryonic developmental stage. Using the same
technique, Nakagawa and Okouchi (in press) have
implemented a method of selecting female
broodstock estimated to be near spawning andthereby obtaining larvae intensively. Broodstock ofNakagawa

this species mate in November of each year (Mori etal. 2003), but they discharge unfertilized eggs if they
do not mate at around the time of spawning. The
new technique has also made it possible to use thecannulation method to select broodstock thatdischarge unfertilized eggs. Consequently, thetechnique for obtaining larvae intensively is nearlycomplete.
As noted here, however, many individuals amonglarvae obtained in large quantities are apparently
deformed or swim unsteadily, accounting for a major
fraction of early mortalities. This phenomenon
occurs immediately after spawning. For that reason,
it might be attributable to some cause that is inherent
in the broodstock. Stabilizing the technique for the
production of juveniles of this species demands
reexamination of the way broodstock are managed.
Development of techniques for minimizing earlymortality rates is also necessary, as are techniques for
identifying abnormal larvae in the embryo stage,
before delivery.

References
Adachi, J. 1993. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1991, Tokyo, p.159–162 [in Japanese].
Adachi, J. 1994. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1992, Tokyo, p.143–145 [in Japanese].
Adachi, J. 1995. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1993, Tokyo, p.153–155 [in Japanese].
Adachi, J. 1996. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1994, Tokyo, p.141–143 [in Japanese].
Adachi, J. 1997. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1995, Tokyo, p.165–167 [in Japanese].
Fisheries Agency & Fisheries Research Agency. 2004.
Materials on the production and release of fingerlings for
sea-farming in 2002, Tokyo, p. 431 [in Japanese].
Kusakari, M. 1995. Studies on the reproductive biology
and artificial juvenile production of kurosoi, Sebastes
schlegeli. Bull. Hokkaido Pref. Fish. Exp. Stn. 47:41–124[in Japanese with English abstract].

25

Lim, S. R., S. M. Choi, X. J. Wang, K. W. Kim, I. S. Shin,
T. S. Min and S. C. Bai. 2004. Effects of dehulledsoybean meal as a fish meal replacer in diets for fingerlingand growing Korean rockfish Sebastes schlegeli.
Aquaculture 231:457–468.
Mori, H., M. Nakagawa, K. Soyano, and Y. Koya. 2003.
Annual reproductive cycle of black rockfish Sebastes
schlegeli, in captivity. Fish. Sci. 69:908–921.
Nagasawa, T. 2001. Early life history of rockfishes(Genus Sebastes) in the Sea of Japan. Bull. Japan Sea
Natl. Fish. Res. Inst. 51:1–132 [in Japanese with English
abstract].
Nakagawa, M. 1998. Juvenile production of black
rockfish. Annual report of the Japan Sea-Farming
Association in 1996, Tokyo, p. 160–161 [in Japanese].
Nakagawa M. 1999a. Juvenile production of black
rockfish. Annual report of the Japan Sea-Farming
Association in 1997, Tokyo, p. 169–170 [in Japanese].
Nakagawa, M. 1999b. Bloodstock management of black
rockfish. Annual report of the Japan Sea-Farming
Association in 1997, Tokyo, p. 34–36 [in Japanese].
Nakagawa, M. 2000. Juvenile production of black
rockfish. Annual report of the Japan Sea-Farming
Association in 1998, Tokyo, p. 176–178 [in Japanese].
Nakagawa, M. 2001. Juvenile production of black
rockfish. Annual report of the Japan Sea-Farming
Association in 1999, Tokyo, p. 158–159 [in Japanese].
Nakagawa, M. 2002. Juvenile production of black
rockfish. Annual report of the Japan Sea-Farming
Association in 2000, Tokyo, p. 44 [in Japanese].
Nakagawa, M. 2003. Juvenile production of black
rockfish. Annual report of the Japan Sea-Farming
Association in 2001, Tokyo, p. 53–54 [in Japanese].
Nakagawa, M., and H. Okouchi. 2003a. The effects ofdensity and feeding frequency on the intermediate cultureof young black rockfish, Sebastes schlegeli. Saibai Giken
30:55–60 [in Japanese with English abstract].
Nakagawa, M., and H. Okouchi. 2003b. Abnormal
spawning of female black rockfish, Sebastes schlegeli, in
the absence of copulation. Saibai Giken 30:75–77 [inJapanese with English abstract].

26

Nakagawa, M., H. Okouchi, and J. Adachi. 2004.
Stocking effectiveness of black rockfish Sebastes schlegeli
released in Yamada Bay evaluated by a fish market census.
In K. M. Leber, S. Kitada, H. L. Blankenship and T.
Svåsand (eds.), Stock enhancement and sea ranching, p.
501–511. Blackwell, Oxford, UK.
Nakagawa, M., and K. Hirose. 2004. Individually specificseasonal cycles of embryonic development in cultured
broodstock females of the black rockfish, Sebastes
schlegeli. Aquaculture 233:549–559.
Nakagawa, M., and H. Okouchi. In press. Examination ofembryonic developmental stage by cannulation tosynchronize the timing of parturition in black rockfish
Sebastes schlegeli broodstock females. Aquacult. Sci. 53[in Japanese with English abstract].
Shimizu, K. 1989. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1987, Tokyo, p. 175–176 [in Japanese].
Shimizu, K. 1990. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1988, Tokyo, p. 194–195 [in Japanese].
Shimizu, K. 1991. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1989, Tokyo, p. 150–151 [in Japanese].
Shimizu, K. 1992. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1990, Tokyo, p. 181–182 [in Japanese].
Shiozawa, S. 1985. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1984, Tokyo, p. 157–170 [in Japanese].
Shiozawa, S. 1986. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1985, Tokyo, p. 178–180 [in Japanese].
Shiozawa, S. 1988. Juvenile production of black rockfish.
Annual report of the Japan Sea-Farming Association in
1986, Tokyo, p. 191–195 [in Japanese].
Thuzaki, T. 1994. Juvenile production of Japaneseflounder. Annual report of the Japan Sea-Farming
Association in 1996, Tokyo, p. 121–123 [in Japanese].
Yamada, J. and M. Kusakari. 1991. Staging and the timecourse of embryonic development in kurosoi, Sebastes
schlegeli. Env. Biol. Fish. 30:103–110.

Nakagawa

Construction of a BAC Library from XY Japanese Flounder
Using Frozen Sperm Genomic DNA
Hiroyuki Okamoto and Hiroyuki Nagoya
Eiichi Yamamoto
Tottori Prefectural Fisheries Experimental Station
Tomari, Tohaku, Tottori 689-0602, Japan
Takashi Sakamoto, Kanako Fuji, and Nobuaki Okamoto
Tokyo University of Marine Science and Technology
Konan, Minato, Tokyo 108-8477, Japan
Kazuo Araki
National Research Institute of Aquaculture
Tamaki, Mie 519-0423, Japan
Ichiro Nakayama
National Research Institute of Fisheries Science
Fukuura, Kanazawa, Yokohama 236-8648, Japan
Keywords: Japanese flounder, Paralichthys olivaceus, bacterial artificial chromosome, cosmid, frozen
sperm, heterozygosity
Abstract
Progress in genomic breeding for aquaculture requires several molecular genetics tools, which will
facilitate analysis of genetic linkage and synteny or to clone genes associated with desirable commercial
traits. Such tools include a recombination map, a physical (e.g., chromosome and radiation hybrid) map andgenomic (bacterial artificial chromosome [BAC] and cosmid) libraries.
We have constructed BAC and cosmid libraries from the frozen sperm of Japanese flounder(Paralichthys olivaceus). The BAC library was generated from an XY male fish, the heterozygosity of which
was confirmed by examination of the male:female ratio of its offspring. This BAC library contains DNA
sequences from both the X and Y chromosome and will constitute a useful tool for the analysis of Y-specificgenes. The sperm had been frozen in liquid nitrogen and stored in a freezer at –80°C for more than one
month. This study suggests that frozen fish sperm can be used in the construction of BAC or cosmid
libraries even after prolonged storage. We have isolated several clones (e.g., major histocompatibility
complex [MHC] class Ia) using the BAC library to analyze the synteny among fish species. Our results
further indicate that freezing does not create a bias in the library.

Introduction
The Japanese flounder (Paralichthys olivaceus)
constitutes a key species for marine culture in Japan.
It has a relatively well understood biology and a
relatively small genome (approximately 0.71
pg/haploid [700 Mb]) (Ojima and Yamamoto 1990),
which is shared among 24 chromosomes. This
genome size is estimated as similar to medaka(Oryzias latipes) (850Mb), red sea bream (Pagellus
bogaraveo or Pagrus major) (900Mb), and yellowtail(Seriola quinqueradiata) (800Mb), twice that of fugu
(Takifugu spp., among others)

Okamoto(390Mb), and one-half of the zebrafish (Danio rerio)(1.6Gb) genomes (Uwa and Iwata 1981, Brenner etal. 1993, Ojima and Yamamoto 1990, Ciudad et al.
2002). Therefore, flounder is a good model tounderstand aquaculture fish and to develop genomic
breeding.
In a project of the Fisheries Research Agency“Genomic Breeding,” we have been producing
genomic resources for Japanese flounder, including
bacterial artificial chromosome (BAC) and cosmid
libraries. The genomic libraries, having large insert

27

DNA, can facilitate rapid construction of a physicalmap and reduce the cost of structural analysis of large
chromosomal regions. They can also help us find a
responsible gene for a desirable commercial trait bypositional cloning.
The original BAC library of Japanese flounder
was constructed using the sperm of an XX
homozygous cloned fish (Katagiri et al. 2000).
However, that former BAC library had lost the Y
chromosome, so we proceeded to construct a BAC
library from an XY heterozygous flounder to getcomplete information from a set of all chromosomes.
For this purpose we had to keep the sperm frozen for
7 months until we received the results of aheterozygosity test. In this study, we describe a BAC
library made from a heterozygous flounder using its
frozen sperm, and also a cosmid library as a DNA
resource from an infectious disease-resistant fish.

Methods and Materials
Animals. Japanese flounders for constructing the
BAC libraries were obtained from the Tottori
Prefectural Fisheries Experimental Station. They
were comprised of five pairs of flounder that weremated one-to-one in June 2003, and whose offspringwere reared until January 2004. For constructing thecosmid libraries, the flounder was an antiEdwardsiella XX clone obtained from the Hyogo
Prefectural Fisheries Experimental Station.
Heterozygosity test of male fish. Five male fish
were designated TY-1 through TY-5 and theiroffspring were subjected to mild estradiol treatment(10 µg/l) for 2 hours every day from 15 June 2003 to
23 July 2003. The fish were observed from bodylengths of 13–50 mm and from 34 to 72 days after
hatching. A preliminary check of the sex ratio of theoffspring was performed in December 2003 byobserving the gonads of the fish. The final check was
performed in January 2004.
Freezing and defrosting of sperm. Semen from theTY-1 through TY-5 fish was centrifuged in June
2003 to obtain a sperm pellet. We dispensed 300–
500 µL of semen into a serum tube or a 2 ml
centrifuge tube. The semen was centrifuged for 10
minutes at 2,000 g at room temperature (preferably at4°C). The supernatant was carefully removed in ice.
The sperm pellet in the tube was immediately frozen
in liquid nitrogen. The tube was stored in a deep
freezer at –80°C until October or December 2003.
To defrost the frozen sperm pellet, we incubated
5 ml of Egami’s Ringers solution for marine fish at

28

30°C in a block incubator. The tube of frozen sperm
was placed in the block incubator, and 1 ml ofpreheated Ringers solution was added. The tube wasgently shaken with the sperm pellet. The thawed
pellet was poured into the tube of the rest of the
Ringers solution. The tube was transferred in ice andgently shaken again to initiate sperm dispersal
without using a pipette. After removing theaggregates from the thawed sperm solution, weconstructed the agarose plugs with the solution
containing 8 × 108 cells/plug. The agarose gel plug
was constructed as described by Peterson et al.
(2000).
Construction and size estimation of BAC andcosmid library. The BAC library was constructedby Geno Tech Co. (Tsukuba, Japan) using the
agarose gel plugs from the frozen sperm of the TY-4
flounder and standard methods. The high molecularweight DNA in the agarose plug was partiallydigested with MboI. After a two-step size selection,
the digested DNA was integrated into the pBAC3.6
vector.
The cosmid library was constructed with the
Copy Control Fosmid library kit (Epicentre) from thefrozen sperm of an anti-Edwardsiella tarda XX
cloned Japanese flounder established at the Hyogo
Prefectural Fisheries Experimental Station in 2002.
Analyses of insert size of BAC and cosmid
clones were performed by pulse field gelelectrophoresis (PFGE) using the fragments
generated by NotI digestion containing the insert
DNAs and BAC or cosmid vector.

Results and Discussion
To check the heterozygosity of the male fish, weused five one-to-one mating pairs in June 2003, andchecked the sex ratio of each offspring 7 months after
fertilization. Before checking the sex ratio, theoffspring were treated with mild estradiol as
previously described. The estradiol treatment did not
influence the XY fish to be male and repressed theXX fish from transforming to male because of watertemperature. According to the data obtained fromchecking the sex ratio of the fish two times, themale:female sex ratio of the offspring of the TY-4
flounder was 53:47; the numbers of male and female
offspring were 61 and 54, respectively.
We concluded the TY-4 flounder was a XY malefish, and proceeded to make a BAC library from thefrozen sperm of TY-4. Because the mating season offlounder is only during spring, we stored the sperm
until we could determine the sex ratio of the offspring

Okamatoseveral months later. We froze the sperm of the fivepaternal fish in June 2003. After checking the sex
ratio of the offspring, we prepared agarose plugs forconstructing a BAC library, and placed an order for alibrary with Geno Tech Co. using the plugs. We
checked that the length of the high molecular weight
DNA of the agarose plug was more than 550 kb,
which should be further evaluated for constructing
the BAC library (Figure 1).

Figure 1. Check length of high-molecular-weight DNA in
agarose gel plug.

The BAC library of TY-4 consisted of 110,592
clones arrayed in 288 microtiter plates with 384
wells. Analysis of insert size of the library was
performed by PFGE using the fragments of BAC
clones, which were produced by NotI digestion.
The insert size distribution is indicated in Figure
2b. The average insert size was calculated to be
140.7 kb. From the average insert size of this library(140.7 kb), the total number of clones (110,592), andthe genomic size of Japanese flounder(approximately 700Mb) (Ojima and Yamamoto1990), we estimated the coverage of our BAC library
to be 22.2 times the size of the genome.
To check the quality of the BAC library, we
preliminarily screened several genes from one-thirdof the BAC library (96 microtiter plates with 384
wells). First, we screened the major
histocompatibility complex (MHC) genes, class Ia,
Ib, IIα and IIβ, by polymerase chain reaction (PCR)
assays from the super pools of BAC clone DNA orcell culture of the library directly. This PCR
screening was performed by the primer sets from thecDNA sequences of MHC genes in Japanese flounder(Surisapoome et al. 2004). After checking each clone
by sequencing, we found that two or three positive
clones for each MHC gene were isolated from onethird of the BAC library (data not shown). Using the
BAC clones, we can develop the microsatellite
markers related with MHC genes from theirsequences, and also analyze the synteny in the MHC
regions between Japanese flounder and another fish
species.

OkamotoFigure 2. Analysis of insert size of BAC clones ofheterozygous Japanese flounder. (A) NotI-digested BAC
DNAs and vector arm on PFGE. (B) Insert size distributionof the representative BAC clones.

Second, we screened 24 microsatellite markers,
which were associated with the 24 linkage groups(Coimbra et al. 2001, 2003). We could isolate atleast one BAC clone for all 24 linkage markers (datanot shown). The results showed that freezing thesperm does not create a chromosome bias. This
study suggests that frozen fish sperm can be used in
the construction of BAC libraries even after
prolonged storage. This library is expected to clone a
Y-chromosome specific DNA sequence, which is
associated with sex determination.
We also constructed a cosmid library using thefrozen sperm of anti-Edwardsiella XX homozygous
clones of Japanese flounder. Edwardsiella tarda
causes an infectious disease in marine fish, andseverely damages the production of aquaculture. The
anti-Edwardsiella fish have been selected after thechallenge of the infectious bacteria and produced the
XX homozygous clone by Hyogo Prefectural
Fisheries Experimental Station in 2002. We
constructed a cosmid library from the diseaseresistant fish using its frozen sperm. Analysis of theinsert size of the library was performed by PFGE
using the fragments of cosmid clones, which were
produced by NotI digestion (Figure 3A), and its size
distribution is indicated in Figure 3B. The analysis
indicated that the mean insert size was 39.5 kb, themaximum insert size was 48 kb, and the minimum
insert size was 33 kb (Figure 3B). The cosmid

29

library consisted of 110,592 clones arrayed in 288
microtiter plates with 384 wells. From the averageinsert size of the library, the total number of clones
and the genomic size of Japanese flounder, theanalyses predicted that the cosmid library would be
6.2 times the size of the Japanese flounder genome.
This library is useful to clone a specific DNA
sequence, which is associated with the disease
resistance.

References
Brenner, S., G. Elgar, R. Sandford, A. Macrae, B.
Venkatesh, and S. Aparicio. 1993. Characterization of thepufferfish (Fugu) genome as a compact model vertebrate
genome. Nature 366:265–268.
Coimbra, M. R .M., O. Hasegawa, K. Kobayashi, S.
Koretsugu, E. Ohara, and N. Okamoto. 2001. Twenty
microsatellites for the Japanese flounder, Paralichthys
olivaceus. Fish. Sci. 67:358–360.
Coimbra, M. R. M., K. Kobayashi, S. Koretsugu, O.
Hasegawa, E. Ohara, A. Ozaki, T. Sakamoto, K. Naruse,
and N. Okamoto. 2003. A genetic linkage map of theJapanese flounder, Paralichthys olivaceus. I 220:203–218.
Ciudad, J., E. Cid, A. Velasco, J. M. Lara, J. Aijón, and A.
Orfao. 2002. Flow cytometry measurement of the DNAcontents of G0/G1 diploid cells from three different teleostfish species. Cytometry 48:20–25.
Katagiri, T., S. Asakawa, I. Hirono, T. Aoki, and N.
Shimizu. 2000. Genomic bacterial artificial chromosome
library of the Japanese Flounder Paralichthys olivaceus.
Mar. Biotechnol. 2:571–576.

Figure 3. Analysis of insert size of cosmid clones of AntiEdwardsiella XX clone fish. (A) NotI-digested cosmid DNAsand vector arm on PFGE. The vector size was estimated tobe 7.4 kb. (B) Insert size distribution of the representative
cosmid clones. The average, maximum, and minimum size ofthe insert DNA are 39.5, 48, and 33 kb, respectively.

Ojima, Y., and K. Yamamoto. 1990. Cellular DNA
contents of fishes determined by flow cytometry. La
Kromosomo II 57:1871–1888.
Peterson, D. G., J. P. Tomkins, D. A. Frisch, R. A. Wing,
and A. H. Paterson. 2000. Construction of plant bacterial
artificial chromosome (BAC) libraries: An illustrated
guide. J. Agricul. Genomics. Online atwww.ncgr.org/research/jag.

These genomic libraries are stored in the
National Research Institute of Aquaculture or
National Research Institute of Fisheries Science ofJapan, and are available upon request for research
purposes.

Srisapoome, P., T. Ohira, I. Hirono, and T. Aoki. 2004.
Cloning, characterization and expression of cDNA
containing major histocompatibility complex class I, IIα
and IIβ genes of Japanese flounder Paralichthys olivaceus.
Fish. Sci. 70:264–276.

Acknowledgments

Uwa, H., and A. Iwata. 1981. Karyotype and cellular
DNA content of Oryzias javanicus (Oryziatidae, Pisces).
Chromosome Information Service 31:24–26.

The authors thank the Hyogo Prefectural
Fisheries Experimental Station for providing theJapanese flounder XX clone fish. We also thank Drs.
Masanori Okauchi, Takanori Kobayashi, andKagayaki Morishima for providing technical support
and Tomoyo Nozaki and Keiko Ikeyama forlaboratory assistance. This research was supported
by a grant from the Genomic Breeding Project by the
Fisheries Research Agency of Japan.

30

OkamatoStatus of Pacific Bluefin Tuna (Thunnus orientalis) Seed Productionin Amami Station of NCSE FRA
Hideki Nikaido, Takayuki Takebe, Kentaro Ide, and Hitoshi Imaizumi
Amami Station National Center for Stock Enhancement
Fisheries Research Agency
955-5, Hyo Setouchi Oshima
Kagoshima 894-2414 Japannikaidoh@fra.affrc.go.jp
Nobuhiro Tezuka
Notozima Station National Center for Stock Enhancement
Fisheries Research Agency
15-1-1 Magarimachi, Notozima
Nanao, Ishikawa, 926-0216, Japan
Syukei Masuma
Miyazu Station National Center for Stock Enhancement
Fisheries Research Agency
1721 Odasyukuno, Miyazu
Kyoto, 626-0052, Japan
Keywords: bluefin tuna, Thunnus orientalis, seed production, stock enhancement
Abstract
We have been developing seed production technology for bluefin tuna since 1995 and succeeded inrearing about 12,000 juveniles (average = 47 mm total length [TL] in 1998). However, the survival rate fromhatching to the juvenile stage was typically only 0.1–0.3%. Three problems exist in seed production of
bluefin tuna: viral nervous necrosis (VNN), 10 days after hatching (DAH) survival, and cannibalism
occurring after 7 mm TL. The VNN was preventable by sterilizing fertilized eggs of bluefin tuna using
residual oxidants in seawater and using oxidants in rearing chambers to sterilize the water. However,reasons and mechanisms of problems of the 10 DAH survival rate and cannibalism remained uncertain.
We presumed that one factor affecting the 10 DAH survival rate is that bluefin larvae sink to the tankbottom during the night and subsequently die, but the cause remains unclear. We circulated the water bycontinuous pumping, which prevented the larvae from sinking. We examined a pump system and an
aeration pipe system to produce water currents. Consequently, the 10 DAH survival rate in 2005 improved
to 46%. The 10 DAH survival rate improved to greater than 60% in 2005.
Survival during rearing of the early larval stage improved greatly by prevention of VNN and improved
the 10 DAH survival. Using these systems, seed production technology of bluefin larvae has been steadily
developing and is anticipated to progress further.

Introduction
Tuna is a marine product that is cherishedworldwide; its popularity is not limited to Japan.
Bluefin tuna, which has a very high market value,
grows quickly and is the largest of the tunas. The
bluefin is an extremely important Japanese fisheryresource. The bluefin tuna is also an international
fishery resource for other countries. For thesereasons, management of bluefin has been entrusted
to international committees. Japan’s government,

Nikaidowhich serves a large market for bluefin, has promoted
the development of stock enhancement techniques for
propagation of wild resources of bluefin tuna.
Three obstacles hinder bluefin tuna seed production:
viral nervous necrosis (VNN), the 10 days after
hatching (DAH) survival rate, and cannibalism
occurring after the larvae reach 7 mm total length(TL). The first of those, VNN, is preventable using
sterilized fertilized bluefin tuna eggs with residual

31oxidants in seawater (Takebe et al. 2004, Imaizumi
et al. in press) and by the use of oxidant-sterilized
seawater as rearing water (Tezuka et al. 2004).
However, the causes and mechanisms associatedwith the problems of 10 DAH survival rate andcannibalism are unclear.
We presumed that one cause of death before 10
DAH is that bluefin larvae sink to the tank bottom
during the night and subsequently die, but the root
cause remains uncertain. To address the problem,
we decided to use a method of circulating watercontinuously using a current generator to prevent
larvae from sinking. We produced currents using apump system and an aeration pipe system. We
examined the 10 DAH survival rate, the current
direction and speed, and the distribution of bluefin
tuna larvae with respect to water depth. This paper
presents the results of those studies with respect toimprovement of 10 DAH survival.

Materials and Methods
Fertilized eggs and hatching method. Takuyo
Co. Ltd. and Kinki University provided fertilized
eggs of bluefin tuna used for seed production; somewere collected from broodstock at Amami Station.
A polycarbonate Artemia hatching tank (200 L) wasused. We sterilized fertilized eggs using residual
oxidants (0.3 mg/L) in seawater before placement in
the hatching tank. The eggs were hatched inoxidant-sterilized seawater.

Figure 1. Photo of pump system. The small arrows show thedirections of flow of sea water and the larger arrows show thecurrent flow.

Aeration pipe system. The aeration pipe system(Figure 2) is comprised of 1 m long, 25 mm diameter
air sprayer hoses. We placed 4–8 pipes on the bottomside of the wall of the rearing tank to produce an
upward current by aeration. We did not attach airstones.

Rearing tanks and seawater. We used octagonal
concrete rearing tanks (50,000 L) at Amami Station
for this study. All seawater used for rearing bluefin
tuna larvae was sterilized using oxidants (0.5 mg/l)
generated in the seawater by electrolysis. We used
activated carbon to absorb residual oxidants.
Current generation method. We tested a methodthat allowed continuous rotation using two devices.
One used a pump, the other used aeration pipes thatare usually used for rearing fish.
Pump system. The pump system (Figure 1) is madeof one pump put in a strainer in the center of therearing tank and crossed plastic pipes with holes(1.0 mm) at 10 cm intervals. Plastic pipes wereplaced on the bottom of the rearing tank, and thenturned upward at 40–50° angles. The rearing
seawater spouts were formed from holes in theplastic pipe, producing an upward current. We used
air stones for aeration.

32

Figure 2. Photo of aeration the aeration system. The small
arrows show air flow directions and the larger arrows show thecurrent direction.

Measurement of current direction and speed. We
divided the rearing tank bottom into 16 areas andmeasured the current direction and speed at the same
point of each area. Depths of the measurement points
were set at 5 cm, 30 cm, 1 m, and 2.5 m from the tankbottom. We used an electromagnetic current meter(ACM-300A, Alec Electronics Co. Ltd.) for
measurements.

NikaidoLarval distribution. We measured the distribution
density of larvae at specific points within the tank.
Depths measurement points were 30 cm, 50 cm, 1m, 1.5 m, 2 m, and 2.5 m from the surface. We
used 16 mm inside diameter plastic pipes for larvae
sampling. The distribution density was calculatedusing the number of larvae per liter of water. The
survey was conducted at 1100 hours and 2100 hours
on 3 DAH and 6 DAH.

Results
In 2005 we reared larvae four times using thepump system and twice using the aeration pipe
system (Table 1). We compared results using
fertilized eggs of identical origin with rearing case2R.

Figure 3. Comparison between survival rates of bluefin larvae
using two ways of generating water currents. (All eggs werefrom the same origin.)

Table 1.

Figure 4. Comparison between survival rates of bluefin larvae
using two ways of generating water currents in tanks. (Originof eggs was not the same in all cases.)

Survival of larvae. The 10 DAH survival rates
using the pump system were 35.9% and 62.4%.
The 10 DAH survival rates using the aeration pipe
system were 5.5% and 6.3%. Rearing results
showed that the pump system provided much higher
survival rates than the aeration pipe system (Figure3). Particularly noteworthy is that the 62.4% resultof 2R-3 became the highest result ever recorded forrearing at Amami Station. The 10 DAH survival
rate ranged from 20.5% to 62.4% for the pumpsystem in all rearing cases including when we used
fertilized eggs of different origin. This result wasconsiderably higher than the 5.5% and 6.3% results
achieved by the aeration pipe system (Figure 4).

NikaidoMeasurement of current direction and speed. We
measured the direction and speed of the current inrearing water (Figure 5). Results for the pump systemshowed a tendency for a strong upward current fromthe bottom of the rearing tank. The current speed wasabout 4–9 cm/s upward. With the aeration pipe
system, the upward current speed from the bottom of
the rearing tank was slower, about 2–5 cm/s, and thecurrent direction was irregular.
Larval distribution. We measured the larval
distribution density for every depth 3 DAH and 6
DAH. With the pump system, the density of larvae at
1100 hours on the third DAH was similar from thesurface to 1.5 m depth. Larvae were present at the 2 mdepth, but distribution density was reduced. Larvae
were almost absent at the bottom of the rearing tankusing the pump system. At 2100 hours, the larvae
were uniformly distributed from the surface to 2.0 m

33

depth and were rare at the rearing tank bottom(Figure 6).

Figure 6. Density of larvae as a function of depth in the tank 3
DAH.

Figure 5. Current direction and speed in rearing tanks
using two methods to generate currents. The arrows show
direction of the currents.

The densities of bluefin larvae at 1100 hours and2100 hours were almost identical from the surface
to 50 cm depth. Few larvae were found at thebottom of the rearing tank (Figure 7).
In the aeration pipe system, larval density at 1100
hours on the third DAH was flat from the surface to
2.0 m depth; larvae were present at the rearing tankbottom but the density was low (Figure 6). The
density of bluefin larvae at 1100 hours and 2100
hours on the sixth DAH with the aeration pipe
system was almost identical from the surface to 50
cm depth and density at the rearing tank bottom waslow (Figure 7).

Discussion
Regarding prevention of the low 10 DAH
survival rate, Tezuka et al. (2005) examined theeffects of light on the surface and current of rearing
seawater from 1999 to 2001. Results showed thenecessity of surface illumination at more than 800
lux and the current speed of the rearing seawater at
1–2 m/s. It is possible to maintain averageilluminance over 800 lux, as Tezuka et al. (2005)

34

Figure 7. Density of larvae as a function of depth 6 DAH.

reported, by taking natural light from the top of therearing tank. Beginning in 2004, we examined theeffects and production methods for providing a current
in the rearing tanks. We succeeded in preventing
bluefin larvae from sinking to the tank bottom byproducing a water current with a pump system. We
demonstrated improvement of the 10 DAH survival
rate. The rate improved to 40% in 2003, but was about
10% in 2000, subsequently improving to a 60% 10
DAH survival rate in 2005.
The purpose of generating the current is to preventthe bluefin larvae from sinking to the bottom of the

Nikaidorearing tanks. Sakamoto and Takashi (2005)
reported that bluefin tuna larvae tend to sink during
the day, and float at night. However, during
rearing, bluefin larvae sink to the bottom of therearing tank and die at night in most cases. Thisphenomenon is not explainable by gravity
fluctuation, as Sakamoto and Takashi (2005)
suggested. They reported that bluefin tuna larvae
are prevented from sinking by swimming actively
for feeding during the day; larvae sink because of
weak swimming during the night. We determinedthat the effect of the pump system prevented bluefin
larvae from sinking to the bottom of rearing tanks
and dying by forcibly pushing them upward using acurrent.
We prevented the VNN problem and solved thelow 10 DAH survival rate, thereby finding solutions
to two of the three salient problems inherent inrearing bluefin larvae. As a result, the survival rate
for rearing early larval stage improved
substantially. The cannibalism problem, however,remains unsolved and will continue as an important
subject for future study. We are reviewing thefeeding protocol to determine if there is a way tomitigate growth differences of larvae and thereby
control cannibalism at TL 7 mm. It appears
possible to delay cannibalism by delaying therotifer period, providing the bluefin larvae withlarval brine shrimp (Artemia nauplii), and delaying
the start of live feeding.
Existing trials show steadily improved results
and present the possibility of mass production of
seedlings. From here, we will examine seedproduction techniques, other measures of mass andhealthy seed production techniques, and secure
genetic diversity, which will be described in futurereports.

NikaidoAcknowledgments
Special appreciation is extended to Takuyo Co.,
Ltd. and the Amami laboratory of Kinki University forproviding valuable fertilized bluefin tuna eggs.

References
Takebe, T., S. Masuma, N. Tezuka, H. Nikaido, and K. Ide.
2004. Hatching management on fertilized eggs of blue fin
tuna thunnus orientalis disinfected by ozonated seawater.
NCSE Technical Report (tentative) 1:80–83.
Imaizumi, H., T. Takebe, H. Nikaido, K. Ide, and S.
Masuma. In press. Effect of low level residual oxidants inseawater on hatching of fertilized eggs of bluefin tuna
Thunnus orientalis. NCSE Technical Report (tentative).
Tezuka, N., S. Masuma, T. Takebe, H. Nikaido, and K. Ide.
2004. Control of Viral Nervous Necrosis (VNN) using
ozonated seawater in seedling production of bluefin tuna,
Thunnus orientalis. NCSE Technical Report (tentative).
1:76–79.
Tezuka, N., M. Koiso, S. Masuma, T. Takebe, H. Nikaido,
and K. Ide. 2005. Effect of water current and light onsurvival of larval bluefin tuna, Thunnus orientalis. NCSE
Technical Report (tentative), 3:41–44.
Sakamoto, W., and T. Takashi. 2005. Gravity changes of
bluefin larvae. The interim research reports of 21st Century
COE Program, Center of Aquaculture Science andTechnology for Bluefin Tuna and Other Cultivate Fish, in
2003–2004, p. 113–116. Fisheries Laboratory & Graduate
School of Agriculture, Kinki University.

35

Visualization Tools to Probe Early Stage Fish Abnormalities
Susumu Uji and Tadahide Kurokawa
National Research Institute of Aquaculture
Research Agency, 422-1
Minami-ise, Watarai
Mie 516-0193, Japanuji@fra.affrc.go.jp
Keywords: abnormality, antibody, histochemical staining, visualization
Abstract
The incidence of malformed fish is an important problem in the seed production of many species. The
abnormal appearance of fish can be judged only when they have become large because examination ormeasurement of the miniscule exterior, jaws, faces, etc., of embryos and larvae is difficult. If abnormalities
occur frequently, then unnecessary expenditures of human resources, money, and time must be undertaken
to raise fish to sufficient size for examination. That waste would be eliminated if we could judge
malformation at an early stage. To this end, we are developing rapid malformation judgment methods forfish in embryo and larval stages.
Malformations are now discovered only at later growth stages, but we infer that they occur mainly
during abnormal organogenesis at early stages. These malformations are detectable in early stages using
visualization tools for specific organs and cells. Antibodies and histochemical staining methods are suitable
for that purpose because they are effective with many fish simultaneously. Many antibodies andhistochemical staining methods are useful for commercial sea fishes. For example, antibodies to acetylated
tubulin, to a cell surface marker (HNK-1), and to Na K-ATPase (a5) are useful, respectively, to visualize thenervous system, neural crest cells, and kidney in fugu (Fugu rubripes), also known as puffer fish, yellowtail(Seriola quinqueradiata), and Japanese eel (Anguilla japonica). Calcein and alkaline phosphatase staining
methods are also useful to visualize bones and intestines. Using these tools to probe abnormalities, we canwork efficiently to solve malformation problems.

Introduction
A considerable number of studies over many
years have examined wild and reared fishabnormalities (Dawson 1964, Dawson 1966, Dawson
1971a, b). Nevertheless, numerous malformed fish
exist in hatchery production throughout the world(Komada 1980, Koumoundouros et al. 1997,
Boglione et al. 2000, Cobcroft et al. 2001 and 2004,
Fraser et al. 2005). Malformed fish are a significant
problem for the aquaculture industry because
malformations are often associated with growthdepression and high mortality rates (BarahonaFernandes 1982, Andrades et al. 1996). Their
incidence engenders unnecessary expenditures ofresources, money, and time required to raise fish in
the hatchery. Fish with malformations also depress
prices and lower market demand.
Despite their importance, the causes ofabnormalities remain poorly understood. Genetic,
environmental, and nutritional causes have beensuggested as possible sources of abnormalities. For
example, genetic factors have been reported to causeUjidevelopmental abnormalities (Kincaid 1976, Gjerde
et al. 2005). Environmental factors such asmechanical or thermal shock, salinity, and oxygen
depletion also have been reported to cause
developmental abnormalities (Alderdice et al. 1958,
Garside 1959, Doroshev et al. 1974, Lee et al. 1981,
Divanach et al. 1997). Several dietary components,
such as fatty acids or vitamins, affect theseabnormalities (Cahu et al. 2003). Although manystudies exist, most examine environmental andnutritional factors, especially at the juvenile stage.
Almost none address maternal factors at early
developmental stages.
This study specifically examines this earlystage—especially maternal factors—until the fish
have a first feeding. Previous studies have shownthat egg quality is a critical factor for survival(Kjørsvik et al. 1990, Furuita et al. 2003, Aegerter et
al. 2004). Most organ systems develop during theearly stages of organogenesis, when organs such as
limbs, eyes, and gills develop (Gilbert 2003).

37

Consequently, aberrant development during that
period creates living but abnormal fish. Duringorganogenesis, which occurs before young fish begin
to feed, the embryo develops using only egg
nutrition. Most humans and domestic animals also
have congenital malformations (Noden et al. 1985,
Moore et al. 1998).
Abnormal development is believed to be
influenced by maternal factors that engender fish
malformation, but no adequate technique exists toassess such problems. The embryo is extremely
small and too transparent to check for abnormalities;
we can obtain little information about abnormalities
using only a microscope. With this consideration, wedeveloped visualization tools to probe for early stage
abnormalities.
This paper represents a compilation of antibody
and histochemical staining methods we used tovisualize tissues of various fish species. We also
propose a concept to facilitate solving malformation
problems.

Antibody Staining Methods
Embryos are extremely small and are tootransparent to allow checking for abnormalities. For
those reasons, we can obtain little information aboutabnormalities merely by using microscopy. To date,assessment of abnormalities has been conducted inmost hatcheries at later stages, such as the larval orjuvenile stages.
Visualization tools are needed to detect
abnormalities at earlier stages such as the embryo or
early larval stages. Visualization tools must also bereadily available for many kinds of fishes. Antibody
and histochemical staining methods meet thoserequirements. Using such methods, we can visualize
their structural information. These methods areuseful for many kinds of fishes. Moreover, we candeal with numerous fish simultaneously.
The availability of antibodies and histochemical
staining methods against specific cell lineages
facilitates the identification and localization ofdifferent organs and cells among species (Table 1).
Furthermore, because of their small size, whole fish
histology can be performed to examine forabnormalities.
Immunohistochemical analyses were performed
using six antibodies: anti-MF-20, anti-HuC/HuD
antibody, anti-HNK-1 antibody, anti-acetylated
tubulin antibody, anti-a5 antibody, anti-myosin heavy
chain, and clone A4 1025 antibody (Figure 1). We
performed experiments on four teleost species: fugu

38

Table 1. Staining method for analysis of abnormal
development in fish.

(Fugu rubripes), zebrafish, (Danio rerio), Japaneseeel, (Anguilla japonica), and yellowtail, (Seriola
quinqueradiata). All these antibodies react in all
teleosts that we examined and antigens derived from
nonteleost species such as mammalian species.
These antibodies probably react in teleosts at least.
For example, anti-HuC/HuD antibody stains
neuronal cells in all organisms that we examined.
The antibody binds specifically to antigens that arepresent exclusively in neuronal cells and are therefore
useful as markers of neuronal cells in tissues. Someabnormal fish phenotypes appear to resemble humanneurocristopathies. Neurocristopathies are neuralcrest-cell diseases in humans. Neural crest cells
originate at the dorsal-most regions of the neuraltube, migrate extensively, and eventually give rise toa remarkable variety of cell types, including neurons
and glial cells, cartilage, pigment cells, andchromaffin cells. Anti-HNK-1 monoclonal antibody
recognizes a carbohydrate epitope on subpopulationsof several cell-adhesion molecules including N-CAM
and MAG. Neural crest cells express this N-CAM.
Consequently, neural crest cells can be stained using
this antibody (Suzuki et al. 2003, Ishikawa et al.
2004). The monoclonal antibody against αacetylated tubulin stains early axons and young
somata of neurons immunohistochemically (Ishikawa
et al. 2004). Anti-a5 antibody recognizes the asubunit of the sodium potassium ATPase (Na, KATPase, Lebovitz et al. 1989). This protein is pivotal
to various physiological processes, including
osmoregulation, cell volume regulation, transport ofcertain amino acids and sugars, and maintenance of
membrane excitability. This method enables thevisualization of kidney and chloride cells.
In addition, MF-20 antibody reacts with all
sarcomere myosin (Bader et al. 1982). It is possible
to view myotomes; thereby, one can speculate thatUji

somites give rise to muscles and the vertebral
column, as well as the epidermal tissues. The antimyosin heavy chain (clone A4 1025) antibody stains
myotomes and cardiac muscle. It allows
visualization not only of myotomes and cardiac
muscle, but also eye muscles such as the superior
oblique, inferior oblique, superior rectus, and inferior
rectus. All six pairs of these eye muscles are visible,
in addition to pharyngeal muscles at later stages.

Figure 1. Examples of whole mount immunohistochemical
staining: a, anti-HuC/HuD antibody reacts in zebrafish
embryo; b, anti-myosin heavy chain, clone A4 1025 antibody
reacts in fugu; c, anti-a5 antibody reacts in Japanese eel; andd, anti-acetylated tubulin antibody reacts in Japanese eel.

human blood tests; using it, we can readily view
blood vessels in whole mounted samples.

Concept to Resolve Fish Malformation
Problems
Several important factors appear to affect the fishin early developmental stages. These factors areclassifiable as genetic, maternal, and environmental.
Perhaps all of them cause malformation. The times
at which these factors exert their respective
influences are also important. First, to approachthese problems, we specifically examine only
maternal factors at the embryo stage. Then we
hypothesize that abnormal development by maternal
factors is a major cause of malformed fish. So far,however, we have only judged malformation whenfish are sufficiently large to verify their shape; at thisstage, it remains unclear whether fish that are being
raised are normal or abnormal. As explained earlier,
it is very inefficient to raise abnormal fish. Somejudgment can be made earlier if the above hypothesis
is correct. Figure 2 shows a simplified scheme of anearly batch checking strategy with fish embryos.

Histochemical Staining Methods
The histochemical staining method is more
useful to assess abnormalities for many kinds offishes. Bone and cartilage are stained, respectively,
using the alizarin red method and alcian blue method.
These well-known histochemical staining methods
are useful as visualization tools to probe fishabnormalities. Calcein is a fluorescent chromophorethat binds specifically to calcium (Du et al. 2001).
This method has an advantage in that it can be used
to follow the development of skeletal systems inliving embryos.
The alkaline phosphatase staining method
visualizes the intestine. Fish exhibit high levels ofendogenous alkaline phosphatase, which renders
them detectable simply by enzymatic staining(Serbedzija et al. 1999). Alkaline phosphatase exists
mainly in endothelial cells. Thus endothelial cells
that line the blood vessel lumen are visible, but wecan see only the intestine and osteoblasts, which havehigh levels of endogenous alkaline phosphatase at thebase of the tooth. That limitation notwithstanding,the method is sufficient because it allows inspectionof the intestines. The sudan black B staining method
shows the blood vessels of intestines. Originally,sudan black B stain was used to stain general fat inUji

Figure 2. Simplified scheme of an early batch checking
strategy.

Because of their small size, the entire checking
procedure can be accomplished within 2 days, andthe cost of this checking procedure is low. If
development could be judged earlier, we could selectgood populations to raise, thereby eliminating thewaste of resources, money, and time in raisingabnormal fish to a sufficient size to allow
examination. Environmental factors could also beassessed, such as mechanical or thermal shocks,
salinity, and oxygen depletion, in terms of theireffects on normal organ development of fish using
these visualization tools. The effects of drugs andchemicals on growth and development could beassessed in detail by examining the size andmorphology of internal organs such as the brain,

39

muscle, intestines, and kidneys using thesevisualization tools.
In summary, this study revealed that many
antibody and histochemical staining methods areuseful for visualization of many, perhaps nearly all,fishes. This paper also presented a new concept toresolve malformation problems.

Acknowledgments
We thank Tohru Suzuki, Hisashi Hashimoto, andKeiichi Mushiake for useful suggestions. The
authors are grateful to Hideki Tanaka and Yoshitomo
Nagakura for providing Japanese eel and yellowtail
samples, respectively. The MF-20 antibody and a5
antibody developed by Donald A. Fischman andDouglas M. Fambrough, respectively, were obtained
from the Developmental Studies Hybridoma Bank
developed under the auspices of the National Instituteof Child Health and Human Development andmaintained by the University of Iowa, Department of
Biological Sciences, Iowa City, Iowa. This study
was supported in part by a grant-in-aid from the BioDesign Program and Development of Seed
Production Technology in Japanese Eel and Japanese
Spiny Lobster Project of the Japanese Ministry of
Aquaculture, Forestry, and Fisheries.

References
Aegerter, S., and B. Jalabert. 2004. Effects of postovulatory oocyte ageing and temperature on egg quality
and on the occurrence of triploid fry in rainbow trout,
Oncorhynchus mykiss. Aquaculture 231:59–71.
Alderdice, D. F., W. P. Wickett, and J. R. Brett. 1958.
Some effects of temporary exposure to low dissolved
oxygen levels in Pacific salmon eggs. J. Fish. Res. Board
Can. 15:229–249.
Andrades, J. A., J. Becerra, and P. Fernandez-Llebrez.
1996. Skeletal deformities in larval, juvenile, and adultstages of cultured gilthead sea bream (Sparus aurata).
Aquaculture 141:1–11.
Bader, D., T. Masaki, and D. A. Fischman. 1982.
Immunochemical analysis of myosin heavy chain during
avian myogenesis in vivo and in vitro. J. Cell Biol.
95:763–770.
Barahona-Fernandes, M. H. 1982. Body deformation inhatchery reared European sea bass Dicentrarchus labrax:
Types, prevalence, and effect on fish survival. J. Fish
Biol. 21:239–249.

40

Boglione, C., F. Gagliardi, M. Scardi, and S. Cataudella.
2000. Skeletal descriptors and quality assessment in larvaeand post-larvae of wild-caught and hatchery-rearedgilthead sea bream (Sparus aurata). Aquaculture 192:1–
22.
Cahu, C., J. Z. Infante, and T. Takeuchi. 2003.
Nutritional components affecting skeletal development infish larvae. Aquaculture 227:245–258.
Cobcroft, J. M., P. M. Pankhurst, C. Poortenaar, and B.
Hickman. 2004. Jaw malformation in cultured yellowtail
kingfish (Seriola lalandi) larvae. N. Z. J. Mar. Freshw.
Res. 38:67–71.
Cobcroft, J. M., P. M. Pankhurst, J. Sadler, and P. R. Hart.
2001. Jaw development and malformation in cultured
striped trumpeter Latris lineata. Aquaculture 199:267–
282.
Dawson, C. E. 1964. A bibliography of anomalies offishes. Gulf Res. Rep. 1:308–399.
Dawson, C. E. 1966. A bibliography of anomalies offishes: Supplement 1. Gulf Res. Rep. 2:169–176.
Dawson, C. E. 1971a. A bibliography of anomalies offishes: Supplement 2. Gulf Res. Rep. 3:215–239.
Dawson, C. E. 1971b. A bibliography of anomalies offishes: Supplement 3. Gulf Res. Rep. 5:35–41.
Divanach, P., N. Papandroulakis, P. Anastasiadis, G.
Koumoundouros, and M. Kentouri. 1997. Effect of water
currents on the development of skeletal deformities in sea
bass (Dicentrarchus labrax) with functional swim bladders
during postlarval and nursery phase. Aquaculture
156:145–155.
Doroshev, S. I., and T. M. Aronovich. 1974. The effectsof salinity on embryonic and larval development ofEleginus navaga (Pallas), Boreogadus saida (Lepechin)and Liopsetta glacialis (Pallas). Aquaculture 4:353–362.
Du, S. J., V. Frenkel, G. Kindschi, and Y. Zohar. 2001.
Visualizing normal and defective bone development inzebrafish embryos using the fluorescent chromophore
calcein. Dev. Biol. 238:239–246.
Fraser, M. R., and R. de Nys. 2005. The morphology andoccurrence of jaw and operculum deformities in cultured
barramundi (Lates calcarifer) larvae. Aquaculture
250:496–503.
Furuita, H., T. Yamamoto, T. Shima, N. Suzuki, and T.
Takeuchi. 2003. Effect of arachidonic acid levels inbroodstock diet on larval and egg quality of Japaneseflounder Paralichthys olivaceus. Aquaculture 220:725–
735.

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Garside, E. T. 1959. Some effects of oxygen in relation totemperature on the development of lake trout embryos.
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Gilbert, S. F. 2003. Developmental Biology, 7th edition.
Sinauer Associates Inc., Sunderland, Massachusetts.
Gjerde, B., M. J. R. Pante, and G. Baeverfjord. 2005.
Genetic variation for a vertebral deformity in Atlantic
salmon (Salmo salar). Aquaculture 244:77–87.
Ishikawa, Y., T. Kage, N. Yamamoto, M. Yoshimoto, T.
Yasuda, A. Matsumoto, K. Maruyama, and H. Ito. 2004.
Axonogenesis in the medaka embryonic brain. J. Comp.
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Egg quality in fishes. Adv. Mar. Biol. 26:71–113.
Komada, N. 1980. Incidence of gross malformations andthe vertebral anomalies of the natural and hatchery
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1989. Molecular characterization and expression of the(Na+ + K+)-ATPase alpha-subunit in Drosophilamelanogaster. Embo. J. 8:193–202.
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development and hatching in grey mullet (Mugil
cephalus). J. Fish Biol. 19:179–188.
Moore, K., and T. V. N. Persaud. 1998. Before we areborn: Essentials of embryology and birth defects. 4thedition. WB Saunders Co., Philadelphia, Pennsylvania.
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and scleroblasts during dorsal fin skeletogenesis inflounder larvae. Dev. Growth Differ. 45:435–448.

Koumoundouros, G., G. Oran, P. Divanach, S. Stefanakis,
and M. Kentouri. 1997. The opercular complex deformity
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Uji

41

Airlift-PolyGeyser Combination Facilitates Decentralized Water Treatment
in Recirculating Marine Hatchery Systems
Ronald F. Malone and Sripavani Gudipati
Department of Civil and Environmental Engineering
Louisiana State University, Baton Rouge, Louisiana 70803-6405
Rmalone@LSU.edu
Keywords: Marine hatcheries, recirculation; biofiltration, airlifts, bead filters, PolyGeyser
Abstract
The nature of reproduction in fecund marine species demands that careful attention be given to theconditioning of broodstock, culture of sensitive larval stages, and production of healthy, properly sized
fingerlings. Hatchery operations generally demand pristine waters and have been found to be particularly
sensitive to biosecurity issues. The trend is toward the design of systems that are capable of meeting more
rigorous water quality expectations in recirculating formats that display a high degree of water reuse.
Traditional treatment sequences consisting of bead filters, fluidized beds, packed columns, and often pureoxygen injection systems have served the research community. These systems have also been successfully
applied in the commercial production of shrimp postlarvae and ornamental fish.
Increasing concerns about biosecurity are encouraging the examination of more decentralized
approaches to water treatment. One such partitioned approach utilizes a pneumatic PolyGeyser floating
bead filter (Aquaculture Systems Technologies LLC, New Orleans, Louisiana) in combination with airlifts tomeet core recirculating needs. Use of airlifts allows the centralization of utilities without the potential ofcross contamination of waters from diverse tank systems. The PolyGeyser filters are pneumatically washedin a manner that permits the recycling of backwash water, thus reducing the need for water replenishment.
Sizing criteria for the bead filters, tanks, and airlift systems are provided to facilitate evaluation in thecommercial sector.

Introduction
It can be anticipated that future marine
production facilities will consist of recirculating
hatcheries coupled with larger scale ponds, tanks, or
net pen operations (Malone 2002). Recirculating
systems are expected to continue to play an importantrole in conditioning broodstock, supporting larvalstages, and rearing fingerlings as marine aquaculture
production expands. These closed marine systems
will have to be operated in a biosecure manner,
thereby assuring the health of broodstock andfingerlings that must be held for extended periods oftime. Water quality expectations will be high,reflecting the high value placed upon healthy,
properly sized fingerlings for stocking growout
facilities. A variety of existing recirculating designs
will be adapted to meet the needs of marine hatcherysystems. Recirculating systems will have to be
designed with a relatively high hydraulic residencetime, minimizing the opportunity for disease
introduction while also minimizing discharge issues.
This paper describes the underlying rationale for an
airlift-floating bead filter combination that isMalonecompatible with the emerging needs of the marine
hatchery systems.
Water quality targets. Malone and Pfeiffer (2006)
proposed a recirculation system classification, laying
a foundation for the establishment of systematicengineering criteria for treatment of recirculating
systems (Figure 1). They identified 17 distinct
recirculating classifications based on salinity,
temperature, and trophic level. This paper
specifically addresses the treatment of marine
systems suitable for use as broodstock maturation andfingerling growout systems in marine hatcheries.
Table 1 presents selected water parameters, the levelsof which characterize water quality expectations forthese hatchery systems. Establishment of these water
quality expectations allows the formulation oftreatment packages that will meet the operator’s
performance expectations. The vast majority ofmarine production needs can be met within theguidelines for oligotrophic and mesotrophic systems;
however, there is some encroachment on the
eutrophic categories.

43

Figure 1. Malone and Pfeiffer (2005) identified 17 distinct
types of recirculating systems.

Table 1. Presumed water quality targets for warm water andcold water marine hatchery systems.

Marine broodstock maturation systems are
generally designed to meet the highest water quality
expectations. These high standards reflect the highvalue of the eggs produced by generally fecundbroodstock. Egg quality often reflects the health andvigor of the broodstock.

44

Conditioning of broodstock often takes months
and few operations can risk loss of the time andmonetary investment associated with death ofbreeders late in the process. Thus filtration systems
are typically oversized, assuring excellent water
quality. Maximum thresholds for toxic compounds(total ammonia nitrogen [TAN] and nitrite-N) are set
at relatively low levels (<0.3 mg-N/L), whilesecondary parameters (biochemical oxygen demand[BOD] and total suspended solids [TSS]) are set atlevels reflective of natural unpolluted marine
systems. Broodstock are generally stocked at a low
density (<15 kg/m3), a practice that, in combination
with the low TAN targets, mandates highrecirculation rates.
Fingerling production systems focus on the
production of healthy fingerlings sized for release or
growout. The value of these fingerlings remains highwhen measured on an individual weight basis andthus justifies recirculating aquaculture systems (RAS)
investment. Fingerlings are generally recognized assensitive to water quality, so toxic water quality
parameters are set at safe but manageable levels (<0.5mg-N/L). Density of these systems is generally
moderate (<30 kg/m3), but feeding rates can be high
(3–7%/day). Water treatment components must be
robust and capable of mitigating high rates of waste
production. Suspended solids removal becomes
critical at this stage. Solids in recirculating systems
are largely organic. Uncontrolled TSS buildups
induce bacterial shifts in the biofilters to thedetriment of nitrifiers (Ohashi et al. 1996, Ling andChen 2005).
Many marine fingerling production systems
approach densities and fingerling sizes that are more
typically associated with freshwater growout. PeakTAN and nitrite levels are allowed to creep upwards(<2.0 mg N/L), a strategy that benefits from pH
depression associated with rising carbon dioxidelevels and the protective nature of chlorides on nitrite
toxicity. Density is allowed to rise to 60 kg/m3 while
the higher TAN targets lessen the importance ofrecirculation rates. For an example of a recirculating
system, see Figure 2. Carbon dioxide removal
becomes critical under these growout conditions.
Rapid alkalinity consumption in combination withelevated carbon dioxide can induce pH declines thatincapacitate nitrifying bacteria.

Current Treatment Strategies
Floating bead filters utilize a packed bed offloating plastic beads. They display the inherent
ability to capture solids across a wide size spectrum.

MaloneFigure 2. Example of a recirculating water treatment system.

Removal efficiencies decline from nearly 100% in
the 30–50 µm size range, but removal in the
problematic 5–10 µm slot is high enough to assure
removal with multiple passes. Thus floating beadfilters are able to provide for both fine and coarse
solids removal irrespective of the degree of waterreuse. In addition, floating bead filters are designed
to encourage biofilm development and naturally
provide biofiltration while they are capturing solids.
Use of the floating bead filters as bioclarifiers
inherently simplifies treatment trains without
sacrificing water quality, laying a foundation forsimplification.
Malone and Beecher (2000) presented design
criteria for warm water systems that have been
widely applied to both freshwater and marine systems
over the last decade. Their criteria support theapplication of pumped (circulation) floating beadfilters (clarification and biofiltration) with blown air(aeration and carbon dioxide stripping) to thecategories of broodstock, fingerlings, and growout
systems. This approach has been applied with many
variations to fingerling and growout systems.
Some engineers, however, reject the concept of
bioclarification and take the traditional approach ofcomplementing the bead filter with a large biofilter.
For example, a pumped (circulation) floating beadfilter (clarification) with a fine sand fluidized bed(biofiltration) is a common combination. The
addition of pure oxygen input through a Speece cone(aeration) and a packed column (carbon dioxide
stripping) completes the treatment train for many
facilities in the shrimp and ornamental fish industry.
A more recent variant utilizes a moving bed reactor
for biofiltration. In the freshwater arena a parallel setof designs are distinguished by the use ofMalonemicroscreens for solids capture instead of a beadfilter. Microscreens are often seen in combination
with fluidized sand or moving bed reactors. These
units are generally complemented by pure oxygen
injection through a low head aerator or Speece conewith packed column carbon dioxide stripping(Summerfelt and Sharrer 2004).
The unique demands of marine hatcheries arestimulating the subtle shifts in system designs (Table
2). Central is the demand of pristine waters, a clear
shift towards the oligotrophic compared to thedemands for high carrying capacity emphasized in
the freshwater sector. Additionally, the heating andcooling needs of the systems, environmental issues
associated with saltwater discharge, and, particularly,
biosecurity issues are encouraging more closure ofsystems. Biosecurity strategies lead to designs thatare more isolated and of smaller scale thancorresponding freshwater growout systems.
Table 2. Water treatment configurations must be adjusted tomeet the demands of marine hatchery systems.

It is becoming increasingly apparent thatbiosecurity issues will force a rethinking ofcommercial marine hatchery configurations.
Biosecurity quickly renders liberal flushing ofsystems with ambient water obsolete. Similarly,
centralized water treatment where mixing of waters
inherently compounds the risk of catastrophic losses
are unlikely to prove biosecure in a commercial
environment. Isolation must be a cornerstone ofbiosecurity strategies seeking to provide protection ofvaluable broodstock and fingerlings from internal andexternal disease vectors. Partitioning of large
assemblies of tanks is dictated. Use of individual or
small clusters of tanks facilitates treatment or, in theworst case, limits losses when treatment isimpracticable. Partitioning also facilitates the45manipulation of individual maturation tanks. Modern
biosecure hatcheries will consist of a large number ofsmall isolated recirculating systems.
The engineering community must recognize thechanging needs of the industry and focus on thedevelopment of treatment strategies appropriate for alarge number of dispersed systems. To that end, thispaper presents a bead filter/airlift combination that iscompatible with these marine hatchery objectives.
The combination addresses the five fundamental
requirements of recirculation (circulation, solids
capture, biofiltration, aeration, and carbon dioxide
stripping) in two devices. Both devices are
essentially passive, allowing the system to be
operated for extended periods of time with periodic
inspections. Water loss is very minimal, providing asuitable format for a biosecure operation with a very
low environmental profile.
PolyGeyser filters. PolyGeyser filters (Aquaculture
Systems Technologies LLC, New Orleans,Louisiana) (Figure 3) are bubble-washed floating
bead filter units. They are designed to operate as afixed bed with intermittent backwashing. The
distinctive difference between these units and earlier
bead filter designs is their ability to recycle their own
backwash waters. The unit’s operation is pneumatic.
Backwashing is implemented automatically by a slow
air feed without the need for electronic timers. Thus
there is no water loss associated with an individual
backwash event. Sludge can be removed from theunit every few days after numerous backwashes haveoccurred. These characteristics make the unit
particularly suitable for marine applications.
A PolyGeyser filter has three distinctive internal
components: a) the bead bed, b) the charge chamber,and c) the sludge basin. The distinguishing
characteristic is the charge chamber. This inverted
compartment is designed to capture and hold a large
bubble of air. The chamber is equipped with atriggering device that releases the captured air when
the charge chamber is filled. Then air bursts throughthe bead bed, rapidly mixing it as it drops. Dirty
wash waters released by the bead bed rush into thecharge chamber, filling it as the air escapes. The
bead bed reforms at the top as the filter refills andwater filtration resumes. This backwash sequence
takes 10–15 seconds. The backwash is so fast that
there is no need to interrupt the influent pump.
During the next few hours, a slow bleed of air
fills the charge chamber for the next backwash event.
This filling process is so slow that the backwash

46

Figure 3. The pneumatic charge chamber stores air to washthe beads then acts as a settling basin recycling backwash
waters.

waters captured in the charge chamber have time tosettle out. A relatively clean stream of water isslowly remixed with the circulating waters as thesolids accumulate in the sludge basin. Sludge isremoved from the sludge basin usually after a great
number of backwash events. Biofilms become
abraded when a bead filter is backwashed. The
frequency and intensity of backwashing is a major
factor that controls the nature of the biofilm, andthus, its biofiltration performance (Golz et al. 1999).
The PolyGeyser filter is normally packed with anenhanced nitrification (EN) media. The EN media
was developed to protect nitrifying bacteria during
backwashing events (Stahl et al. 2000). It is also
more porous than spherical beads. A bed of EN
media can hold about twice as much bacteria as aspherical bead bed without displaying excessive headloss.
In the mid-1990s a number of studies confirmedthat a frequent but gentle backwashing strategy
created conditions optimum for nitrification.
Wimberly (1990) found the best performance of his
hydraulically washed unit was eight times a day.
Sastry et al. (1998) coaxed the best performance outof his bubble-washed units at a backwash frequency
of five times a day. Finally, DeLosReyes et al.

Malone(1997) found his unit performed the best with abackwash interval of only two hours. The
nitrification performance of filters packed with EN
media is comparable to top ranked biofilters such as
the moving bed reactor and fluidized sand filters.
The headloss displayed by EN media beds
subject to a high frequency washing strategy is very
low even when subject to heavy loading. Headloss
through the bed itself is typically only a few
centimeters. This favorable headloss profile
facilitates water circulation with airlift pumps.
Airlift pumps. An airlift pump aerates and strips
carbon dioxide from the water as it is circulated. Of
the five basic processes that form the treatment coreof any recirculating system, three of them can besubstantially achieved with the simple combinationof a piece of PVC pipe and an air blower. Theenergy input that is normally reserved for thecirculating function alone is used to simultaneously
effectuate the important gas transfer functions. This
strategy can result in extremely simple yet robust
system configurations that are very energy efficientin comparison to more traditional pumped
configurations.
More subtly, in contrast to centralized water
pumps, use of airlift pumps allows the mechanicalsource of power to be centralized without thecorresponding cross contamination issues that occur
when waters from different tanks are mixed. This is
an important biosecurity distinction for marine
hatcheries. A central source of air, usually a low
head rotary vane blower, can be located in a dry
location away from the corrosive effects of saltwater.
The air is distributed pneumatically allowing for
precise localized control of the dispersed tank andfilter systems without the potential for crosscontamination. At the same time, centralized air
pumping dramatically reduces upkeep requirements
in comparison to multiple near tank centrifugal
pumps.
Within the context of recirculating applications,the operational performance of an airlift pump is
controlled by the characteristics of the rotary vane
blower that drives it. Blowers common to theindustry are generally designed for air injection
above a depth of 1.5 m, a depth that safely avoids gas
bubble disease concerns in most applications. The
flow performance of an airlift is defined by the ratioof the lift to the submergence, with flow delivery at a
fixed air input increasing dramatically as the lift
decreases. Given the constraints on depths ofinjection, recirculating airlift pumps are most cost

Maloneeffective when they can be operated at relatively low
lifts. Loyless and Malone (1998), for example,
presented flow performance curves for a 5.05 cm
airlift with a 91.4 cm submergence that showed a
50% decrease in flow delivery as the lift is increased
from 7.62 to 30.5 cm.
This issue is further complicated by gas transfer
efficiencies. The gas transfer characteristics arecontrolled by changes in the rate of air injection (gas)as it relates to the water flow (liquid). It is desirablefrom an energy consumption perspective to operate
the airlifts at low gas to liquid ratios (G:L). Air
delivery rates are set to meet flow demands only. If
additional aeration or degassing capacity is
demanded, air is directed to the tank where bothoxygen and carbon dioxide are more efficiently
transferred (Loyless and Malone 1998).
Gudipati (2005) developed several rules of
thumb for the sizing and installation of airlifts forwater flow in recirculating applications. This work
concluded that airlifts should be designed to operatewith submergence to lift ratios in the range of four tofive, keeping the G:L below two. The author also
observed that lift pipes should be conservatively
sized to support a water flow of 0.3 m/sec. Velocity
recommendations for approach pipes were higher, in
the range of 0.6–0.9 m/sec, representing a balance ofscour and headloss concerns. These simple rules
facilitate the use of airlifts with a wide variety of low
head filtration devices. Figure 4 illustrates an airlift
configuration that has been evaluated with
PolyGeyser filters ranging in size from 28 L to 1.4
m3.

Figure 4. Most PolyGeyser filters operate well with a screen
placement 30.4 cm and an airlift air injection 122 cm below
the tank water level.

The physical positioning of the PolyGeyser filter
relative to the water level in the tank is critical.
Typically, it is necessary to place the effluent screen47

(or pipe) 25–28 cm below the tank water level. This
placement assures that there is sufficient waterpressure to push the water through the tank screens,through the tank drain lines and up though the bead
bed itself under the worst case conditions. Under avariety of applications, the RAS airlifted PolyGeyser
tends to operate with a cumulative head loss fromtank to filter effluent in the range of 15–38 cm. This
low headloss range is compatible with airlifts thathave submergence depths in the range of 120–150
cm. Various engineering issues relating to the
PolyGeyser filter are presented in Table 3.

Table 4. These interim guidelines can be used as airlifted
PolyGeysers are introduced to support recirculating
aquaculture systems (metric version).

Table 3. Engineering issues to be aware of in a complex
airlifted PolyGeyser recirculation system.

Airlift/PolyGeyser treatment. Table 4 provides
some interim sizing guidelines for RAS airlifted beadfilters. The table presents filter feed levels andapproximate tank sizes for a wide distribution of
PolyGeyser sizes. The feed loading indicated,coupled with reasonable management of the
PolyGeyser as a biofilter, should provide water
quality conditions that correspond to the water
quality levels indicated in Table 1 for the different
loading zones. Sizing for the approach pipes and thelift tubes associated with the airlift recirculation areprovided. A dual lift tube option is also sized since
the use of two tubes facilitates energy conservation
during periods of light loading. Finally the airdelivery pressure and flow are provided to allow for
selection of air pumps.

48

These criteria are applicable for a temperature
range of 15–30°C. Application of these criteria underpeak loading outside that range should be undertakenwith caution. Below a temperature of about 10°C,
the overall nitrification capacity of the bioclarifier
can be expected to decline. At these lowtemperatures, systems failure will most probably be
indicated by chronic nitrite accumulation. Failure attemperatures above 30°C are most likely to be firstevidenced by low dissolved oxygen levels in therearing tank as decreasing oxygen saturation levels
undermine the aeration capacity of the airlifts.
Supplemental, in-tank aeration may be appropriate at
this point.
Individuals installing larger units should seek
professional assistance at the design and installationlevels to assure that the elevations for tank and filter
are properly selected and that secondary problems
such as filter flotation under high groundwaterconditions are avoided (Table 4). The air delivery
requirements of an individual unit arestraightforward. However, facilities that use multiple
airlift units should seek professional advice to assure
air delivery systems are properly designed.
Unexpected changes in air delivery pattern can occur
as multiple units randomly backwash. Air
distribution systems must be rationally laid out toMaloneassure that air distribution remains predictable.
Multiple airlifts serving a single PolyGeyser unit
should return discharge water just above the tankwaterline to avoid flow reversal between competingairlifts.
There are a number of secondary processes thatcan be used to complement the PolyGeyser/airlift
combination that form the core of the treatment
sequence (Table 5). For example, it is common
practice to equip broodstock conditioning tanks with
an ultraviolet light to suppress expression of marine
infectious diseases or parasites.
Table 5. The PolyGeyser/airlift treatment core can beenhanced by the addition of compatible technologies.

Summary and Conclusions
The special needs of marine hatchery systems areforcing engineers to reevaluate design strategies.
Although current design configurations are serving
the industry well, increasing biosecurity concerns
dictate greater isolation and encourage high reusedesigns that minimize exposure to external disease
vectors. Internally, marine hatcheries benefit fromthe decentralization of treatment, a strategy that
reduces the potential for internal disease propagation.
The ability of the PolyGeyser floating bead filter
to recycle its own backwash waters facilitates theoperation of recirculating systems with highhydraulic retention times. This feature also permits
the implementation of a high frequency backwashingstrategy that avoids headloss buildups in the beadbed. The low headlosses associated with PolyGeyser
bioclarifier operation permit airlift circulation.
Airlifts provide substantial aeration and degassing as
they circulate, laying the foundation for a simplified
treatment. An airlift PolyGeyser combination
operates completely pneumatically with no moving
parts. Operation is robust and requires minimal
supervision, desirable features in hatchery operations
with multiple isolated tank modules. Recently
developed airlift sizing and established beadfilter
guidelines have been merged to define interim design
guidelines for airlifted PolyGeyser filters. These
guidelines will be refined as commercial
introductions of the airlifted PolyGeyser continue.

Acknowledgments

Addition of a temperature control loop facilitates
temperature manipulations used for condition inbroodstock. Moving bed reactors and rotating
biological filters are examples of two biologicalfilters that can be used in an airlift recirculating
system. A number of foam fractionator designs are
air driven and compatible with the pneumatic
operation of the airlifted PolyGeyser. Foam
fractionators are most frequently installed on heavily
loaded fingerling systems, or on broodstock systems
that are operated with high hydraulic residence times(>5 days). Denitrification also becomes necessary
when water reuse extends hydraulic residence times.

Malone

The authors acknowledge the contributions madeby commercial and other research and development
units toward the refinement of this strategy. Funding
for baseline studies was derived in past years fromthe Louisiana and national Sea Grant College
programs. Commercial development was fostered bya Department of Commerce grant and several U.S.
Department of Agriculture Small Business Innovative
Research Grants conducted by Aquaculture Systems
Technologies Inc., New Orleans, Louisiana, under
the direction of Douglas Drennan. Burt Nichols at
Water Garden Gems Inc., Marion, Texas, has been
active through donations of equipment forcommercial demonstrations.
An early commercial demonstration (circa 1995)
of the airlift technology with a paddle-washer was
conducted by Steve Abernathy of Tiltech Inc.,
Robert, Louisiana. That demonstration, which
continues today, formed the driving force for thecontinued research efforts. Waddell Mariculture
Center in South Carolina formed an early test base49

for small-scale airlift evaluations, as did the Centro
de Investigacion Cientifica y de Educacion Superior
de Ensenada (CICESE) in Baja, Mexico. Hubbs-Sea
World Research Institute in San Diego, California,
has an ongoing evaluation of a 25 cubic foot airlift
unit.
Most notably, Mote Aquaculture Park, Sarasota,
Florida, is conducting the largest demonstration
project consisting currently of four 1.5 m3 (50 ft3)
PolyGeyser units on freshwater sturgeon, while
evaluating several PolyGeyser Moving Bed
combinations on their marine broodstock systems.
These pioneers identified the limitations of ourknowledge as this emerging technology was
introduced.

References
DeLosReyes Jr., A. A., L. E. Beecher, and R.F. Malone.
1997. Design rationale for an air-driven recirculating
system employing a bubble-washed bead filter. In M. B.
Timmons and T. Losordo (eds.), Advances in aquacultural
engineering: Aquacultural Engineering Society
Proceedings III, ISTA IV, Orlando, FL, 9–12 November
1997, p. 287–294. NRAES, New York.
Golz, W. J., K. A. Rusch, and R. F. Malone. 1999.
Modeling the major limitations on nitrification in floatingbead filters. Aquacult. Eng. 20:43–61.
Gudipati, S. 2005. Distributed airlift systems design withapplication to recirculating soft shell crawfish shedding
systems. Master’s thesis. Louisiana State University,
Baton Rouge.
Ling, J., and S. Chen. 2005. Impact of organic carbon on
nitrification performance of different biofilters. Aquacult.
Eng. 33:150–62.

Malone, R. F., and L. E. Beecher. 2000. Use of floating
bead filters to recondition recirculating waters inwarmwater aquaculture production systems. Aquacult.
Eng. 22:57–73.
Malone, R. F. 2002. Engineering for a responsible
aquaculture with focus on growout facilities forcommodity foodfish. In R. L. Creswell and R. Flos (eds.),
Perspectives on responsible aquaculture for the newmillennium, p. 94–111. World Aquaculture Society, Baton
Rouge, Louisiana, and European Aquaculture Society,
Oostende, Belgium.
Malone, R. F., and T. J. Pfeiffer. 2006. Design andselection of biological filters for freshwater and marine
applications. Aquacult. Eng. 34(3):389-402.
Ohashi, A., D. G. Viraj de Silva, B. Mobarry, J. A.
Manem, D. A. Stahl, and B. E. Rittmann. 1996. Influenceof substrate C/N ratio on the structure of multi-species
biofilms consisting of nitrifiers and heterotrophs. Water
Sci. Technol. 32(8):75–84.
Sastry, B. N., A A. DeLosReyes Jr., K. A. Rusch, and R.
F. Malone. 1999. Nitrification performance of a bubblewashed bead filter for combined solids removal andbiological filtration in a recirculating aquaculture system.
Aquacult. Eng. 19:105–117.
Summerfelt, S. T., and M. Sharrer. 2004. Design
implication of carbon dioxide production within biofilters
contained in recirculating salmonid culture systems.
Aquacult. Eng. 32:171–182.
Wimberly, D. M. 1990. Development and evaluation of alow-density media biofiltration unit for use in recirculatingfish culture systems. Master’s thesis. Louisiana State
University, Baton Rouge.

Loyless, J. C., and R. F. Malone. 1998. Evaluation ofairlift pump capabilities for water delivery, aeration, anddegasification for application to recirculating aquaculture
systems. Aquacult. Eng. 18:117–133.

50

MalonePreparation of Marine Silage and its Potential for Industrial Use
Motoharu Uchida
National Research Institute of Fisheries and Environment of Inland Sea
2-17-5 Ohno, Hatsukaichi
Hiroshima, Japan 739-0452
uchida@affrc.go.jp
Keywords: hatchery diet, lactic acid fermentation, marine silage, seaweed
Abstract
Marine silage (MS) as used here is a dietary material prepared from algae by a fermentation process.
The objective of this paper is to report on the method used to prepare MS from seaweeds and discuss its
potential for industrial use. The fermentation of seaweeds can be performed by the enzymaticsaccharification by cellulase, followed by fermentation using lactic acid bacteria and/or yeast. The method
can be applied to any kind of seaweed containing cellulose. Red sea bream that were challenged withiridovirus and fed a diet containing MS at 10% (w/w) of the diet showed a significantly improved survival
percentage compared with those fed a control diet. The use of MS in the diet may allow for the productionof cultured fish that are free of drugs.
In the case of Asian kelp (Undaria pinnatifida), the fronds are fragile and easily decomposed during thesaccharification process. The particle size of the product obtained (single cell detritus-marine silage, or
SCD-MS) is ca. 5~10 µm in diameter, similar to a microalgae diet. The mass culture of the SCD-MS can be
performed in a plastic tank, with concentrations reaching ca. 107–108 cells/mL. Supplemental aeration,
temperature control, and light control are not necessary.
Feeding trials with pearl oyster (Pinctada fucata martensii) spat demonstrated the dietary value of the
SCD-MS to bivalves. The SCD-MS diet may replace a part of microalgae diets in the future. The
development of the SCD-MS diet also suggests a new style of aquaculture. We are attempting to link the
production of nuisance algae such as Ulva spp. to fish production utilizing the marine silage system. We
believe that aquaculture based on algal fermented materials is ecofriendly.

Introduction
Fermentation has a long history and plays animportant role in agriculture and the livestockindustry for preparing compost and silage. However,fermentation is not extensively used to feed fish
except for some limited applications in ingredient
manufacturing. Particularly lacking has been theapproach of fermenting algae, which are plentiful inmarine ecosystems. In fact, there have been no
fermented food products made from marine algae(Figure 1). Recently, the author developed a method
to perform lactic acid fermentation on seaweeds(Uchida and Murata 2002). The method contains twoelements (Figure 2a). The first is the enzymatic
decomposition and saccharification of seaweeds. The
second is lactic acid fermentation using
microorganisms. These two processes can be
performed simultaneously by adding polysaccharidedecomposing enzymes; usually cellulase, and startermicroorganisms; usually lactic acid bacteria (LAB)or yeast to initiate the process. This method can be

Uchidaapplied to any type of seaweed that contains cellulose(Table 1, Uchida and Murata 2004).

Figure 1. Categories of fermented food products.

51

Table 1. Results of the fermentation test for various kinds ofseaweed with the addition of cellulase and the microbial
starter mixture (Uchida and Murata 2002).

Figure 2. Fermentation process of seaweed. Lactic acidfermentation (a) and lactic acid fermentation with single cell
detritus (SCD-MS) products (b).

Materials and Methods

The author is promoting the use of this fermented
material from seaweed as a fish dietary ingredientwith the proposed name marine silage (MS).
Furthermore, in the case of Asian kelp (Undaria
pinnatifida), the observation was made that the frond
tissue can be decomposed to one-cell units bycellulase during fermentation (Figure 2b). As aresult, large numbers of small detrital particles (ca.
5–10 µm in diameter) are produced. The author
termed these particles single cell detritus-marine
silage (SCD-MS) and studied the optimal culture
conditions for preparing the material (Uchida and
Murata 2002).
This paper reports on the suitable culture
conditions for performing lactic acid fermentation ofseaweed and for preparing SCD-MS with highefficiency. Furthermore, to demonstrate the potentialof the MS as fish dietary ingredient, a feeding trial
was conducted with red sea bream. Since the size of
the SCD-MS is analogous to microalgae diets such as
Chaetoceros calcitrans and Pavlova lutheri, afeeding trial with young pearl oysters (Pinctada
fucata martensii) was also conducted to demonstrate
the dietary value of the SCD-MS as a replacement for
microalgae diets (Uchida et al. 2004a).

52

Preparation of SCD-MS for determining
appropriate culture conditions. To determine theappropriate culture conditions for SCD-MS, sterile
500 mL polycarbonate bottles with screw caps
(Nalgene) were used for a small scale fermentation
trial. A commercial product of dried particles of U.
pinnatifida (Wakamidori <74 µm, Riken Co.) was
dispensed in the amount of 10.0 g each into bottles
containing 180 mL of autoclaved 3.5% w/v NaClsolution and cellulase (R-10, Yakult PharmaceuticalInd. Co.) (Table 1) and mixed well. The culture
bottles were capped and incubated at 20°C withshaking several times a day to improve dispersion.
Initially, the preparation of SCD in cultures without
the addition of a microbial mixture was conductedunder various reaction temperatures and cellulase
concentrations. The number of detrital particles of
Undaria obtained was counted using a Coulter
Multisizer (Coulter Electronics Ltd.) with a 140 µm
orifice. The detrital particles in the fractions of 5.8–
11.5 µm in diameter were tentatively regarded as
SCD-MS products. The weight distribution was
calculated from the distribution of the detrital
particles on the assumption that the detrital particles
were of a spherical form with a specific gravity of
1.0.
For testing the effect of the use of microbial
elements as a starter, cultures containing different
levels of cellulase were incubated with an inoculationof the microbial mixture. The microbial mixture wascomposed of one type of LAB Lactobacillus brevis
strain B5201 (FRA 000033) and two types of yeast:
Debaryomyces hansenii strain Y5201 (FERM BP7302) and Candida sp. strain Y5206 (FERM BP7303). The bacterium and yeast cells were

Uchidapreincubated in MRS broth (Merck) and YM broth(Difco), respectively, collected by centrifuge, washedtwice, and suspended in sterile 0.85% NaCl solution
at a concentration of OD 660 nm = 1.0. The
suspension mixture of 2 mL of L. brevis (containing
1.0 × 109 cells) and 1 mL each of D. hansenii (5.3 ×
105 cells) and Candida sp. (1.2 × 107 cells) was used
for inoculation of the microbial mixture. All cultures
were prepared in duplicate and the average values ofdata were determined.
Mini preparation of SCD-MS. A sterile 50 mL
volume polypropylene centrifuge tube provided witha screw cap (Iwaki) was used to prepare thefermentation cultures. To prepare NaCl-added
cultures (No. 1a–14a), 2.0 g of the Undaria productwere mixed with 40 mL of autoclaved 3.5% NaClsolution, 40 mg of cellulase 12S (Yakult
Pharmaceutical), and 0.4 mL of bacterial cellsuspension and mixed. The bacterial cell suspension
was prepared for the 14 LAB strains: L. brevis FRA
000033, L. brevis IAM 12005, L. plantarum ATCC
14917T, L. plantarum IAM 12477T, L. casei IFO
15883T, L. casei FRA 000035, L. rhamnosus IAM
1118T, L. zeae IAM 12473T, L. acidophilus IFO
13951T, L. kefir NRIC 1693T, L. fermentum ATCC
14931T, L. delbrueckii bulgaricus ATCC 11842T,
Streptococcus thermophilus NCFB 2392, andLeuconostoc mesenteroides IAM 13004T. The strains
were precultured with MRS medium, collected bycentrifuge, resuspended to make a concentration of
OD 660 nm = 1.0 (containing 7.3 × 107–1.1 × 109
CFU/mL), and used. To prepare non-NaCl cultures(No. 1b–14b), autoclaved distilled water was usedinstead of 3.5% NaCl solution. Cultures without
LAB inoculation were prepared as No LAB controls(No. 15a and 15b). The triplicate cultures wereincubated at 20°C for 11 days mixing several times
by hands every day to help dispersion.
Mass preparation of SCD-MS. Sterile 10 L
polycarbonate bottles with screw caps (Nalgene)
were used for the preparation of MS. One kg of U.
pinnatifida Wakamidori was dispensed into bottles
containing 9 L of autoclaved 3.3% NaCl solution and
10 g cellulase R-10 and mixed well. The cultures
were inoculated with the microbial mixture atquantity of 5.0 × 1010 CFU (L. brevis), 5.5 × 107 CFU(D. hansenii) and 1.2 × 108 CFU (Candida sp.). Eachculture bottle was capped, incubated at 20°C andmixed several times a day to improve dispersion for
the first eight days. The culture bottles were thenpreserved at 20°C for 18 months.

UchidaRearing trial for red sea bream challenged withiridovirus. The Ecklonia-MS was prepared byfermenting the Ecklonia maxima (a dried productimported from South Africa) at 10°C for one monthafter the addition of 0.15% (as a final conc.) cellulase
3S (Yakult Pharmaceutical Ind. Co.), 0.035%
pectinase G (Amano Enzyme Co.), and 8 × 106
CFU/mL L. plantarum (TOA Pharmaceuticals Co.).
The Ecklonia-MS diet was prepared by adding the
Ecklonia-MS to a control diet at 10% w/w. The
composition of the control diet was 40 g brown meal,
20 g white meal, 10 g northern krill meal, 7 g sardine
oil, 8 g dextrin, 10 g wheat gluten, 3 g guar gum, 1 g
vitamin mixture, 0.1 g mineral mixture, 0.66 g
CaCO3 , 0.23 g CaPO4, and 0.01 g Hospitane. For therearing trial, red sea bream (mean ±SD of bodyweight = 32.9 g ±3.2 g) were challenged with 50 µL
each of iridovirus stock suspension (TCID50 =105.8/mL). Then 50 individuals were held in each of
three 200 L tanks and reared at 25°C. The rearing
water was changed at the rate of 1L/min. The fish
were fed to satiation twice a day.
Rearing trial for young Japanese pearl oysters.
For the preparation of MS for the rearing test, 20 g of
the Undaria product was dispensed into 500 mL
polycarbonate bottles containing 360 mL of 3.5%
NaCl solution and 2 g cellulase R-10 and mixed well.
The cultures were inoculated with 2 mL of the cellsuspensions of the previously described three types of
microorganisms and incubated for 8 days at 20°C
(SCD-MS2). A culture that was prepared without
addition of the microorganisms and incubation wasused as a control diet (C-MS2). The SCD-MS2 andC-MS2 were preserved at 5°C during the rearing test
and filtered through 6.3 µm nylon mesh just before
use. Early stationary phase cells of Chaetoceros
calcitrans were prepared by culture at 20°C (14 h:10
h light:dark cycle) in a modified Erd-Schreiber
medium. For the rearing trial, 15 young Japanese
pearl oysters (mean of hinge length = 4.47–4.95 mm)
were held in a 5 L tank and reared for 12 days. The
rearing water was supplied with air and kept at atemperature of 27°C and a salinity of 32–33 ppt.
Oysters were fed once a day and the rearing water
was changed every day with fresh, sand-filteredseawater. The control groups were an unfed group, a
Chaetoceros group (3 × 104 cells/mL/day), and a 1/10
Chaetoceros group (3 × 103 cells/mL/day). The test
groups were those fed the SCD-MS at 2 × 104
cells/mL/day (SCD-MS2) and 5 × 104 cells/mL/day(×2.5 SCD-MS2). The combined feeding was tested
by feeding the MS2 at 2 × 104 cells/mL/day with
Chaetoceros at 3 × 103 cells/mL/day (SCD-MS2 +53

1/10 Chaetoceros). The test groups using the C-MS2
diet were presented with the same feeding conditions
as the SCD-MS2 groups. Shell length of the spat’s
hinge lines (hinge length) were measured and used
for calculating growth rates. The survival rate of thespat was obtained by counting the number of living
individuals at the end of the rearing trials.

Results and Discussion
Suitable culture conditions for preparing the
SCD-MS. The relationship between the culture
temperature and the number of SCD-MS products
was studied with a culture containing 3.5% NaCl and
1.0% cellulase. The volume proportion of SCD-MS
increased gradually and reached a maximum value inthe range of 60.1–69.0% on the fourteenth day(Figure 3). The production efficiency of SCD-MS
was maximum at 2°C and almost constant in therange of 20–50°C based on the profile of Figure 3.
The production efficiency of SCD-MS was maximum
at 2°C and almost constant in the range of 20–50°C
based on the profile of Figure 3.

odors and were judged to have rotted. In contrast, theculture media containing cellulase at 0.25–5.0% had
ester-like preferable odors and were judged to befermented.
For the cultures without cellulase, microbial flora
after 5 days of culture varied greatly, implying
unstable culture conditions from the viewpoint of
microbial composition. One culture contained
predominantly lactic acid bacteria (80.5% ofheterotrophic microorganisms) while the othercontained predominantly marine heterotrophic
microorganisms (92.9%). For the fermented cultures,
lactic acid bacteria were predominant at a 108
CFU/mL level and the ratio of lactic acid bacteria toheterotrophic microorganisms was in the range of
8.5–11%, while the proportions of marine
heterotrophic against heterotrophic microorganisms
were in the range of 3.6–8.0%. It was observed thatthe addition of 0.25% cellulase to the culture media
was enough to obtain SCD-MS without spoilage if
the starter microorganisms were added (Table 2). In
contrast, the addition of cellulase more than 0.5%
was necessary to obtain SCD-MS without spoilage if
the starter microorganisms were not added.

Figure 4. Relationship between cellulose concentration andproduction efficiency of SCD-MS.
Figure 3. Relationship between culture temperature andproduction efficiency of SCD-MS.

Figure 4 shows a profile of the relationshipbetween cellulase concentration and the number of
SCD-MS in Undaria cultures prepared in thepresence of starter microbes. The number of SCDMS increased from 2.5 × 107/mL (18.4% based onvolume proportion) at the start to 3.5–5.9 × 107/mL(63.0–69.1% based on volume proportion) at 6 daysof incubation and the maximum SCD-MS products
were obtained with a cellulase concentration at 0.5%
(59.1% based on volume proportion). Culture media
without the addition of cellulase had unacceptable

54

Table 2. Cellulase concentration necessary for successful
fermentation.

The starter microorganisms were first obtained
from a naturally fermented material of Ulva spp.
Three species of microorganisms, namely L. brevis,

UchidaD. hansenii and Candida sp., were isolated as
predominant microorganisms in the fermented Ulva.
We observed that the addition of the individuallycultured cell suspensions of these microorganisms asa starter for fermentation of seaweed was effective inthe production of SCD-MS (Uchida and Murata
2004). However, we also found that lactic acidbacteria alone can be used as a starter (Uchida et al.
2004b). Results of fermentation with NaCl-added
cultures are shown in Figure 5a. The predominanceof the inoculated species in the cultures was 100% for
the cases of L. casei IFO 15883T and L. casei FRA
000035; 96.7% for the cases of L. brevis FRA
000033, L. brevis IAM 12005, and L. plantarum IAM
12477T; 93.3% for the case of L. plantarum ATCC
14917T; 90.0% for the case of L. rhamnosus; 13.3%
for the case of L. fermentum; and less than 3.3% for
the cases of L. zeae, L. acidophilus, L. kefir, L.
delbrueckii bulgaricus, S. thermophilus, andLeuconostoc mesenteroides. The LAB became
predominant (>90.0%) for 13 cases out of 14, while
the inoculated species became predominant for 7
cases out of 14.
Results of fermentation with NaCl-less culturesare shown in Figure 5b. The predominance of thestarter species was 100% for the cases of L. casei IFO
15883T, L. casei FRA 000035, and L. rhamnosus;
96.7% for the cases of L. brevis FRA 000033 and L.
plantarum ATCC 14917T; 90.0% for the case of L.
plantarum IAM 12477T; 63.0% for the case of L.
brevis IAM 12005; 3.3–20.0% for the cases of L.
zeae, L. kefir, L. fermentum, and Leuconoscoc
Mesenteroides; and less than 3.3% for the cases of L.
acidophilus, L. delbrueckii bulgaricus, andStreptococcus Thermophilus. The LAB became
predominant (>90.0%) for 10 cases out of 14, while
the inoculated species became predominant for the 6cases out of 14. L. brevis, L. plantarum, L. casei, andL. rhamnosus predominated in the MS cultures.
Promotion of survival rate of fish against viraldisease. Red sea bream challenged with iridovirus
and fed a diet containing the MS showedsignificantly better survival rate (86.5% ±1.6%)
compared with those fed the control diet (73.1%±6.3%). The use of MS diet may help increase thesupply of fish that are produced without the use ofdrugs, which is an important consideration for
consumers today.

Figure 5. Microbial compositions after fermentation of
Undaria with inoculation of different starters in NaCl-added
cultures (a) and cultures lacking NaCl (b). Starter strains No.
1-14 are: L. brevis FRA 000033, L. brevis IAM 12005, L.
plantarum ATCC 14917T, L. plantarum IAM 12477T, L.
casei IFO 15883T, L. casei FRA 000035, L. rhamnosus IAM
1118T, L. zeae IAM 12473T, L. acidophilus IFO 13951T, L.
kefir NRIC 1693T, L.fermentum ATCC 14931T, L.
delbrueckii subsp. bulgaricus ATCC 11842T, Streptococcus
thermophilus NCFB 2392, and Leuconostoc mesenteroides
IAM 13004T. Culture 15a and 15b are control cultures
prepared without starter inoculation.

the results of a feeding trial conducted on young pearl
oysters. The feeding of Chaetoceros calcitrans at 3×104 cells/mL/day resulted in a shell growth of 168µm/day and survival of 66.7%, while unfed controls
showed negative shell growth (–10 µm/day) and
53.3% survival. Feeding with a SCD-MS preparedfrom U. pinnatifida at 2 × 104 cells/mL/day resulted
in a shell growth of 23 µm/day and survival of
53.3%. When the SCD-MS was supplied with C.
calcitrans at 3 × 103 cells/mL/day, shell growth was
130 µm/day and survival was 80%. This study
demonstrates the dietary value of SCD-MS to bivalve
mollusks and shows that SCD-MS can partiallyreplace microalgal diets in hatcheries.
Table 3. Results of feeding trials of SCD-MS2 and C-MS2
with young pearl oyster (Uchida et al. 2004a).

SCD-MS as a replacement hatchery diet for
microalgae diet. Since the size of the SCD-MS isabout 5–10 µm in diameter, it has a potential toreplace microalgal diets in hatcheries. Table 3 shows

Uchida55

New Aquaculture Style Based on Marine
SilageThe development of the MS preparation
technology and its widespread use may change theway aquatic animals are fed in the future. Figure 6
presents the concept of aquaculture based on asustainable and ecofriendly marine silage system.
There has been an increase in nuisance blooms of
Ulva spp. and other green algae in the eutrophic
coastal waters of many countries. The blooms are
believed to be the result of excessive nutrients suchas nitrogen and phosphate in coastal waters.
Therefore, collection and utilization of algal fronds
could contribute to decreases in coastal water nutrient
levels. Another likely advantage in the use of SCDMS diets is that the microorganisms present in the
SCD-MS media may have probiotic or prebiotic
effects on the animals being reared. We are currently
working on that potential for SCD-MS.

References
Uchida, M., and M. Murata. 2002. Fermentative
preparation of single cell detritus from seaweed, Undaria
pinnatifida, suitable as a replacement hatchery diet forunicellular algae. Aquaculture 207:345–357.
Uchida, M., and M. Murata. 2004. Isolation of a lactic
acid bacterium and yeast consortium from a fermented
material of Ulva spp. (Chlorophyta). J. Appl. Microbiol.
97:1297–1310.
Uchida, M., K. Numaguchi, and M. Murata. 2004a. Masspreparation of marine silage from Undaria pinnatifida andits dietary effect for young pearl oyster. Fish. Sci. 70:456–
462.
Uchida, M., H. Amakasu, Y. Satoh, and M. Murata.
2004b. Combinations of lactic acid bacteria and yeast
suitable for preparation of marine silage. Fish. Sci.
70:508–518.

Figure 6. Concept of eco-friendly aquaculture based on amarine silage diet.

56

UchidaTechniques for Live Capture of Deepwater Fishes with Special Emphasison the Design and Application of a Low-cost Hyperbaric Chamber
Jeffrey E. Smiley and Mark A. Drawbridge
Hubbs-SeaWorld Research Institute2595 Ingraham Street
San Diego, CA 92109
JSmiley@hswri.org
Keywords: hyperbaric chamber, rockfish, gas bladder
Abstract
Following rapid depressurization from depth, physoclistous fish suffer from overinflation of their gas
bladder. The decreased pressure also causes bubbles to form in the circulatory system and causes trauma tointernal tissues resulting in hemorrhage, swelling, and death. Our research has shown that Cowcod(Sebastes levis) can only survive a few hours after rapid depressurization from depths as shallow as 90 m. To
overcome this problem, we developed a relatively simple, low cost hyperbaric chamber to aid in thecollection of adult rockfish to hold as broodstock. This system was designed to quickly recompress fish once
brought to the surface on hook and line, and allow for decompression over a period of days. The hyperbaric
chamber is capable of continuous stable operation at <10.2 atmospheres and can accommodate fish up to
91.4 cm in length and 26.8 cm in diameter. Pressure in the chamber is maintained by a Goulds booster
pump that delivers continuous pressure and supplies flows of 3.8–7.6 L/min to as many as four chambers.
The hyperbaric chamber operated very effectively and allowed us to successfully decompress 14 cowcod andtwo vermillion rockfish (Sebastes miniatus) from capture depths of 90.2-146.3 m.

Introduction
When fish are captured at depth, rapid
depressurization can cause serious injuries, including
gas bubbles in the blood vessels, gills, skin, andbrain; exophthalmia; internal and external
hemorrhage; everted stomachs; disoriented
swimming; altered behavior; and death (Gotshall1964, Beyer et al. 1976, Feathers and Knable 1983,
Rogers et al. 1986, Gitschlag and Renaud 1994).
Specialized collection techniques are needed tominimize barotrauma among deepwater fishes thatare collected live for public display or breeding
programs. Collection techniques will vary based on
the depth of capture and sensitivity of the target
species.
Techniques used to collect fish from depth
typically include cages, trawls, and hook and line
fishing. Without special handling, fish collectedusing these methods are typically either moribund or
dead. Special handling techniques include gradualdecompression and gas bladder deflation (Gotshall1964, Lee 1992, Keniry 1996). Gas bladder deflation
has enabled collection of some specimens from thesea, but mortality is highly variable and increases
with depth. To increase survivability, more timemust be taken to decompress fish. In order toaccomplish this, specialized pressure-controlled

Smileychambers have been developed. These hyperbaric
chambers are typically designed to trap fish at depth
and return them to the surface under pressure(Jannasch et al. 1973, Yayanos 1978, Macdonald andGilchrist 1978, Wilson and Smith 1985, Koyama etal. 2002).
Hyperbaric fish traps have made it possible tocollect specimens from the deep sea (600–5,700 m)
but they are very costly to design and build, thereby
creating the need for a low cost solution, especiallyfor fish at shallower depths. To meet this need, wedeveloped a portable hyperbaric chamber that is
capable of recompressing fish brought to the surface,then maintaining them at pressures up to 10.2
atmospheres for weeks. We targeted several speciesof rockfish (Sebastes sp.) with the goal of holding thefish for breeding purposes.

Hyperbaric Chamber Design andConstruction
Design criteria. The hyperbaric chamber describedin this paper was designed based on the following
criteria:
1. Portability to allow manageable movement offish from the field to the laboratory undercontrolled conditions.

57

2. Low cost using standardized parts that arecommon and relatively inexpensive.
3. Pressure stabilization at 150 psi to allow
complete recompression of fish caught at
depths of 102.7 m.
4. Temperature control to maintain constantconditions from the point of capture thereby
reducing stress on the fish.
5. Visual monitoring to ascertain fish health andmake decisions for safe decompression
schedules.
6. Water quality monitoring to ensure stableseawater temperature and adequate dissolved
oxygen concentrations.
Designing the rest of the apparatus to meet our
portability goals required a multipart decompression
system that could be broken apart in manageable
sections. These sections included a primary andsecondary manifold and chamber. The primarymanifold was responsible for maintaining stablepressures for the secondary manifolds, the secondary
manifolds were responsible for maintaining stablepressures and flows in the chambers, and thechambers were responsible for specimen containment
and pressure isolation for greater than 30 minutes; thetime allowed for transport from the boat back to thelab.
Principle component: Primary manifold. Pressurein the primary manifold was created by a 3.0 hp
Goulds booster pump (25GBS3014P4) that delivered
94.6 L/min of seawater to the apparatus. Pressure in
the system was controlled by a 2.54 cm pressure
relief valve that vented excess seawater back to thesump and created stable backpressure in the system.
A portion of the backpressured seawater was diverted
before the pressure relief valve and served as thesource for the secondary manifolds. Four 1.9 cm ballvalves operating in parallel from the primarymanifold controlled the flow of seawater to four
secondary manifolds through sections of 1.3 cm
flexible hose. The hose had a maximum operatingpressure of 20.4 atmospheres and ranged in lengthfrom 4.6 to 6.1 m. Pressure in the primary manifold
was monitored on a 10.1 cm face gauge up to 13.6
atmospheres. Two 5.1 cm ball valves were also
installed on the primary manifold and opened during
startup procedures to reduce initial water-hammering.
For portability the primary manifold and pump wasbolted to a hand truck (Figure 1).
Principle component: Secondary manifold. A
secondary manifold was constructed for each of the58

Figure 1. Portable primary manifold and booster pumpattached to a hand truck for ease of transport.

four chambers that allowed individual pressure andflow control, safety, and bypass loops. Water fromthe primary manifold was diverted through a checkvalve and a needle valve designed to control theamount of water delivered to the chamber. Water
could enter or bypass the chamber through a 1.9 cm
ball valve. This bypass allowed operation of thesecondary manifold without seawater flowing
through the chamber. After seawater was routed
through the chamber or bypass, it was pushed
through a t-strainer before being routed through a 0.6
cm pressure relief valve. Downstream of the pressure
relief valve, a 1.0 cm normally closed solenoid valve
was installed that operated as a safety valve whensystem power failed. Seawater that had passed thesolenoid valve was routed through a rotary flow
indicator and two 0.6 cm labcock valves used todivert outgoing flow to a cylindrical reservoir wherewater quality measurements could be taken. Twoother 1.3 cm ball valves were also used to bypass thesolenoid and pressure relief valves in the event ofcomponent failure or operation at ambient pressure(Figure 2).

Figure 2. Schematic of secondary manifold and chamber
control valves.

SmileyPrinciple component: Chamber. The hyperbaric
chamber was constructed from 30.5 cm schedule 80
PVC pipe with an internal diameter of 28.6 cm. It
was cut 91.4 cm in length and had two 30.5 cm
flanges glued to each end, increasing internal length
to 95.3 cm. On one end a 5.1 cm-thick acrylic
window and gasket were bolted to the flange with 12
stainless steel bolts. Two 1.3 cm bulkhead fittings
were installed in the side of the cylinder 50.8 cm
apart. A 1.3 cm ball valve was connected to each of
the bulkhead fitting with two 5.1 cm face pressuregauges located on opposite sides of the valves
coming in and out of the chamber. A wood cradle
was designed for the chamber allowing it to be
strapped to a 600 lb capacity hand truck (Figure 3).
The secondary manifold was strapped beneath thechamber. Connections from the chamber to thesecondary manifold were made with a 1.3 cm flexible
hose rated at 20.4 atmospheres. A PVC blind flange
and gasket were attached to the open end of thecylinder to complete the chamber. Pressure in thesecondary manifold and chambers reached amaximum of 9.5 atmospheres.

Pressurization sequence. Prior to receiving a fish,
each chamber was set on its end (open side up) andfilled with seawater (Figure 4). Water pressure wasregulated and stabilized in the primary manifold at
10.9 atmospheres and in the chamber bypass line of
the secondary manifold at 8.8 atmospheres. Once afish was caught and brought to the surface, it wasplaced inside the chamber head down. A blind flange
and gasket assembly was then attached to the top of
the chamber with 12 stainless steel bolts. The bolts
were torqued in a star pattern to 13.1 m/kg. Oncesealed, the valves isolating the chamber were opened,
which equalized pressure from the secondary
manifold. This began the recompression sequence.
Once at the maximum pressure, the valve bypassing
the chamber on the secondary manifold was closed
forcing seawater to flow through the chamber.

Figure 4. Hyperbaric chamber set on end prior to fish
insertion.

Figure 3. Hyperbaric chamber and secondary manifold (notin view) attached to a hand truck for ease of operation andportability.

Hyperbaric Apparatus Operation
Seawater supply. Seawater supply to the apparatus
depended on its location—either in the field or thelaboratory. During field operation, the primarymanifold was supplied with chilled water from thebait well onboard the vessel using a 0.5 hp Flotec
submersible sump pump. In the laboratory, seawater
was pumped from Mission Bay (San Diego,
California), filtered using rapid sand filters, andchilled in a recirculating sump. The temperature
regulation systems at both locations were computer
controlled, resulting in consistent supply
temperatures.

SmileyMonitoring environmental conditions andchamber water quality. Subsurface sea conditions
were monitored using a Hydrolab DataSonde. The
data were used to identify target water quality
parameters for chamber operations and broodstock
maintenance. The Sonde was factory fit with depth(±0.03 m), dissolved oxygen (±0.01 mg/L),
temperature (±0.01°C), pH (±0.01 unit), salinity(±0.01 ppt), and PAR (±1). Measurements wererecorded every 30 seconds as the probe was lowered
to depth at targeted fishing areas. A Reefnet Sensus
Pro depth recorder was used to record changes inpressure as the fish were being caught by hook andline, recompressed, and decompressed in thechamber. The depth recorder was programmed to log
data every 10 seconds when attached to thefisherman’s line, and every 30 seconds when placed59

in the chamber. The Reefnet logger measured depth
to the nearest 0.3 meters.
Water quality was monitored externally andinternally of the chamber. Externally water quality
was monitored downstream of each chamber using an
Oxyguard Handy Gamma probe. The probe wasfactory fit with temperature (±0.1°C) and dissolved
oxygen (±0.1%). Measurements were recorded athourly intervals while on the boat and every few
hours in the laboratory during the daytime.
Measurements and observations were also recorded
at night between staged decompression plateaus. For
continuous in-chamber measurements an Onset
Tidbit temperature probe was deployed to record
temperature every 30 seconds to the nearest 0.01°C.
No other water quality parameters were measured forthe chambers.
Decompression sequence. Fish were stabilized at
depth for 20 to 25.5 hours prior to beginning theirdecompression sequence. Four to six hours into theinitial stabilization period the chambers were isolated(valves closed and disconnected from the water
source on the boat) and transported back to thelaboratory. Transportation times took no longer than
25 minutes, at which point water flow wasreestablished to the chambers from the laboratory
supply system. After the stabilization period, fish
were decompressed manually in stages typical of
those performed by scuba divers practicing a safety
stop. This routine allowed for a relatively rapidchange in pressure with an extended period of time todecompress at the new pressure. This extended
stabilization period allowed fish to equalize internal
gas concentrations with those of the environment,
reducing the possibility of bubble formation in thecirculatory system.
Ascent rates used to calculate minimum timerequired to “off gas” varied from 1.8 to 0.9 m/min
depending on depth. Ascent rates were: 1.8 m/min
below 61.0 m, 1.5 m/min between 30.5 and 61.0 m,
1.2 m/min between 15.2 and 30.5 m, and 0.9 m/min
between 0 and 15.2 m.
Termination of decompression. At the end of eachdecompression sequence, the chamber was set
vertically (blind flange up) and the blind flange wasremoved. The fish was then sluiced into a custombuilt, vinyl sling and placed into a 1.5 m diameter
pool. The chambers were cleaned with freshwaterand scrubbed as needed between uses. Maintenanceof the system was also conducted at this time.

60

Field Trials
Our apparatus was used to recompress anddecompress 14 cowcod. The fish were recompressedto pressures equivalent to 84.7 m ±2.2 m in depth.
The average time taken to place the fish in thechamber and seal the lid on the chamber was 8.0
minutes ±1.4 minutes and the average time taken to
recompress the fish to the desired pressure was 1.8
minutes ±0.9 minutes. Recompression rates ranged
from 20.5 to 164.3 m/min with an average of 63.6 cm±37.6 m.
Following recompression, the cowcod wereallowed to stabilize for 20 to 25.5 hours. After that
period fish were decompressed at a rate of 1.4 m/min
to sequential target stabilization depths (plateaus).
The stabilization depths typically were 75.8 m ±0.6m, 60.4 m ±0.5 m, 47.0 m ±0.2 m, 31.5 m ±0.5 m,
18.0 m ±0.2 m, 11.1 m ±0.4 m, 4.1 m ±0.3 m, and 3.6
m ±1.0 m. Stabilization times at those depths ranged
from 5.4 to 11.0 hours depending on scheduling andinitial stabilization depth. Total recompression
decompression treatments lasted from 73.4 to 150.4
hours and produced healthy viable specimens (Figure
5).

Figure 5. Decompression profile for one of 14 cowcod
successfully collected by HSWRI. Pressure readings
converted to actual or simulated depths with watertemperature also shown.

Water temperature. Variation in water temperaturein the chamber was dependent on the operation ofchillers both on the boat and in the laboratory. On
the boat the initial temperature in the chamber was
warmer than desired because chilled seawater was
not circulated through the chambers while waiting forfish to be captured. During the recompression
sequence chilled water was circulated and thetemperature inside the chamber quickly decreased
from a maximum of 18°C to the target temperature of

Smileyapproximately 12°C. This usually occurred in thefirst 30 minutes of operation and thereafter thetemperature stayed relatively constant.

Conclusion
The hyperbaric chamber was used successfully torecompress, transport, and decompress a species
never before maintained in captivity. The apparatus
is portable and capable of stable operation under avariety of field and laboratory conditions. The
system is also relatively inexpensive to build,
operate, and maintain. Further modifications to thehatch design are being evaluated to expedite theprocess of inserting the fish and initiating therecompression sequence. This modification may also
facilitate future attempts to use the chamber underwater, so that fish do not need to ascend all the way
to the surface before being placed in the chamber. In
the future we plan to collect other species forbreeding purposes. We also plan to use the chambersin the laboratory to conduct studies to determine
species and size-specific differences in susceptibility
to barotrauma.

References
Beyer, D. L., B. G. D’Aoust, and L.S. Smith. 1976.
Decompression-induced bubble formation in salmonids:
Comparison to gas bubble disease. Undersea Biomed.
Res. 3(4):321–338.
Feathers, M. G., and A. E. Knable. 1983. Effects ofdepressurization upon largemouth bass. N Am. J. Fish.
Manag. 3:86–90.
Gitschlag, G. R., and M. L. Renaud. 1994. Field
experiments on survival rates of caged and released redsnapper. N. Am. J. Fish. Manag. 14:131–136.
Gotshall, D. W. 1964. Increasing tagged rockfish (genus
Sebastodes) survival by deflating the swim bladder. Calif.
Fish Game 50:253–260.
Jannasch, H. W., C. O. Wiren, and C. L. Winget. 1973. A
bacteriological pressure-retaining deep-sea sampler andculture vessel. Deep-Sea Res. 20:661–664.
Keniry, M. J., W. A. Brofka, W. H. Horns, and J. E.
Marsden. 1996. Effects of decompression and puncturing
the gas bladder on survival of tagged yellow perch. N.
Am. J. Fish. Manag. 16:201–206.
Koyama, S., T. Miwa, M. Horii, Y. Ishikawa, K.
Horikoshi, and M. Aizawa. 2002. Pressure-stat aquarium
system designed for capturing and maintaining deep-seaorganisms. Deep-Sea Res. 49:2095–2102.
Lee, D. P. 1992. Gas bladder deflation of depressurized
largemouth bass. N. Am. J. Fish. Manag. 12:662–664.
Macdonald, A. G, and I. Gilchrist. 1978. Further studies
on the pressure tolerance of deep-sea crustacean, withobservations using a new high-pressure trap. Mar. Biol.
45:9–21.
Rogers, S. G., H. T. Langston, and T. E. Targett. 1986.
Anatomical trauma to sponge-coral reef fishes captured bytrawling and angling. Fish. Bull. 84:697–704.
Wilson, R. R. Jr., and K. L. Smith Jr. 1985. Live capture,
maintenance, and partial decompression of a deep-sea
grenadier fish (Coryphaenoides acrolepis) in a hyperbaric
trap-aquarium. Deep-Sea Res. 32:1571–1582.
Yayanos. A. A. 1978. Recovery and maintenance of live
amphipods at a pressure of 580 bars from an ocean depthof 5700 meters. Science 200:1056–1059.

Smiley61

A Preliminary Report on the Use of Porphyra Protoplast as a Food Substitutefor Culturing Aquatic Animals
TakaoYoshimatsu
National Research Institute of Aquaculture
Fisheries Research Agency
Mie 516-0193, Japan
takaoyos@fra.affrc.go.jp
Alok Kalla and Toshiyoshi Araki
Mie University
Graduate School of Bioresources
Mie 514-8507, Japan
Dong-Ming Zhang
Kyushu University
Graduate School of Bioresource and Bioenvironmental Sciences
Fukuoka, 812-8581, Japan
Shuichi Sakamoto
Oriental Yeast Industry Co. Ltd.
Tokyo 174-8505, Japan
Keywords: Porphyra, protoplast, feed, red sea bream, Manila clams
Abstract
Protoplasts are plant cells that have had their cell wall removed using either mechanical or enzymaticmeans. As protoplasts have no cell walls they are easily digestible when ingested by animals as foodcomponents. In the present experiment we investigated the availability of Porphyra protoplasts (PP) as afood supplement or live food substitute for culturing red sea bream and Manila clams in the laboratory.
With respect to red sea bream, a PP supplemented diet improved the feed utilization efficiency and enhanced
fish growth, although no remarkable effect was noted on carcass composition in either treatment group.
Survival rate was improved by incorporating 5% PP in the diet. In Manila clams, high survival was
obtained in both PP and diatom fed group. There appeared to be no difference in carcass composition
between two adult shellfish groups fed on either PP or a natural diet. According to the experimental results
obtained in these trials, Porphyra protoplasts proved to be a good food substitute candidate for culturing
aquatic animals like finfish and bivalve spat.

Introduction
Purple laver, Porphyra spp., is known to be oneof the most nutritious edible macroalgae (red algae)
and its thin sheet-like processed products are wellknown as a wrapping material for a famous Japanesedish, sushi-rolls. Porphyra contains very high levelsof various kinds of minerals, and vitamins such asvitamins A, B complex, C, and E. Laver is also agood source of digestible protein (Resource Council
2000). In addition, it is notable that Porphyra
contains a high amount of taurine, an importantamino acid for larval fish (Kim et al. 2005).
Recently, efficient biotechnological techniques for

Yoshimatsu

producing protoplasts from macroalgae using purified
polysaccharases isolated from various bacteria weredeveloped (Araki et al. 1987, 1998, 2000; Aoki et al.
1990). As a result, the practical mass production of
PP became a reality.
Protoplasts are plant cells that have had their cell
walls completely removed using either mechanical or
enzymatic means. Spheroplasts are generally calledsomething other than protoplasts because the cellwall tends not to be destroyed completely by
enzymatic treatment and some parts of the outer cellwall still remain intact. As protoplasts have no cell63

walls, they are easily digestible when ingested byanimals as a food ingredient. In the present
experiment we investigated the availability ofPorphyra protoplasts (PP) as a live food substitutefor culturing two types of aquatic animals: finfish andbivalve molluscs.

fish were weighed individually after being
anesthetized with 2-phenoxyethanol (500 mg/L).
Table 1. Ingredient content and proximate analyses of theexperimental diets for red sea bream.

Materials and Methods
The PP were prepared in the laboratory as
follows. As the cell wall of this alga is composed ofthree kinds of polysaccharides (β-1, 4-mannan, β-1,3-xylan, and porphyran), three enzymes (β-1, 4mannanase; β-1, 3-xylanase; and agarase) were
produced from various types of bacteria which havebeen isolated from marine environments (Araki et al.
1987, 1998, 2000; Aoki et al. 1990). Suitableconditions for preparing a large amount of PP in thelaboratory were determined before the onset ofrearing experiments. Included were determinationsof the proper pH of the reaction mixture,
concentration of each enzyme, and time andtemperature of the reaction process, among others.
Since it is particularly difficult to distinguish andseparate protoplasts from spheroplasts in a wholereaction mixture, we use only the term protoplasts
here when referring to combined enzymatic-treatedproduct from Porphyra. After obtaining theprotoplasts, they were subjected to freeze drying so
the nutrient qualities were retained. Freeze-dried PP
were ground and smashed into powder form
manually using a mortar pestle. The particle size ofPP after grinding was varied from several µm toseveral tens of µm depending on the level ofenzymatic reaction that had occurred.
One hundred liter polycarbonate aquaria were
stocked with 13 fish each (initial average bodyweight = 15.4 g ±0.2 g) that were fed theexperimental diets for 6 weeks. Each aquarium wasaerated (400–600 mL/min) and supplied with flow
through temperature-controlled seawater (32–34 ppt,
25°C) at 8 L/min under the artificial photoperiod of
12L:12D. Two kinds of semipurified diets (Table 1)with and without supplementation of 5% PP were
formulated using brown fish meal as a major protein
source (crude protein level after formulation: 48.7–
51.0%. Every morning the diets were processed into
moist material by adding 60% w/w of freshwater.
They were fed ad libitum three times a day at 1000,
1400, and 1800 hours by hand. The daily feeding
ration was about 6% of the mean wet body weight
estimated from the growth curve obtained duringbiweekly measurement when all the experimental

64

At the end of the feeding trials, in addition to thebody weight and length measurements, hepato- andviscero-somatic indices were calculated from theweights of individual bodies, livers, and viscera.
Proximate analyses of liver and dorsal muscle werecarried out from pooled samples of each groupobtained after termination of the experiment. The
biological parameters used for evaluating rearing
results were defined and calculated as follows:
Condition factor = body wet weight (g) x
1000/body length3 (cm).
Hepatosomatic index (HSI) = liver wet weight(g) x 100/body wet weight (g).
Viscerosomatic index (VSI) = whole visceral wet
weight (g) x 100/body wet weight (g).
Daily growth rate = (ln Wf-ln Wi) x 100/rearing
period (days), where Wf and Wi are final andinitial mean wet body weights (g),respectively.
Feed efficiency = body wet weight gain x
100/dry weight of feed fed (g).
Protein retention rate = dorsal muscle protein
gain (g) x 100/protein fed (g).
Protein efficiency ratio = body wet weight gain(g)/protein fed (g).
The trials with bivalves were conducted asfollows. Manila clam (Ruditapes philippinarum) spat
were obtained from a hatchery. Spat (6-months-old,mean shell length 5.34 mm) were randomly
distributed in small stainless steel mesh cages (10 cm
diameter), with 20 individuals each in six replicatesreceiving each dietary treatment and reared in a
flowthrough system of temperature controlled
seawater (32–34 ppt, 20°C) for 6 weeks. Two diets

Yoshimatsu(PP and condensed natural diatoms [Chaetoceros
gracilis] Tasaki Shinju Co. Ltd., Japan) were fed ad
libitum to the clams four times a day (0800, 1200,
1600, and 2000 hours) by stopping the water flow
until clams complete consuming the food. Adultclams (mean shell length 35.3 mm) were reared under
the same conditions as those used with spat and forthe same period. Before feeding, the PP was mixed
with sea water using an electric mixer and offered tothe clams in liquid form. The dietary performancewas evaluated by growth of the shells, bodycomposition and survival.

PP and control fish showed reduced moisture content
and increased crude protein and lipid content.
However, there were no significant differences inproximate analyses data between them (p > 0.05).
Table 3. Effect of Porphyra protoplast supplementation on
the growth and feed utilization by red sea bream.

Results
Proximate compositions of dried Porphyra andthe PP are shown in Table 2. After enzymatic
processing of Porphyra the protein and lipid contents
increased while crude ash content decreased due
mainly to the digestion of the cell wall
polysaccharides.
Table 2. Proximate composition of Porphyra protoplasts.

The rearing results of red sea bream fed on test dietswith (PP) or without PP supplementation (control)
are summarized in Table 3. After 6 weeks of feeding
the fish reached 65.9 g ±3.5 g in the PP group, but
only 57.0 g ±2.1 g in the control group. There wasmore than a 60% difference in the percentage of
average weight gained between the PP and control
groups (p < 0.01). As a result, a significantly better(p < 0.01) daily growth rate (3.47% ±0.16%) was
obtained in the PP group compared with the control
group (3.11% ±0.11%). In addition, survival was
better in PP group (94.9–100%). As for feed
utilization, the PP group showed better results in feed
efficiency, protein retention rate, and protein
efficiency than the control group, although the twotest diets had no major differences in their proximate
compositions (Table 2). There was no significant
difference in condition factor between the PP andcontrol groups. Nevertheless, higher values wereobtained in both HSI and VSI for the PP group. In
Table 4, proximate compositions of the dorsal muscle
sampled from test fish after the termination of thefeeding trial are shown. After 6 weeks feeding, both

Yoshimatsu

Table 4. Proximate compositions of the dorsal muscle of red
sea bream fed on Porphyra protoplast.
Figure 1. Increase in shell length of spat fed PP as comparedwith diatoms over a 6 week experimental period.

A review of preliminary trial data on length
increase indicated that clams fed on natural diatoms
grew faster than the clams fed the PP diet (Figure 1, p
< 0.01). Nevertheless, over-all mortality was low(97.5–99.2%) and independent of the experimental
treatments. Also, no significant variation wasobserved in proximate carcass composition after thefeeding trial for adult clams (Table 5). After 6 weeks
rearing, both PP and natural diatom-fed groups
showed reduced moisture content and increased
crude protein and lipid contents; the same result asobtained in red sea bream.

65

Figure 1. Increase in shell length of spat fed PP as comparedwith diatoms over a six week experimental period.

Table 5. Proximate composition of Manilla clam fed on
Porphyra protoplast and Chaetoceros gracilis.

Discussion
In red sea bream, a PP supplemented diet
improved feed utilization efficiency and enhanced
fish growth (Table 3), although no notable effects
were obtained with respect to carcass composition of
the fish used in the two treatments (Table 4). Dietary
protein utilization was improved significantly bysupplementing PP as shown in Table 3. As a feed
ingredient, protein is the most important dietary
element and its efficient utilization must be thoughtof as the first priority when formulating a fish food.
The experimental results obtained in this feeding trial
show that PP has great potential as a food supplementfrom the viewpoint of efficient utilization of dietary
protein. Survival rate was also improved by the
supplementation of 5% PP to the diet.
Nakagawa (2005) summarized information
indicating that small amounts of various kinds ofmacroalgal supplementation to the diet improves fish
growth and feed efficiency, mainly due to an
improvement in lipid metabolism. Algal
supplementation to fish feed was previously reported
to be effective either by enhancing the immune

66

system or activating fat accumulation/mobilization as
well as improving absorption, assimilation, andretention (Mustafa et al. 1995). Nevertheless, reportsof the supplemental effect of microalgal protoplasts
have apparently not been published to date.
Enhanced feed efficiency of red sea bream obtainedin the present experiment, when fed with dietsupplemented with 5% PP, may also be attributed tothe probable presence of a food attractants, vitamins,
minerals, essential amino acids, and taurine. Also,
physiological changes induced through dietary PP
supplementation might have affected the feeding
behavior and efficiency of the test fish. At the
moment, we do not know the mechanism by which
PP supplementation improved the dietary value of thefeed for red sea bream in this feeding trial. Further
study is needed to clarify this point.
As for the results obtained in the clam
experiment, PP proved to be a good food source forculturing not only grown shellfish but also spat.
Under the experimental conditions employed, growthin PP group of spat was inferior to that of spat fed
diatoms (Figure 1). Nevertheless, final survival washigh and almost same in the PP-fed group as innatural food group. As there appeared to be nodifference in carcass composition between the twogroups, it should be possible in the future to improvethe dietary effect by manipulating feeding techniques
and ingredients added to PP diet to enhance its
quality.
According to the experimental results obtained inthese culture trials, the protoplasts of Porphyra
proved to be a good food substitute for culturing
aquatic animals like finfish and bivalve spat. We
should continue investigating the applicability of PP
and determine what kind of nutrients must be
supplemented to enhance the dietary value to meetthe nutritional requirements of these aquatic animals
and develop the feeding techniques that mightenhance the performance of the PP.

Acknowledgment
This study was supported by a grant program of
the Agriculture, Forestry, and Fisheries ResearchCouncil in Japan (Research project for utilizing
advanced technologies in agriculture, forestry, andfisheries. No.1681, 2004-2006, Studies on theeffective utilization of low-grade Porphyra by
enzymatic means).

YoshimatsuReferences
Aoki, T., T. Araki, and M. Kitamikado. 1990.
Purification and characterization of a novel beta-agarase
from Vibrio sp. AP-2. Eur. J. Biochem. 187:461–465.
Araki, T., T. Aoki, and M. Kitamikado. 1987. Preparation
and regeneration of protoplasts from wild-type ofPorphyra yezoensis and green variant of P. tenera.
Nippon Suisan Gakk. 53:1623–1627.
Araki, T., Z. Lu, and T. Morishita. 1998. Optimization ofparameters for isolation of protoplasts from Gracilaria
verrucosa (Rhodophyta). J. Mar. Biotechnol. 6:193–197.
Araki, T., S. Hashikawa, and T. Morishita. 2000.
Cloning, sequencing, and expression in Escherichia coli of
the new gene encoding β-1, 3-xylanase from a marine
bacterium, Vibrio sp. strain XY-214. Appl. Environ.
Microbiol. 66:1741–1743.
Halver, J. E. 1957. Nutrition of salmonoid fishes–III.
Water-soluble vitamin requirements of Chinook salmon. J.
Nutr. 62:225–243.

Yoshimatsu

Kim, S.K., T. Takeuchi, A. Akimoto, H. Furuita, T.
Yamamoto, M. Yokoyama, and Y. Murata. 2005. Effectof taurine supplemented practical diet on growth
performance and taurine contents in whole body andtissues of juvenile Japanese flounder Paralichthys
olivaceus. Fish. Sci. 71:627–632.
Mustafa, M. G., S. Wakamatsu, T. Takeda, T. Umino, and
H. Nakagawa. 1995. Effects of algae meal as feed
additive on growth, feed efficiency, and body composition
in red sea bream. Fish. Sci. 61:25–28.
Nakagawa, H. 2005. Effective utilization of macroalgal
waste as feed ingredients for aquaculture. In M. Sakaguchi
and T. Hirata (eds.), Advanced effective utilization of fish
and fisheries products, p. 292–300. NTS Press, Tokyo,
Japan.
Resource Council. 2000. Algae. In Standard tables offood composition in Japan (Fifth revised edition), p. 152159. Science and Technology Agency, Tokyo, Japan.

67Aquaculture and Stock Enhancement Technologies Basedon Recently Discovered Calcium-sensing Receptors in Finfish
Steve Jury, H. William Harris, Timothy Linley,David Russell, Marlies Betka, and Susan Anderson
MariCal Inc.
Portland, Maine USA (www.marical.biz)
sjury@marical.biz
Shinji Harakawa and Akikuni Hara
Hakuju Institute for Health Science, Tokyo, Japan (www.hakuju.co.jp)
Keywords: calcium sensing receptor, salinity, enhancement, salmon, cobia, aquaculture
Extended Abstract
Recent discoveries that calcium-sensing receptors (CaRs) act as salinity sensors and are involved insensory reception in finfish provides a basis for the development of multiple technologies designed to
improve aquaculture and stock enhancement efforts in the U.S. and Japan. The combination of molecularcloning, cell biology, and physiological experiments show that CaRs play key roles in finfish osmoregulationand physiological adaptation to varying salinity environments. Important CaR-mediated roles in finfish
appear to involve not only osmoregulatory acclimation via gill, renal and intestinal tissues, but also changes
in endocrine as well as olfactory responses. Taken together, these data provide support for CaRs as
regulators of the multiple physiological and behavioral changes occurring during smoltification in salmonid
species.
Based on knowledge of the ionic pharmacology of CaRs, juvenile salmonids can be pre-adapted to seawater
while they remain in freshwater. Alternatively, marine fish species normally restricted to seawater can be
maintained under near freshwater conditions. In this regard, the SuperSmolt and SeaReady processes are currently
being licensed to salmon aquaculture and stock enhancement producers. Both of these technologies are applied tofish reared in tanks where mineral salts are added to the rearing water and fish are fed a diet containing elevated
NaCl and a naturally occurring CaR-reactive amino acid for an interval of 3–6 weeks. Thus the resulting biologicalchanges are induced by manipulation of natural processes and do not involve genetic modifications. These changes
are monitored using enzymatic (e.g., gill Na+K+ATPase) and physiological (e.g., seawater challenge, plasma
chloride level) tests to determine development of seawater tolerance. In farmed Atlantic salmon (Salmo salar), theSuperSmolt process reduces the deleterious effects of incomplete smoltification after the transfer of smolts toseawater. This improves both the growth and feed conversion ratios while reducing mortalities during growout inseawater. In Chinook, coho, and sockeye salmon (Oncorhynchus tshawytscha, O. kisutch, O. nerka), the SeaReady
process improves the osmoregulatory and growth performance in the early seawater rearing environment andpreliminary data suggest that this may increase the number and performance of adult returns. The SuperSmolt
process has been recently tested and endorsed by EWOS, a major aquaculture feed producer. By contrast, CaR
modulation in marine fish such as cobia (Rachycentron canadum) enables their production under very low salinity
conditions, providing inland growers or those utilizing recirculation technologies the option to produce high valuemarine species.
Research by MariCal and Hakuju has also focused on the roles of CaRs in sensory biology, including salinity
reception, olfaction, and the response of fish to electric fields via lateral line or specific electroreceptor tissues.
Since a gradient of charged ions (Na+, Cl-, Mg2+ or Ca2+) is likely involved in salinity as well as electric field
detection, CaRs in specific tissues may provide a molecular means to “sense” and integrate changes in both theionic and electric field environments that fish may encounter. While much ofthis research is ongoing, we conclude that this CaR-based approach has considerable potential to improveaquaculture and stock enhancement technologies in the U.S. and Japan.

Jury

69

Background. CaRs are part of a family of G-protein
coupled receptors that are closely related tometabotropic glutamate and pheremone receptors andare highly conserved between all species tested(Breitwieser et al. 2004, Ingleton et al. 1999, Figure1). CaR proteins were first identified in humanparathyroid glands and their role in calcium
homeostasis has been firmly established (Brown and
MacLeod 2001, Breitwieser et al.2004, Chang andShoback 2004). CaRs are responsive tophysiologically and environmentally relevantchanges in extracellular divalent cations such as Ca2+and Mg2+ (Breitwieser et al. 2004), but are also
modulated by other extracellular conditions including
NaCl (Quinn et al. 1998, Loretz et al. 2004), specificamino acids (Conigrave et al. 2004), several heavy
metals; e.g., Pb2+ and Al3+ (Handlogten et al. 2000,
Quinn et al. 2004), and pH (Doroszewicz et al. 2005,
Quinn et al. 2004). CaRs have been found in
multiple organs associated with osmoregulation andsalinity detection in several fish species including
Atlantic salmon (Salmo salar)(Nearing et al. 2002,
Nearing et al. submitted), sea bream (Sparus aurata)(Flanagan et al. 2002), and tilapia (Oreochromis
mossambicus)(Loretz et al. 2004). We have alsocharacterized a full-length CaR cDNA from dogfish(Squalus acanthias) kidney (SKCaR) that is localizedin kidney, olfactory lamellae, epithelial cells andvessels in the gastrointestinal tract, gills, and rectal
gland (Nearing et al. 2002, Hentschel et al. 2003).
Several studies have shown CaR in other fish species
without molecular cloning of their full-lengthtranscripts or proteins. These species include theflounders, Platichthys flesus (Ingleton et al. 1999)
and Pleuronectes americanus (Nearing et al. 2002),
and goldfish, Carassius auratus (Hubbard et al.
2002).
The potential role of CaRs as regulators of Ca2+
homeostasis in fish has been emphasized (Hubbard etal. 2000, 2002, Ingleton et al. 1999, Radman et al.
2002, Fellner and Parker 2002, Loretz et al. 2004,
Nearing et al. submitted), but many of these studies
have also suggested the involvement of CaRs in
salinity sensing in fish (Nearing et al. 2002, Hubbard
et al. 2002, Loretz et al. 2004, Nearing et al.
submitted).
Thus CaRs are present and functional in various
tissues of multiple teleost species, and we proposethat they are important in the integration of several
physiological and behavioral processes of concern tothe aquaculture and stock enhancement industries.
We have used the approach of studying receptor
biology to address problems of concern in hatcheryproduction of anadromous and marine species. In

70

Figure 1. Calcium-sensing receptors (CaR) are integrators of
extracellular and environmental signals leading to various
intracellular responses.

this report we describe applications developed using
this approach as well as an introduction to someresearch to develop additional technologies. These
applications are: 1) SeaReady, 2) growing marine
fish in near freshwater, and 3) the role of CaR insensory biology.
SeaReady. Anadromous species, including
salmonids, are able to survive in either freshwater orseawater due to osmoregulatory adjustments intissues including gill, skin, kidney, andgastrointestinal tract. These are the same tissues
where CaRs are localized (Nearing et al. submitted)
and may be differentially expressed during seawater
acclimation. The process of smoltification insalmonids raised under hatchery production
conditions prepares salmon for the remodeling of
their tissues after seawater transfer. Thesephysiological changes must be accomplished within alimited interval of time and be matched to the
movement of fish to higher salinity environments.
These changes may occur naturally during a normal
smolt timing window that is dependent on the species
concerned. The complex changes that occur duringsmolting determine how ready salmonids are for aseawater existence. Smoltification has traditionallybeen manipulated under hatchery rearing conditions
with varying degrees of success by changes inphotoperiod, temperature, and feeding (McCormick
et al. 1998). While the factors that initiate andcontrol smoltification continue to be the subject ofJurymany studies, they are not entirely understood. The
variability in the effects of different methods, as well
as logistic problems associated with hatchery rearing
often leads to a gradient of incomplete smoltificationand subsequent osmotically induced lethal andsublethal effects upon transfer of fish to seawater.
These effects can include significant elevations inplasma ionic content resulting in the dysfunction ofvarious organ systems, impaired ion homeostasis,
abnormal swimming behavior, morphologicalchanges, impaired feeding behavior, and often
increased mortality.
Based on our understanding of the distribution
and function of calcium-sensing receptors relative toosmoregulation, as well as the logistic andsocioeconomic constraints of hatchery operations,
Marical has developed the SeaReady (U.S. patent#6,748,900) and SuperSmolt (U.S. patent#6,463,883) processes as methods to enhancesmoltification in salmonid fishes while they are stillin the hatchery prior to release into rivers or transfer
to seawater for growout. This process consists ofmanipulating the ionic environment (e.g., Ca2+ andMg2+) as well as application of feed addititives (NaCland CaR reactive amino acids) over a period of 4–6
weeks (Figure 2).

Figure 2. SeaReady process overview.

SeaReady serves to preadapt salmon to seawaterand allows for improved performance upon transfer
to seawater. In terms of stock enhancement, we
propose that this improved early seawater
performance will lead to enhanced ocean survival(Linley 2000) and subsequent improved returns.
Benefits include the ability to transfer fish toseawater at smaller sizes, transferring of smolts
outside of their traditional species specific smolt
timing windows (e.g., fall entry), reduced feeding
inhibition upon seawater transfer, and improved

Jurygrowth during seawater rearing. This process has
been successfully tested on multiple species (Atlantic
salmon, Chinook, coho, and sockeye salmon) in
multiple locations on over 3 million smolts. We
believe that there may be additional effects of thispreadaptation process, including priming for
imprinting, manipulating residualization, decreased
estuarine residency time (and thus decreased
predation in early marine life history), as well as
improved ocean survival and subsequest improved
returns of released fish (Linely 2000). However,these effects remain to be quantified.
Growing marine fish in near freshwater.
Acclimating marine species to grow in freshwater ornear freshwater conditions offers multiple benefits
for hatchery production. For certain applications,freshwater recirculating systems are more feasible on
a commercial scale to raise marine species, thentransfer them to marine sites for growout. This
would allow for raising fish to a larger size undercontrolled hatchery conditions, potentially at a lower
cost. The reasons for using freshwater systems
include: 1) coastal property is very expensive forshore based tank farms and political/environmentalpressures are restrictive along the coast in many
areas, 2) environmental regulations limit the amountof saline water discharge and sludge disposal fromrecirculating facilities, 3) higher value marine species
may be grown in systems currently culturing lower
value species, and 4) freshwater culture of marine
species may mitigate some disease issues.
We have patented (U.S. patent # 6,463,882)
methods to grow marine species in near freshwaterby manipulating the ionic composition of the rearing
environment and adding CaR compounds to the feed
to induce compensatory changes in the plasma. The
fish are subjected to a gradual or step-wise decreasein salinity for a period of time prior to transfer tofreshwater, while being fed a modified diet. The fish
are then maintained in a near freshwater low salinity
environment with an optional long day photoperiod
for a sufficient period of time to increase or maintainexpression or sensitivity of calcium-sensing
receptors.
MariCal has been conducting feasibility studies
with cobia (Rachycentron canadum) and has
determined that cobia grow well in salinities as low
as 2 ppt and can be successfully transferred fromthese conditions back to 35 ppt seawater. MariCalplans to grow cobia to market size in future large
scale trials.

71Role of CaRs in sensory biology. CaRs are
expressed in most osmoregulatory tissues in salmon,
but they are also found throughout the peripheral andcentral nervous systems (Nearing et al. 2002, Nearing
et al. submitted). In particular CaRs have been foundin the olfactory system (olfactory epithelia, olfactorybulb, olfactory nerve) as well as in electroreceptive
organs (e.g., electric organs, ampullae of Lorenzini)of teleosts and elasmobranchs. Based on extracellular recordings of the olfactory nerve, we havedetermined that Atlantic salmon are responsive tocalcium, magnesium, gadolinium, and strontium atconcentrations that are consistent with CaR activation
or modulation (Nearing et al. 2002, Nearing et al.
submitted). On this basis, we propose that CaRs playa role in multiple osmoregulatory, and sensory
functions in salmon which experience large changes
in the concentration and composition of the ionicenvironments encountered during their normal life
history. This has many implications for stockenhancement of salmonids including the role of CaRs
in olfactory mediated behaviors such as imprinting,homing, mate selection, and predator avoidance. Our
research programs have focused on the function of
CaR in marine and aquatic organisms. Using
information from these studies, we plan to continue
developing applications useful to the stockenhancement and aquaculture industries.
Conclusions. The identification of CaRs in multiple
tissues of many species of finfish is an importantdiscovery opening an area of research on the
integration of environmental and extracellularsignalling of cations. Exposure of finfish to variable
ionic environments under naturally occurring
conditions (e.g., the typical life history ofanadromous species) or under hatchery conditions(e.g., SeaReady) influences CaR signaling in specifictissues (e.g., gill, olfactory epithelia, electric organ).
CaRs are likely to be involved in many physiological
functions in the salmonid life cycle, and using an
evolving understanding of the role of these receptors,
we have developed technologies beneficial to thestock enhancement and aquaculture industries in the
U.S. and Japan.

72

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Biological Studies on the Digestive Mechanism in Abalone
Kentaro Niwa and Hideaki Aono
National Research Institute of Fisheries Science
6-31-1 Nagai, Yokosuka
Kanagawa 238-0316, Japan.
niwaken@affrc.go.jp
Tomoo Sawabe
Laboratory of Microbiology
Hokkaido University
3-1-1 Minato-cho, Hakodate
Hokkaido 041-8611, Japan
Keywords: abalone, Vibrio halioticoli, alginate lyase
Abstract
Abalone are one of the most important marine gastropods in Japan’s coastal fishery. Recent studies on
abalone suggest that the gut microbes work together with polysaccharide degrading enzymes of the host to
digest seaweeds in the stomach. Genes encoding alginate lyase have been cloned from the digestive
diverticulum of Haliotis discus hannai and H. d. discus. The bacterium, Vibrio halioticoli, with alginate
degrading activity has also been isolated from the digestive fluid of various abalone species. However, theprocess by which the bacterium colonizes gut of abalone is unknown. Therefore, our goal is to clarify whencolonization occurs and to what extent the bacterium contributes to digestion of polysaccharides.

Introduction
Abalone are economically important marine
gastropods that bring moderate to high prices on theworld market (Oakes and Ponte 1996). In Japan,
there are six species of abalone, Haliotis discus
discus, H. discus hannai, H. madaka, H. gigantea, H.
diversicolor diversicolor, and H. diversicolor
aquatilis, that are targets of coastal fisheries andaquaculture. Abalone ingest mainly algae, with apreference for brown algae. Abalone are distributed
along the coastal reefs in Japan, where many kinds ofkelp in the order Laminariales (brown algae) growabundantly. Laminaria sp. is known to be theoriginal source of alginate. Alginate accounts formore than 30% of the dry weight of the thallus insome algae. It is known that the cell walls of brownalgae are formed by linkages of alginate and cellulose(Kloareg and Quatrano 1988). Alginate molecules
consist of two homopolymeric structures:polymannuronate (polyM) block and polyguluronate(polyG) block, and a heteropolymeric part, which is
randomly connected to manuronate to guluronate(MG random block). These polysaccharides are
difficult to degrade by vertebrate digestive enzymes.
However, some kinds of herbivorous arthropods,
nematodes and molluscs can degrade thosepolysaccharides. Many researchers have reported the

Niwa

existence of alginate lyase in the digestive
diverticulum of abalone (Tsujino and Saito 1962,
Nakada and Sweeny 1967, Ohnishi et al. 1985,
Boyen et al. 1990). Uki and Watanabe (in Shepherd1992) investigated the nutritional requirements forthe development of artificial feeds for abalone andsuggested that abalone can efficiently use alginic acidas an energy source. It has been reported thatalginate lyase detected from the digestive tract of
abalone preferentially degraded the polyM block(Nakada and Sweeny 1967, Boyen et al. 1990).
Alginate lyase specific for polyM was cloned fromthe digestive diverticulum of H. discus hannai(Shimizu et al. 2003). Recently, we succeeded incloning of the equivalent gene from H. discus discus(in preparation). Furthermore, 68% of all alginolytic
bacteria isolated from H. discus hannai showed astrong preference for the polyG block (Sawabe et al.
1998). Therefore, it is reasonable to speculate thatthe polyM part of ingested alginate is degraded by thedigestive enzyme of the abalone, and the remaining
polyG part is metabolized by these bacteria.
Sawabe et al. (1998) isolated a unique alginolytic
marine bacterium from the gut of H. discus hannai.
The bacterium was identified as Vibrio halioticoli,
which was recognized as a new species (Sawabe et75

al. 1995). The bacterium produced at least six typesof alginate lyases, two of which were observed to be
specific for polyM and four of which were specificfor polyG.
V. halioticoli was detected from the gut of three
species of the Japanese abalone, H. discus discus, H.
diversicolor aquatilis and H. diversicolor
diversicolor, and the South African abalone, H.
midae, with a range of 40–65% among thosemicrofloras (Sawabe et al. 2003). The bacterium wasalso found in the gut of a turban shell, Turbocornutus, although the occupation rate wassignificantly smaller than that found in Haliotis spp.
Furthermore, acetic acid and formic acid weredetected as major fermentation products of alginate in
the V. halioticoli strains. The acetate is absorbed
from the gut tissue of abalone and metabolized as anoxidative energy source (Breznak 1982). It issuggested that the abundant populations of V.
halioticoli in the gut of Haliotis abalone may have agreat role for converting alginate to acetic acid
(Sawabe et al. 2003).
The population of V. halioticoli in the gut of
juvenile abalone increased when the diet was
switched from diatoms to laminaria (Tanaka et al.
2003). Although the finding seems to suggest thatthe main source of the bacterium is thalli of
laminaria, V. halioticoli has never been isolated fromthat alga. The main route and timing of gut
colonization are not yet known. Therefore, we plan
to clarify when V. halioticoli colonize the gut of theabalone and to what extent the bacterium contributes
to digestion of polysaccharides.

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