U.S. DEPARTMENT OF COMMERCE

National Oceanic and Atmospheric Administration

National Marine Fisheries Service

NOAA Technical Memorandum NMFS-F/SPO-85

Aquaculture and Stock

Enhancement of 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

© 2002 Natalie Fobes, all rights reserved

Aquaculture and Stock Enhancement

of 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
ii

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.
iii

Table of

Contents

Introduction

Species Reports

Seriola

 Current Situation of Technical Developments in Seed 

 Production of Yellowtail (Seriola quinqueradiata) in Japan

 Keiichi Mushiake, Hideki Yamazaki, and Hiroshi Fujimoto

Sciaenids

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

 Recirculating System

 Reginald B. Blaylock, Angelos Apeitos, Jason T. Lemus, and

 Jeffrey M. Lotz

 Disease Surveillance in Wild and Cultured Stocks of White 

 Seabass (Atractoscion nobilis)

 Mark S. Okihiro

Sebastes

 Juvenile Mass Production of Black Rockfish (Sebastes 

 schlegeli) in FRA Miyako Station: Present Status and Problems

 Masahiro Nakagawa, Junichi Adachi, Ken Shimizu, and

 Satoshi Shiozawa

Paralichthys

 Construction of a BAC Library from XY Japanese Flounder 

 Using Frozen Sperm Genomic DNA

 Hiroyuki Okamoto, Hiroyuki Nagoya, Eiichi Yamamoto, Takashi 

 Sakamoto, Kanako Fuji, Nobuaki Okamoto, Kazuo Araki, and

 Ichiro Nakayama

Scombrids

 Status of Pacific Bluefin Tuna (Thunnus orientalis) Seed 

 Production in Amami Station of NCSE FRA

 Hideki Nikaido, Takayuki Takebe, Kentaro Ide, Hitoshi Imaizumi, 

 Nobuhiro Tezuka, and Syukei Masuma

Genetics and Reproduction

 Visualization Tools to Probe Early Stage Fish Abnormalities

 Susumu Uji and Tadahide Kurokawa

Special Topics

 Airlift-PolyGeyser Combination Facilitates Decentralized 

 Water Treatment in Recirculating Marine Hatchery Systems

 Ronald F. Malone and Sripavani Gudipati

 Preparation of Marine Silage and its Potential for Industrial 

 Use

 Motoharu Uchida

1

5

11

21

27

31

37

43

51
iv

Special Topics (continued)

 Techniques for Live Capture of Deepwater Fishes with Special 

 Emphasis on the Design and Application of a Low-cost 

 Hyperbaric Chamber

 Jeffrey E. Smiley and Mark A. Drawbridge

A Preliminary Report on the Use of Porphyra Protoplast as a 

 Food Substitute for Culturing Aquatic Animals

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

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

57

63

69

75
v

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

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

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

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

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

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

exchange.

Under the auspices of the UJNR Aquaculture Panel, U.S.-Japan scientists have 

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

alternated between the two countries. In November 2005 the meeting (comprised 

of 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 agreement 

of 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 States
vi
Mushiake 1

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 the

Fisheries 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 is

late April to early May), which caused the smaller size of released juveniles, was solved by developments in 

advanced 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 to

that of the wild stock.

Introduction

Yellowtail (Seriola quinqueradiata), which is a 

large migratory species that moves north and south in

waters adjacent to the Japanese archipelago, is an

important species of the fishery stock in western

Japan and the coastal areas of the Sea of Japan. In

2002, the annual catch was 50,000 tons and the

aquaculture 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 Fisheries

Research Agency (FRA), started to develop the

techniques 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 ground

is 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).
2 Mushiake

Brief history of seed production in yellowtail

Collection of eggs and culture of larvae and

juveniles were started in order to obtain basic 

information in 1960. A marked decrease of catch

quantity in 1970s led NCSE to begin the technical

development for broodstock management in 1977

and seed production in 1978.

In NCSE, the brief history of the technical

developments for yellowtail culture was as follows

(Shiozaw and Yamasaki 2006):

1. 1977–1989. NCSE was engaged in seed

production 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 introduction

of formulated diets and developments in the 

improvement of survival rates during the 

early stages.

3. 1996–2005. Since 1996 advanced egg

collection and early seed production

techniques have been implemented.

In the present paper, some technical developments in

seed production since 1990 are presented, as well as

the details of both improvement techniques for 

survival in seed production and advanced spawning

of broodstock.

Improvement techniques for survival in seed

production 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 a

total length of about 15 mm until they grew to

juvenile size. Those were the two major causes of 

mortality, and some measures have been taken to

counteract the problems.

One approach was aeration which led to a high

survival rate during the early larval stages. In the 

past, the sinking of hatched larvae, seen before and

after 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 high

mortalities due to the sinking of larvae. Since 1995,

the problem has been eliminated by employing

stronger aeration using air-block pipes set in each

corner at the bottom of the tanks. This method

enabled the tank water to have a stronger upwelling

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, the 

survival 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 the 

surface at night, so grading is carried out during a

single 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 mm 

mesh 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 the 

large and small sized fish that were selected by this

method. The fish were clearly divided into two

groups 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 survival

rate, 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% to

78.9% 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 graded

with a 3 mm mesh net at night when the fish averaged 15 mm

TL.
Mushiake 3

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 chorionic 

gonadotropin (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, the

size 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 delay

in 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 in

order to induce earlier maturation. These 

manipulations were followed by HCG injection and

spawning was successfully induced between

February and March (Mushiake et al. 1994, 1998).

Now eggs are available for seed production in the

hatchery 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 a 

similar size and almost the same growth as their wild

counterparts, as indicated by open circles. In fact, the

hatchery-reared juveniles were released with a brand

mark into the Seto Inland Sea and collected at a fish

market in December 1999. The body weight of the

wild and the released fish was 1.2 kg and both types

traded for the same price in the fish market. The seed

production at present is capable of producing fish of a 

similar 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 seed

production techniques were the optimal ones

and solve the various problems faced in the

past, so the techniques have been already

established for yellowtail.

2. With regard to the advanced egg collection

method during February and March, the 

hatchery-reared juveniles that were produced

by advanced spawning showed growth rates

to their wild counterparts. These developed

techniques were effective for the promotion

of 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 the 

fundamental management of yellowtail

aquaculture. According to our studies, there

was no difference between yellowtail

produced in advanced spawning and those 

hatched 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).
4 Mushiake

References

Fujimoto, H., H. Yamasaki, and A. Suda. 2006. Release

for stock enhancement. In M. Matsuyama, Y. Hiyama, K.

Mushiake, and H. Yoshiaki (eds.), Biology, stock

enhancement 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 water

temperature. Nippon Suisan Gakkaishi, 72(2):186-192.

Mushiake K. 1994. Studies on technical development of

broodstock management in striped jack and yellowtail.

Doctoral thesis. Hiroshima University, Higashi-hiroshima.

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 the

photoperiod and water temperature. Fish. Sci. 64:727–731.

Shiozawa, S., and H. Yamasaki. 2006. Seed production of

juveniles. In M. Matsuyama, Y. Hiyama, K. Mushiake,

and H. Yoshiaki (eds.), Biology, stock enhancement and

aquaculture of buri [Japanese yellowtail (Seriola

quinqueradiata)].
Blaylock 5

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 rearing

of larvae in earthen ponds containing estuarine water and mixed wild zooplankton. We have designed a

recirculating system in which to rear spotted seatrout larvae and juveniles intensively. As a test of the

intensive 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 were

transferred 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 ±1.93

mm 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 unsustainable 

past 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, the

Magnuson-Stevens and Sustainable Fisheries acts

required that plans for management and restoration of

overfished species be put in place. Typically, the

plans 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 effectiveness

combined with concerns over maladaptive behaviors,

artificial genetic selection, and disease problems in

cultured fish created skepticism about the desirability

of the approach. The advent of new hatchery and

tagging technologies, the continued decline of 

managed stocks, and an accumulation of data from 

studies 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 stock

enhancement is possible through carefully planned

research that incorporates principles of fish culture,

ecology, health, genetics, and assessment.

The spotted seatrout (Cynoscion nebulosus) is an

estuarine and nearshore species ranging from New

York to Texas (Robins and Ray 1986). In the 

southeastern U.S. and in the Gulf of Mexico, the 

spotted seatrout is widely esteemed among

recreational anglers. In Mississippi waters, the 

spotted 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 potentially

vulnerable to depletion. It is from this perspective

that the Gulf Coast Research Laboratory in

cooperation with the Mississippi Department of
6 Blaylock

Marine Resources and recreational angler

Organizations founded SPEC, the Seatrout

Population Enhancement Cooperative, a program to

determine 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 for 

spawning and rearing spotted seatrout in the

laboratory. Colura et al. (1976) and Tucker (1988) 

described experimental methods of fingerling

production for spotted seatrout. Although Arnold’s

original production system included early larval

rearing in which he achieved as high as 30% survival

through 30 days, his system evolved into the

extensive 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 for 

transitioning to pond rearing was inconsistent and

low 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 of 

available 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 of 

technology for capturing, transporting, quarantining

and maintaining broodstock; 2) Spawning—the 

development 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 in

intensive, indoor recirculating systems; 4) tag and

release—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 the 

information to assess the effectiveness of the

program.

Broodstock development. Broodstock development

is patterned directly after the Texas Parks and

Wildlife program (David Abrego, personal

communication). Wild animals are caught locally on

hook-and-line using barbless hooks. Seatrout are 

delicate animals and must be handled with great care.

Soft catch-and-release nets or standard nets lined

with plastic are required for landing the fish. Fish are

transferred from the hook into holding pens that serve 

as a reservoir to contain all fish caught during a 

collecting event. Upon completion of the collecting

event, fish are transferred using the plastic-lined nets

into hauling tanks for transport to the lab. Stress Coat

and supplemental oxygen are added to the hauling

tank. 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 in

a staging area that allows acclimatization and

recovery from the stress of capture and transport.

After a brief acclimatization period, fish enter the 

quarantine area. Because our goal is to maintain fish

in optimal condition for long-term holding and

because all wild fish have parasites, we initially

quarantine the fish to eliminate diseased fish and

parasites that are likely to cause problems in captivity

(i.e., parasites with direct life cycles). We practice

standard animal care facility sanitation practices such

as 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 minute 

freshwater dip that detaches many of the

ectoparasites, particularly Amyloodinium ocellatum,
Blaylock 7

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 a 

minimum of 10 days, during which they are observed

for health problems (and treated if necessary) and

transitioned 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 the 

maturation facility. During the move, the fish are

anesthetized, weighed, measured, sexed, tagged

according to sex, and fin clipped for DNA analysis.

Fish are stocked into 12,000 L circular tanks fitted

with egg collectors in a temperature and photoperiod

controlled environment. Water quality is maintained

using 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 three 

times 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 are 

fed shrimp (≈36%), squid (≈45%) and cigar minnows

(≈18%). Postspawning animals are fed at the same 

rate a diet consisting of approximately 16% shrimp,

42% squid, and 42% cigar minnows. The

temperature and photoperiod cycle mimics the

natural 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 other 

marine fishes including the red snapper (Ogle and

Lotz 2005). Seatrout spawn at night. The eggs are 

buoyant and are carried with the current out of the 

tank into an egg collector where they are retained.

From the egg collector they are placed in a conical

incubator and agitated with air. Viable eggs float,

and 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 and

cultured 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 and

rotifers 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 and

size-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 consists

of 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 of 

mortality. We did two things in the nursery to try and

minimize the problem. First, we used the adjustable 

spray bars and pumps to create currents sufficient to

keep the juveniles active. Second, we manipulated

the density. Not all species respond to changes in

density 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 was

slowly 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 of

juveniles going into the nursery. Originally, the 

nursery phase was intended to last for at least 14

days, however concerns over our ability to maintain

water quality in the high density tanks led to

harvesting 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%), the

variability was such that we can not summarily

exclude a high density nursery as an option for future

batches of juveniles. Although fish stocked at the

highest density were smaller than those stocked at
8 Blaylock

other densities, major size discrepancies within tanks

were not observed.

Growout. Growout for this batch was accomplished

in 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 were 

approximately 80–100 mm, and we estimated

approximately 75% survival to day 60. There was

more size variability than in the nursery, perhaps due

to unequal access to food created by poor circulation

in the raceway.

Tagging and release. At the time of the hurricane,

plans for experimental tagging with coded-wire and

elastomer tags to assess handling and insertion

procedures, tag placement, and tag retention were 

being developed. Acoustic tags will be explored in

the future. Growth rates and feed conversion ratios

were being monitored. We anticipate that release

procedures will involve a fish pumping device like 

those widely used in the aquaculture industry for 

stocking and harvesting netpens.

Seatrout are known to vary genetically among

localities (Gold et al. 2003, Wiley and Chapman

2003), thus, the test group of juveniles from Texas

could not have been released in Mississippi. A

project to assess genetic differences among fish from 

different localities in Mississippi is in development to

provide information critical to the development of a 

sound release strategy. Until that information is

available, we restrict ourselves to releasing juveniles

only in the area from which the broodstock were 

acquired. Initial issues addressed by the release will

include survival after release, percent recovery, and

movement. More complicated issues such as the 

effect 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 for

spawning 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, and

competition in cultured predatory fish larvae: Biotic and

abiotic 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 an

experimental 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 to

Atlantic Coast fishes of North America. Houghton

Mifflin, Boston, Massachusetts.
Blaylock 9

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.

Taniguchi, A. K. 1981. Survival and growth of larval

spotted seatrout (Cynoscion nebulosus) in relation to

temperature, prey abundance, and stocking densities.

Rapp. P-V Reun. J. Cons. Int. Explor. Mer. 178:507–508.

Tsukamoto, K. 1993. Marine fisheries enhancement in

Japan and the quality of fish for release. Eur. Aquacult.

Soc. Spec. Publ. 19:556.

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.

Tucker, J. W., Jr. 1988. Growth of juvenile spotted

seatrout on dry feeds. Prog. Fish-Cult. 50:39–41.

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.

Okihiro 11

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 been

due, 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 of 

proven 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 an

effective 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, in

Central 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 and

commercial fishermen started to decline dramatically.

The fishery was essentially nonexistent by the late 

1970s.

In response to the collapse of wild stocks, the 

Ocean Resources Enhancement and Hatchery

Program (OREHP) was formed in 1983. OREHP is a

cooperative effort between the California Department

of 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 to

help restore marine fish populations in California 

waters. OREHP works actively to culture several

marine finfish species, but the major focus of 

remediation efforts is on WSB; WSB are currently

the only cultured marine fish being released into

California waters. As part of the OREHP program, a 

dedicated marine fish hatchery was constructed in

1995. The Leon Raymond Hubbard Jr. Marine Fish

Hatchery (also known as Carlsbad Hatchery), located
12 Okihiro

in Carlsbad, California, has produced more than

100,000 juvenile WSB for release into the Pacific in

each 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 netpen

sites. 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. Gill

flukes (Anchoromicrocotyle guaymensis) plagued

four netpen sites. As of November 2005, there was a 

severe herpes virus outbreak at the Carlsbad Hatchery

involving 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 winter 

was one of the wettest on record for California, and

water quality early in 2005 was marginal at best with

wide fluctuations in temperature, salinity, and

particulate loads. Although no specific testing was

done, contaminant loads (i.e., xenobiotics and

bacteria) from rainwater runoff were assumed to be 

high throughout Southern California.

Disease outbreaks are an unfortunate, and

sometime inevitable, consequence of almost all highdensity marine aquaculture and finfish enhancement

programs. To maximize mitigation efforts, while at

the 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; all

cultured fish are thoroughly evaluated prior to

release. At a minimum, cultured WSB are inspected

twice. The initial health check is done at the hatchery

prior 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 health

assessments are done as needed if fish are behaving

abnormally or if there is an increase in daily

mortality.

The third arm of OREHP’s health assessment

and disease prevention program is an ongoing effort

to survey wild stocks of marine fish species—with

special emphasis on those species targeted for

mitigation or aquaculture. The goal of this disease

assessment 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 a 

veterinary or fisheries background, but should be 

qualified in the diverse array of subjects required for 

disease prevention, detection, control, and

management. Some of the required fields of 

expertise (or at least familiarity) include: general

biology, marine biology, fish diseases, general

pathology, fish pathology, cytology, hematology,

serology, histology, histopathology, general anatomy,

fish anatomy, and molecular biology. Some 

background in toxicology, microbiology, fish

physiology, surgery, and genetics is also helpful.

While finding a fish pathologist with a 

comprehensive and diverse educational background

is important, equally important is finding one with

extensive practical experience with a variety of fish

diseases (infectious and noninfectious) in a variety of 

fish 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 is

already familiar with the particular fish species in

use. 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 and

experience to the species at hand and to the unique

disease problems associated with that species.

Eventually, given sufficient time and examination of 

a 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 is

located and gains experience with the particular fish

species being cultured, the next major hurdle is to
Okihiro 13

ensure that this disease knowledge base (often

painstakingly acquired over a period of years) is not

lost when the fish pathologist leaves, retires, or gets

eaten by a shark while diving. Knowledge transfer to

the next pathologist can be simplified by 1) having a

comprehensive and detailed file of pathology reports,

2) having detailed written protocols for most (if not

all) 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 mount

cytology photos, light micrographs of histologic 

lesions, as well as transmission electron micrographs

of ultrastructural lesions and pathogens. A

comprehensive and well-organized photographic 

record of diseases, for a given fish species, will go a 

long 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 and

background information, 2) clinical assessment of

live 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 a

thorough history is the first step in obtaining a 

reliable diagnosis. Background information should

include specifics on the species, age, size, and

number 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 were 

transported. The number of transport trips that were 

needed, as well as any problems encountered during

transport, are also important pieces of information.

Daily mortality (both raw numbers and percentage of

the population) and mortality patterns over the 

preceding two weeks are important indicators of 

health, 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 and

health. 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 of

tank bottoms and cleaning of end screens are the 

primary maintenance items to look for. Inconsistent

care—because of personnel shortages or sickness—

should also be looked for and noted. The majority of

growout facilities have logbooks, which can be

accessed to determine if pen maintenance was

consistent 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 health

and 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 to

days) often precede outbreaks of infectious disease.

Clinical assessment. Knowledge of normal behavior 

for the fish species, age group, and enclosure is

important if deviations from the norm are to be 

detected. With WSB, it is important to know how the 

different age groups (larvae, juvenile, subadult, and

adult) act when they are in round versus rectangular 

tanks, concrete raceways, fiberglass raceways, or 

netpens. Do fish school? At what age do they

school? In what types of enclosures do they school? 

Are fish normally up near the surface, in the middle 

of the water column, or near the bottom? Answers to

all of these questions are important to define what is

normal behavior for a given species, age group, and

tank setup. This knowledge can only be acquired over 

time, so it is always important to take the time to

observe living fish in both sickness and health. Some

general clinical signs to look for in unhealthy fish

are: 1) weight loss (head out of proportion to the 

body), 2) anorexia (sick fish rarely feed), 3) lethargy

(sick fish swim slowly) and absent or reduced escape 

reflex, 4) weakness (sick fish are pushed towards end

screens or pulled towards drains), and 5) abnormal

pigmentation (sick fish are usually darker than

normal).

In addition to these nonspecific signs of disease,

there are other more specific clinical indicators of ill

health. With WSB, even heavily parasitized fish

rarely flash (i.e., attempt to scratch themselves on

fixed objects). Still, when flashing is observed, it is

wise to check for external parasites. Terminal

spinning or spiraling in juvenile WSB is usually

associated 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 is

often indicative of Ichthyobodo infestation, while

focal white areas of depigmentation are characteristic 

of P. salmonis. There is never 100% uniformity with

respect to the clinical presentation, but with careful
14 Okihiro

observation, patterns will usually emerge and will

help indicate from what the majority of fish are 

suffering.

Necropsy. A thorough necropsy is the most

important step in determining why fish are dying. A

good necropsy, performed by an experienced

pathologist, will often quickly reveal what disease is

involved 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 the 

appropriate fish is critical. Live moribund fish are

always preferable to dead fish, fresh dead is preferred

to a fish that has been dead for several hours, and a 

dead fish several hours old is preferable to one that

has been frozen. A good pathologist will always

select his or her own fish to examine because for the

uninitiated, 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, then

fish should be caught from areas where sick fish

congregate. 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 to

sample six fish per group. The number of fish

sampled may be as few as four, if the cause of

disease is obvious, or as many as 400, if there is need

for a large number of different diagnostic assays.

Moribund WSB are euthanized with MS222

(tricaine methane sulfonate). Total length (TL) and

standard 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 gross

external exam is the next step and involves careful

assessment of the skin, fins, gills, pseudobranch, oral

cavity, tongue, and eyes. The skin and fins are

checked for irregular pigmentation, erosion, and

ulcers. The gills and pseudobranch are assessed for

color, filament damage, and parasites. The oral

cavity and tongue are checked for parasites, lesions,

and deformities. Deformities of the maxilla and

mandible are relatively common among some groups

of WSB. The eyes are evaluated primarily for 

evidence of emphysema associated with exposure to

gas supersaturation. Following the gross external

exam, scrapings are taken from the gills and skin for

cytological assessment of parasite and bacterial loads.

For the gross internal exam, the fish is placed in

right lateral recumbency (i.e., right side down, head

to the left). The entire left abdominal body wall is

removed 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 >12

cm TL can be sexed grossly. After sex is determined,

the heart, left liver lobe, pyloric cecae, stomach, and

swim bladder are examined. The left liver lobe and

stomach are then reflected ventrally to expose the 

spleen, intestinal tract, right liver lobe, gall bladder,

and endocrine pancreas. The last step is to pull the 

swim bladder away from the spine and to examine 

the head, trunk, and tail kidney.

For the majority of cultured WSB, the brain and

otoliths are not examined during routine health

checks. Brains are dissected and sampled only when

hatchery fish exhibit neurologic signs, have suffered

cranial trauma, or when viral nervous necrosis

(VNN) is suspected. For wild WSB, otoliths are 

routinely 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 not

deep 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 are 

located in the semicircular canals located below the

brainstem.

Sample Collection, Storage, Transport, and

Analyses

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, ensures

that the maximum amount of information will be

obtained.

Cytology. Wet mount cytology is used for the 

diagnosis of many common bacterial and parasitic 

infections in WSB. Cytological samples include gill

and skin scrapings and occasional squash

preparations used to assess nodular masses. Gill and

skin scrapings are collected using a dull scalpel to

minimize 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 15

the first or second gill arch. With skin samples, the

base 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 are

transferred onto glass slides and spread into a thin

layer by applying a glass coverslip. Coverslipped

samples are examined immediately via dark field

light microscopy for parasites and bacteria.

The usefulness of the exam is dependent on how

long the fish has been dead, when the sample was

taken, and the thickness (between the slide and

coverslip) of the sample. Ideally, samples are taken

as soon as possible after the fish has died and

examined as soon as possible after the slide has been

prepared. If a fish has been dead for too long, and

the 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 to

determine etiologies of ocular infections. Squash

preps are made by excising a small (1 mm3) piece of

the 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 to

adding 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 and

coverslip). Thin preparations allow clear 

visualization of bacteria and smaller protozoa.

Thicker preparations (e.g., those with large scales or

particulates) 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 or 

desired. 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.

Histology is also a faster and cheaper diagnostic 

alternative for some viral infections (e.g., VNN) and

rickettsial pathogens (e.g., P. salmonis).

For histology to be effective, a key factor is to

obtain samples as quickly as possible after euthanasia 

or natural death. Autolysis, from bacterial or host

enzymes, and bile, will rapidly degrade organs and

tissues, 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 normal

architecture. Samples from larger parenchymal

organs are cut with Teflon-coated razor blades to a

thickness of 1 cm (or thinner) to ensure rapid and

thorough 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, the

abdominal 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 used

with smaller larvae to prevent fish from slipping

through slots in cassettes and being lost during

processing. Embedded tissues are sectioned at 5–7

μm; sections are mounted on glass slides and stained

with hematoxylin and eosin (HE). All HE slides are 

evaluated in house by the OREHP fish pathologist.

Slides are assessed to determine which organs are

affected, number and types of lesions, lesion severity,

and presence of microbial pathogens. If etiologic 

agents are not readily apparent, special stains may be

ordered and HE slides may be sent out for review by

other fish pathologists.

Electron microscopy. EM is used to confirm the

identity 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, and

immersion 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 from

either formalin-fixed material or from paraffin

histology blocks. Formalin-fixed material is

transferred to Karnovsky’s, while paraffin-processed

material is excised from blocks using scalpel blades,
16 Okihiro

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 the

presence of viral particles in the nucleus or 

cytoplasm. For nonviral pathogens, lesions are

scanned to locate, identify, and characterize 

rickettsial or protozoan parasites.

Samples for direct EM examination are used to

identify gastrointestinal (GI) parasites that are shed

into the lumen of the GI tract. Direct EM is

specifically used by OREHP to try and identify a 

suspect herpes viral pathogen. Direct EM samples

are taken from pyloric cecae and intestine; the 

stomach is avoided to prevent sample degradation

from gastric acids. The gall bladder (closely

associated with the intestine in WSB) is similarly

avoided to prevent damage from bile salts. Contents

of 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 viral

particles.

Microbiology. Microbiology includes the fields of

mycology, 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 made

via cytology, but occasionally, fungal colonies will

be isolated on Sabouraud-dextrose agar. Major

bacterial pathogens of WSB are Vibrio spp. and

Flexibacter 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 and

brain.

Samples are ideally taken from freshly

euthanized fish, but can be taken from euthanized or 

dead fish that have been stored at 4°C (or on ice) for

up to 12 hours. Sterile technique, employing flame 

sterilized metal loops or prepackaged sterile plastic

loops, is used to take initial samples. Flexibacter is

cultured 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 have

been 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 all

liver 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 a 

suspect herpes virus that has only been identified

using TEM. Samples for virus isolation (VI) assays

include: eye and brain (for VNNV), spleen and

kidney (for VHSV) and pyloric cecae and intestine 

(for herpes virus). The liver has also occasionally

been sampled for VI. With liver, the left lobe is

collected (the right lobe and associated gall bladder 

are not sampled). Splenic samples are taken by

freeing 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 dissected

by grasping conjuctival tissues with forceps, applying

traction and using scissors to cut periocular

connective 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 Davis

on ice. VNNV is cultured on a snakehead fish cell

line (SNN-1), while VHSV is cultured on a carp cell

line (EPC). Both VNNV and VHSV have 

characteristic cytopathic effect when grown on the

appropriate cells lines. Positive cultures are

confirmed with either PCR or TEM. Attempts have

been made to culture the herpes virus using several

fish cell lines, but have to date been unsuccessful.
Okihiro 17

Enzyme-linked immunosorbent assay. ELISAs

have been developed by Dr. Ronald Hedrick’s

laboratory at UC Davis to assess pathogen exposure

in WSB to three major pathogens: VNNV, VHSV,

and P. salmonis. ELISAs work by detecting

antibodies specific for the three pathogens in

circulating blood. Although ELISAs detect exposure

and 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 to

the anal fin, to access the large vertebral vein located

ventral 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 collected

from 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 Davis

for final ELISA assessment.

ELISAs are layered assays conducted in

multiwell plastic plates. The initial antigen layer is

derived from viral or rickettsial pathogens grown in

cell culture. The second layer is serum from wild or 

cultured WSB that has varying concentrations of 

antibodies 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 fourth

layer is a horseradish peroxidase (HRP) streptavidin,

which binds selectively to biotin on the rabbit

antibodies. The final layer is tetramethylbenzidine, a 

substrate specific for the HRP enzyme. Positive 

samples change color from yellow to blue. Degree of 

optical density (OD) is determined by an automated

plate reader. Degree of OD is dependent on the

amount 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 

controlled laboratory exposures or from confirmed

disease outbreaks among hatchery fish.

Polymerase chain reaction. PCR assays have been

developed 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 specific 

primers 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 viral

diseases 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 of

juvenile 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 a 

transient, benign infection of the gills by an

Epitheliocystis sp.; the other, piscirickettsiosis, is a 

lethal 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 gills

of 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 probable 
18 Okihiro

myxosporidian parasite commonly found in

mesonephric 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 most

part, untreatable. As such, these three diseases pose 

the greatest challenge to OREHP, both from a

hatchery production standpoint and from a disease 

risk standpoint to wild fish stocks.

Viral nervous necrosis. VNN is a lethal nodaviral

disease of larval WSB (Curtis et al. 2001). Prior to

the spring of 2003, VNN epizootics were a regular 

occurrence at the Carlsbad Hatchery. Since 

installation of an ozone water treatment system at the

hatchery 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 have

characteristic necrotizing and cystic lesions.

Presumptive diagnosis is made via observation of 

larval fish with classic clinical signs. Confirmation

of 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 (<4

months) hatchery seabass exposed to ambient lagoon

waters during fall and winter months. There have 

been two confirmed outbreaks and both occurred at

the Carlsbad Hatchery: one in the fall of 2002, the 

second in the fall of 2005. Mortality can exceed

3,000/day if HVGE occurs simultaneous with

Flexibacter. Typical clinical signs (when

uncomplicated with Flexibacter) are: 1) high

mortality among fish with no cutaneous lesions, 2)

yellow-white stringy fecal casts in the water, and 3)

terminal spiraling prior to death. Target organs are 

stomach, 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 is

multifocal necrosis of mucosal epithelium of pyloric

cecae and intestine, with minimal inflammation.

Some epithelial cells may have marginated chromatin

and an absence of nucleoli. The diagnosis is

confirmed with TEM. There is no PCR assay

available and the virus has not been successfully

grown 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 have 

occurred twice since the hatchery opened. The first

outbreak 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 the 

heart, and multifocal, pale tan to yellow, nodular

masses in the liver. The diagnosis can be confirmed

with 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 with

the 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 high

percentage 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 to

humans, surviving stocks would either be sold or 

eaten. But since this is an enhancement program,

OREHP has to consider the relative risk of disease 

transfer to the wild fish stocks they are seeking to

help.

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 19

which lethal infectious diseases are naturally

occurring or endemic to wild stocks. The rationale is

that if a disease is endemic among wild fish, then the

relative 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), and

cowcod 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, 43

vermillion 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 and

100+ 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 have

been 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 from

wild WSB have failed. A small number of PCR

samples have been assessed and these have also

turned up negative for virulent pathogens known to

occur in cultured WSB. Results from the majority of 

PCR samples, however, are still pending. Of the 

more than 100 intestinal content samples taken, 50

have been analyzed via direct EM for herpes virus. A

single direct EM sample had suspect hexagonal

profiles, 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 fish

survey lead to three conclusions. First, VNNV is

probably 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 Hatchery 

Operations

The impact wild fish disease survey data have on

hatchery operations is that OREHP can now make

informed decisions regarding what to do when

virulent disease outbreaks occur. With VNNV

epizootics, the hatchery can reasonably decide to

salvage survivors and release them at a later date 

when they are healthy. In contrast, with P. salmonis,

the assumption has to be that this disease does not

occur among wild WSB, and that any cultured

seabass infected or exposed to this pathogen should

be euthanized. With herpes and VHSV, there still is

not enough information to determine whether or not

these 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 is

definitive proof, OREHP policy will be to operate as

if 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 and

experimental 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-like

bacterium 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 of

juvenile white seabass, Atractoscion nobilis,cultured in

southern 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 guide

to Pacific Coast fishes of North America, p. 218–222.

Houghton Mifflin Company, Boston, Massachusetts.
20 Okihiro

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 viral

hemorrhagic septicemia virus. Dis. Aquat. Organ. 55:221–

220.
Nakagawa 21

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, Japan

mnakagaw@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 the 

cultivation 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 to

fluctuate 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 early

mortality.

Introduction

Juveniles of six rockfish species (Sebastes

schlegeli, S. inermis, S.s pachycephalus, S.s vulpes, S.

thompsoni and S. oblongus) are currently produced in

Japan. 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

(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 juveniles

of the species in Hokkaido, Akita, Yamagata, Miyagi,

Hiroshima, Fukushima, Niigata and Ehime 
22 Nakagawa

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. Development

of the juvenile production technique at Miyako

Station in recent years has therefore been aimed at

reducing the unit cost of juvenile production. On the

other 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 of

juvenile production at Miyako Station.

Subsequently, we describe the tasks that remain to be 

accomplished.

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.

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 and

deterioration of rearing water were feared because

fish eggs were used as food.

Second period (1994–1998). An automatic 

feeding machine was developed that was capable of 

dispensing a formula feed consisting of minute 

particles (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 thought

possible 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 and

Artemia 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 and

Artemia 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 of

rearing was 12.4 mm ±1.1 mm in Group I larvae and

12.6 mm ±0.7 mm in Group II. No significant

difference (p > 0.05) was found between the two

groups. The average survival rates were 65.1% in

Group I and 64.9% in Group II. Therefore, two types

of 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

For juvenile production, it is most important to

ensure that few individual larvae are deformed or 

swim with marked instability. The larvae were

quantified, and healthy larvae were transferred using

a 50 mm diameter siphon hose to 50,000 L square

concrete 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 heat

from a boiler, a ventilation device with a blower, an

automatic 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 was

controlled at a tank temperature of 22°C and

eggshells and Artemia nauplii were separated 42 h

after hatching. Artemia nauplii were enriched for 24

h at 20°C by addition of nutrients (Plus-Aqualan,
Nakagawa 23

BASF Japan Ltd.) at a density of 50 g/1,000 L. One 

liter of freshwater chlorella concentrate (Chlorella

sp., 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 water

exchange 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 and

nutrient enrichment of Artemia nauplii. The tank

floors were cleaned using an automatic bottom 

vacuum cleaner every day and dead larvae were

counted at that time. The total length of the larvae or

juveniles was measured every five DAB, and

specimens (fixed in 10% formalin) were collected to

determine their developmental stages.

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 for

each 1,000 L of rearing tank (hereafter called the unit

production number) because the number of tanks

used varied among years. As indicated in the 

classification above, 1982–1993 was defined as the 

first 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 were

compared using Scheffé’s F certification method.

Results showed no significant differences among the

periods in the unit production numbers of juveniles

(p > 0.05), but the total lengths of juveniles at the end

of production and the rearing times differed

significantly between the first and third periods

(p < 0.05). An increase in mortality rates as a result

of 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 the 

harvest, which was intended to raise the survival rate 

after harvesting. Survival rates were converted into

variables and compared using Scheffé’s F

certification method, but no significant differences

were 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 in

juvenile production and improvement in production

efficiency became possible through a reduction in the

variety and type of feed required for juvenile 

production.

Table 2. Results of mass production breeding experiments in

the black rockfish, Sebastes schlegeli.

Growth and Development

Figure 1 shows the growth of larvae and

juveniles reared in 50,000 L tanks. The equation Y=

5.6609e

0.184x (r2 = 0.9967) represents the relationship

between the total length and DAB. Variations

became greater as rearing progressed.

Table 3 shows results of observations of the 

developmental stages of larvae and juveniles reared

in 50,000 L tanks. Larval development was

classified into seven stages according to the method

of 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 in

which 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 was

considered 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 the 

growth of the larvae and their delivery, but its
24 Nakagawa

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 deviation

of total length.

Table 3. Developmental-stage composition (%) in larval and

early 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 were 

observed 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 the

mortality rate were observed in larvae and juveniles

from 0–10 DAB, 18–22 DAB and 30–35 DAB, a

pattern that was common to all years. It is

noteworthy 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 the 

average 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 ±0.2

mm, 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 dead

fish reached developmental stage D or E, with no

significant difference in average total length.

Numerous food organisms were found in the 

digestive 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 that

was far more inflated than those of normal fish,

implying death from gas-balance disorder. Aerating

the seawater immediately before it is poured into the 

rearing tanks minimizes mortality. Mortalities of 

larvae from 30 to 35 DAB occurred from cannibalism 

that was attributed to differences in growth rates.

Unit Cost of Juveniles

Based on rearing data for 2003, we calculated the

unit 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 of

broodstock rearing and juvenile production by the

number of fish produced. That result was 8.5 yen.

Itemization revealed that wages accounted for 39.6%
Nakagawa 25

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 for

intermediate rearing as indicated by Nakagawa and

Okouchi (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. Each

day, more than 1 million newborn larvae of multiplespawning fish such as Japanese flounder are now

obtainable on a stable basis, thanks to advances in the 

techniques used for broodstock rearing (Thuzaki

1994). However, the black rockfish, which is

viviparous, 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 been

difficult for most juvenile production agencies to

obtain larvae of the species in large quantities at one 

time. Using a cannulation method, Yamada and

Kusakari (1991) developed a technique to estimate

the birth dates of larvae from examination of 

embryonic developmental stage. Using the same 

technique, Nakagawa and Okouchi (in press) have 

implemented a method of selecting female

broodstock estimated to be near spawning and

thereby obtaining larvae intensively. Broodstock of

this species mate in November of each year (Mori et

al. 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 the

cannulation method to select broodstock that

discharge unfertilized eggs. Consequently, the 

technique for obtaining larvae intensively is nearly

complete.

As noted here, however, many individuals among

larvae 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 early

mortality 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].
26 Nakagawa

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 dehulled

soybean meal as a fish meal replacer in diets for fingerling

and 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 of

density and feeding frequency on the intermediate culture

of 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 [in

Japanese with English abstract].

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 specific

seasonal 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 of

embryonic developmental stage by cannulation to

synchronize 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 Japanese

flounder. 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 time

course of embryonic development in kurosoi, Sebastes

schlegeli. Env. Biol. Fish. 30:103–110.
Okamoto 27

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 and 

genomic (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-specific 

genes. 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)

(390Mb), and one-half of the zebrafish (Danio rerio) 

(1.6Gb) genomes (Uwa and Iwata 1981, Brenner et

al. 1993, Ojima and Yamamoto 1990, Ciudad et al.

2002). Therefore, flounder is a good model to

understand 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
28 Okamato

DNA, can facilitate rapid construction of a physical

map 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 by

positional 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 get

complete 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 a

heterozygosity 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 were

mated one-to-one in June 2003, and whose offspring

were reared until January 2004. For constructing the 

cosmid 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 their 

offspring 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 body

lengths of 13–50 mm and from 34 to 72 days after 

hatching. A preliminary check of the sex ratio of the 

offspring was performed in December 2003 by

observing the gonads of the fish. The final check was

performed in January 2004.

Freezing and defrosting of sperm. Semen from the

TY-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 at

4°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

30°C in a block incubator. The tube of frozen sperm 

was placed in the block incubator, and 1 ml of 

preheated Ringers solution was added. The tube was

gently 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 and

gently shaken again to initiate sperm dispersal

without using a pipette. After removing the 

aggregates from the thawed sperm solution, we 

constructed 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 and

cosmid library. The BAC library was constructed

by Geno Tech Co. (Tsukuba, Japan) using the

agarose gel plugs from the frozen sperm of the TY-4

flounder and standard methods. The high molecular 

weight DNA in the agarose plug was partially

digested 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 the 

frozen 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 gel

electrophoresis (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, we

used five one-to-one mating pairs in June 2003, and

checked the sex ratio of each offspring 7 months after

fertilization. Before checking the sex ratio, the 

offspring were treated with mild estradiol as

previously described. The estradiol treatment did not

influence the XY fish to be male and repressed the 

XX fish from transforming to male because of water

temperature. According to the data obtained from 

checking the sex ratio of the fish two times, the 

male: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 male 

fish, and proceeded to make a BAC library from the

frozen sperm of TY-4. Because the mating season of

flounder is only during spring, we stored the sperm 

until we could determine the sex ratio of the offspring
Okamoto 29

several months later. We froze the sperm of the five

paternal fish in June 2003. After checking the sex

ratio of the offspring, we prepared agarose plugs for 

constructing a BAC library, and placed an order for a

library 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), and

the genomic size of Japanese flounder 

(approximately 700Mb) (Ojima and Yamamoto

1990), 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-third

of 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 or 

cell culture of the library directly. This PCR 

screening was performed by the primer sets from the

cDNA 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 their

sequences, and also analyze the synteny in the MHC

regions between Japanese flounder and another fish

species.

Figure 2. Analysis of insert size of BAC clones of

heterozygous Japanese flounder. (A) NotI-digested BAC

DNAs and vector arm on PFGE. (B) Insert size distribution

of 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 at

least one BAC clone for all 24 linkage markers (data 

not shown). The results showed that freezing the

sperm 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 the 

frozen sperm of anti-Edwardsiella XX homozygous

clones of Japanese flounder. Edwardsiella tarda

causes an infectious disease in marine fish, and

severely damages the production of aquaculture. The

anti-Edwardsiella fish have been selected after the

challenge 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 the

insert 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, the 

maximum insert size was 48 kb, and the minimum

insert size was 33 kb (Figure 3B). The cosmid
30 Okamato

library consisted of 110,592 clones arrayed in 288

microtiter plates with 384 wells. From the average

insert size of the library, the total number of clones

and the genomic size of Japanese flounder, the

analyses 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.

Figure 3. Analysis of insert size of cosmid clones of AntiEdwardsiella XX clone fish. (A) NotI-digested cosmid DNAs

and vector arm on PFGE. The vector size was estimated to

be 7.4 kb. (B) Insert size distribution of the representative

cosmid clones. The average, maximum, and minimum size of

the insert DNA are 39.5, 48, and 33 kb, respectively.

These genomic libraries are stored in the 

National Research Institute of Aquaculture or

National Research Institute of Fisheries Science of 

Japan, and are available upon request for research

purposes.

Acknowledgments

The authors thank the Hyogo Prefectural

Fisheries Experimental Station for providing the 

Japanese flounder XX clone fish. We also thank Drs.

Masanori Okauchi, Takanori Kobayashi, and

Kagayaki Morishima for providing technical support

and Tomoyo Nozaki and Keiko Ikeyama for 

laboratory assistance. This research was supported

by a grant from the Genomic Breeding Project by the

Fisheries Research Agency of Japan.

References

Brenner, S., G. Elgar, R. Sandford, A. Macrae, B.

Venkatesh, and S. Aparicio. 1993. Characterization of the

pufferfish (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 the

Japanese 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 DNA

contents of G0/G1 diploid cells from three different teleost

fish 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.

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 at

www.ncgr.org/research/jag.

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.

Uwa, H., and A. Iwata. 1981. Karyotype and cellular

DNA content of Oryzias javanicus (Oryziatidae, Pisces).

Chromosome Information Service 31:24–26.
Nikaido 31

Status of Pacific Bluefin Tuna (Thunnus orientalis) Seed Production 

in 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 Japan

nikaidoh@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 in

rearing about 12,000 juveniles (average = 47 mm total length [TL] in 1998). However, the survival rate from

hatching 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 tank 

bottom during the night and subsequently die, but the cause remains unclear. We circulated the water by

continuous 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 cherished

worldwide; 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 fishery

resource. The bluefin tuna is also an international

fishery resource for other countries. For these 

reasons, management of bluefin has been entrusted

to international committees. Japan’s government,

which 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
32 Nikaido

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 associated

with the problems of 10 DAH survival rate and

cannibalism 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 water 

continuously using a current generator to prevent

larvae from sinking. We produced currents using a

pump 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 to

improvement 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; some

were collected from broodstock at Amami Station.

A polycarbonate Artemia hatching tank (200 L) was

used. We sterilized fertilized eggs using residual

oxidants (0.3 mg/L) in seawater before placement in

the hatching tank. The eggs were hatched in

oxidant-sterilized seawater.

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 method

that allowed continuous rotation using two devices.

One used a pump, the other used aeration pipes that

are usually used for rearing fish.

Pump system. The pump system (Figure 1) is made 

of one pump put in a strainer in the center of the

rearing tank and crossed plastic pipes with holes

(1.0 mm) at 10 cm intervals. Plastic pipes were

placed on the bottom of the rearing tank, and then

turned upward at 40–50° angles. The rearing

seawater spouts were formed from holes in the

plastic pipe, producing an upward current. We used

air stones for aeration.

Figure 1. Photo of pump system. The small arrows show the

directions of flow of sea water and the larger arrows show the

current 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 bottom 

side of the wall of the rearing tank to produce an

upward current by aeration. We did not attach air 

stones.

Figure 2. Photo of aeration the aeration system. The small

arrows show air flow directions and the larger arrows show the

current direction.

Measurement of current direction and speed. We 

divided the rearing tank bottom into 16 areas and

measured 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 tank

bottom. We used an electromagnetic current meter 

(ACM-300A, Alec Electronics Co. Ltd.) for 

measurements.
Nikaido 33

Larval distribution. We measured the distribution

density of larvae at specific points within the tank.

Depths measurement points were 30 cm, 50 cm, 1

m, 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 calculated

using 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 the

pump system and twice using the aeration pipe 

system (Table 1). We compared results using

fertilized eggs of identical origin with rearing case2R.

Table 1.

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 (Figure 

3). Particularly noteworthy is that the 62.4% result

of 2R-3 became the highest result ever recorded for

rearing at Amami Station. The 10 DAH survival

rate ranged from 20.5% to 62.4% for the pump

system in all rearing cases including when we used

fertilized eggs of different origin. This result was

considerably higher than the 5.5% and 6.3% results

achieved by the aeration pipe system (Figure 4).

Figure 3. Comparison between survival rates of bluefin larvae

using two ways of generating water currents. (All eggs were

from the same origin.)

Figure 4. Comparison between survival rates of bluefin larvae

using two ways of generating water currents in tanks. (Origin

of eggs was not the same in all cases.)

Measurement of current direction and speed. We 

measured the direction and speed of the current in

rearing water (Figure 5). Results for the pump system

showed a tendency for a strong upward current from

the bottom of the rearing tank. The current speed was

about 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 the

current 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 the 

surface to 1.5 m depth. Larvae were present at the 2 m 

depth, but distribution density was reduced. Larvae 

were almost absent at the bottom of the rearing tank

using the pump system. At 2100 hours, the larvae

were uniformly distributed from the surface to 2.0 m 
34 Nikaido

depth and were rare at the rearing tank bottom 

(Figure 6).

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 and

2100 hours were almost identical from the surface

to 50 cm depth. Few larvae were found at the

bottom 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 tank

bottom 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 was

low (Figure 7).

Discussion

Regarding prevention of the low 10 DAH

survival rate, Tezuka et al. (2005) examined the 

effects of light on the surface and current of rearing

seawater from 1999 to 2001. Results showed the

necessity 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 average

illuminance over 800 lux, as Tezuka et al. (2005) 

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

DAH.

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

reported, by taking natural light from the top of the 

rearing tank. Beginning in 2004, we examined the 

effects and production methods for providing a current

in the rearing tanks. We succeeded in preventing

bluefin larvae from sinking to the tank bottom by

producing 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 prevent

the bluefin larvae from sinking to the bottom of the 
Nikaido 35

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 the 

rearing tank and die at night in most cases. This

phenomenon 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 determined

that 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 a 

current.

We prevented the VNN problem and solved the

low 10 DAH survival rate, thereby finding solutions

to two of the three salient problems inherent in

rearing 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 the 

feeding protocol to determine if there is a way to

mitigate growth differences of larvae and thereby

control cannibalism at TL 7 mm. It appears

possible to delay cannibalism by delaying the 

rotifer period, providing the bluefin larvae with

larval 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 seed

production techniques, other measures of mass and

healthy seed production techniques, and secure 

genetic diversity, which will be described in future 

reports.

Acknowledgments

Special appreciation is extended to Takuyo Co.,

Ltd. and the Amami laboratory of Kinki University for

providing 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 in

seawater 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 on

survival 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 and

Technology for Bluefin Tuna and Other Cultivate Fish, in

2003–2004, p. 113–116. Fisheries Laboratory & Graduate

School of Agriculture, Kinki University.

 

 

 

Uji 37

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, Japan

uji@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 or

measurement 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 for 

fish 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 and 

histochemical 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 the 

nervous 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 can 

work efficiently to solve malformation problems.

Introduction

A considerable number of studies over many

years have examined wild and reared fish

abnormalities (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 growth

depression and high mortality rates (BarahonaFernandes 1982, Andrades et al. 1996). Their

incidence engenders unnecessary expenditures of

resources, 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 of 

abnormalities remain poorly understood. Genetic,

environmental, and nutritional causes have been

suggested as possible sources of abnormalities. For

example, genetic factors have been reported to cause 

developmental abnormalities (Kincaid 1976, Gjerde

et al. 2005). Environmental factors such as

mechanical 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 these

abnormalities (Cahu et al. 2003). Although many

studies exist, most examine environmental and

nutritional factors, especially at the juvenile stage.

Almost none address maternal factors at early

developmental stages.

This study specifically examines this early

stage—especially maternal factors—until the fish

have a first feeding. Previous studies have shown

that 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 the 

early stages of organogenesis, when organs such as

limbs, eyes, and gills develop (Gilbert 2003).
38 Uji

Consequently, aberrant development during that

period creates living but abnormal fish. During

organogenesis, 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 to

assess 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, we

developed visualization tools to probe for early stage 

abnormalities.

This paper represents a compilation of antibody

and histochemical staining methods we used to

visualize tissues of various fish species. We also

propose a concept to facilitate solving malformation

problems.

Antibody Staining Methods

Embryos are extremely small and are too

transparent to allow checking for abnormalities. For 

those reasons, we can obtain little information about

abnormalities merely by using microscopy. To date,

assessment of abnormalities has been conducted in

most hatcheries at later stages, such as the larval or 

juvenile stages.

Visualization tools are needed to detect

abnormalities at earlier stages such as the embryo or

early larval stages. Visualization tools must also be 

readily available for many kinds of fishes. Antibody

and histochemical staining methods meet those

requirements. Using such methods, we can visualize 

their structural information. These methods are

useful for many kinds of fishes. Moreover, we can

deal with numerous fish simultaneously.

The availability of antibodies and histochemical

staining methods against specific cell lineages

facilitates the identification and localization of 

different organs and cells among species (Table 1).

Furthermore, because of their small size, whole fish

histology can be performed to examine for

abnormalities.

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

Table 1. Staining method for analysis of abnormal

development in fish.

(Fugu rubripes), zebrafish, (Danio rerio), Japanese 

eel, (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 are

present exclusively in neuronal cells and are therefore 

useful as markers of neuronal cells in tissues. Some

abnormal fish phenotypes appear to resemble human

neurocristopathies. Neurocristopathies are neuralcrest-cell diseases in humans. Neural crest cells

originate at the dorsal-most regions of the neural

tube, migrate extensively, and eventually give rise to

a remarkable variety of cell types, including neurons

and glial cells, cartilage, pigment cells, and

chromaffin cells. Anti-HNK-1 monoclonal antibody

recognizes a carbohydrate epitope on subpopulations

of 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 of 

certain amino acids and sugars, and maintenance of

membrane excitability. This method enables the

visualization 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 that
Uji 39

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; and

d, anti-acetylated tubulin antibody reacts in Japanese eel.

Histochemical Staining Methods

The histochemical staining method is more

useful to assess abnormalities for many kinds of

fishes. 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 fish

abnormalities. Calcein is a fluorescent chromophore

that 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 in

living embryos.

The alkaline phosphatase staining method

visualizes the intestine. Fish exhibit high levels of 

endogenous 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 we 

can see only the intestine and osteoblasts, which have 

high levels of endogenous alkaline phosphatase at the 

base of the tooth. That limitation notwithstanding,

the method is sufficient because it allows inspection

of 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 in

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 fish

in early developmental stages. These factors are 

classifiable 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 approach

these 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 when

fish are sufficiently large to verify their shape; at this

stage, it remains unclear whether fish that are being

raised are normal or abnormal. As explained earlier,

it is very inefficient to raise abnormal fish. Some 

judgment can be made earlier if the above hypothesis

is correct. Figure 2 shows a simplified scheme of an

early batch checking strategy with fish embryos.

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, and

the cost of this checking procedure is low. If

development could be judged earlier, we could select

good populations to raise, thereby eliminating the 

waste of resources, money, and time in raising

abnormal fish to a sufficient size to allow

examination. Environmental factors could also be 

assessed, such as mechanical or thermal shocks,

salinity, and oxygen depletion, in terms of their

effects on normal organ development of fish using

these visualization tools. The effects of drugs and

chemicals on growth and development could be 

assessed in detail by examining the size and

morphology of internal organs such as the brain,
40 Uji

muscle, intestines, and kidneys using these

visualization tools.

In summary, this study revealed that many

antibody and histochemical staining methods are 

useful for visualization of many, perhaps nearly all,

fishes. This paper also presented a new concept to

resolve malformation problems.

Acknowledgments

We thank Tohru Suzuki, Hisashi Hashimoto, and

Keiichi 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 and

Douglas M. Fambrough, respectively, were obtained

from the Developmental Studies Hybridoma Bank

developed under the auspices of the National Institute 

of Child Health and Human Development and

maintained 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 adult

stages 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 in

hatchery reared European sea bass Dicentrarchus labrax:

Types, prevalence, and effect on fish survival. J. Fish

Biol. 21:239–249.

Boglione, C., F. Gagliardi, M. Scardi, and S. Cataudella.

2000. Skeletal descriptors and quality assessment in larvae

and post-larvae of wild-caught and hatchery-reared

gilthead sea bream (Sparus aurata). Aquaculture 192:1–

22.

Cahu, C., J. Z. Infante, and T. Takeuchi. 2003.

Nutritional components affecting skeletal development in

fish 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 of

fishes. Gulf Res. Rep. 1:308–399.

Dawson, C. E. 1966. A bibliography of anomalies of

fishes: Supplement 1. Gulf Res. Rep. 2:169–176.

Dawson, C. E. 1971a. A bibliography of anomalies of

fishes: Supplement 2. Gulf Res. Rep. 3:215–239.

Dawson, C. E. 1971b. A bibliography of anomalies of

fishes: 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 effects

of salinity on embryonic and larval development of

Eleginus 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 in

zebrafish embryos using the fluorescent chromophore

calcein. Dev. Biol. 238:239–246.

Fraser, M. R., and R. de Nys. 2005. The morphology and

occurrence 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 in

broodstock diet on larval and egg quality of Japanese

flounder Paralichthys olivaceus. Aquaculture 220:725–

735.
Uji 41

Garside, E. T. 1959. Some effects of oxygen in relation to

temperature on the development of lake trout embryos.

Can. J. Zool. 37:689–698.

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.

Neurol. 476:240–253.

Kincaid, H. L. 1976. Inbreeding in rainbow trout (Salmo

gairdneri). J. Fish. Res. Board Can. 33:2420–2426.

Kjørsvik, E., A. Mangor-Jensen, and I. Holmefjord. 1990.

Egg quality in fishes. Adv. Mar. Biol. 26:71–113.

Komada, N. 1980. Incidence of gross malformations and

the vertebral anomalies of the natural and hatchery

Plecoglossus altivelis. Copeia 1:29–35.

Koumoundouros, G., G. Oran, P. Divanach, S. Stefanakis,

and M. Kentouri. 1997. The opercular complex deformity

in intensive gilthead sea bream (Sparus aurata)

larviculture: Moment of apparition and description.

Aquaculture 156:165–177.

Lebovitz, R. M., K. Takeyasu, and D. M. Fambrough.

1989. Molecular characterization and expression of the

(Na+ + K+)-ATPase alpha-subunit in Drosophila

melanogaster. Embo. J. 8:193–202.

Lee, C. S. and B. Menu. 1981. Effects of salinity on egg

development and hatching in grey mullet (Mugil

cephalus). J. Fish Biol. 19:179–188.

Moore, K., and T. V. N. Persaud. 1998. Before we are

born: Essentials of embryology and birth defects. 4th

edition. WB Saunders Co., Philadelphia, Pennsylvania.

Noden, D. M. and A. D. Lahunta. 1985. Embryology of

domestic animals: Developmental mechanisms and

malformations. Williams & Wilkins Co., Baltimore,

Maryland.

Serbedzija, G. N., E. Flynn, and C. E. Willett. 1999.

Zebrafish angiogenesis: A new model for drug screening.

Angiogen. 3:353–359.

Suzuki, T., Y. Haga, T. Takeuchi, S. Uji, H. Hashimoto,

and T. Kurokawa. 2003. Differentiation of chondrocytes

and scleroblasts during dorsal fin skeletogenesis in

flounder larvae. Dev. Growth Differ. 45:435–448.

Malone 43

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 the 

conditioning 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 pure 

oxygen 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 to

meet core recirculating needs. Use of airlifts allows the centralization of utilities without the potential of

cross contamination of waters from diverse tank systems. The PolyGeyser filters are pneumatically washed

in 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 the

commercial 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 important

role in conditioning broodstock, supporting larval

stages, 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 and

fingerlings that must be held for extended periods of

time. 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 hatchery

systems. Recirculating systems will have to be

designed with a relatively high hydraulic residence 

time, 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 is

compatible 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 systematic 

engineering 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 and

fingerling growout systems in marine hatcheries.

Table 1 presents selected water parameters, the levels

of which characterize water quality expectations for 

these hatchery systems. Establishment of these water

quality expectations allows the formulation of 

treatment packages that will meet the operator’s

performance expectations. The vast majority of

marine production needs can be met within the 

guidelines for oligotrophic and mesotrophic systems;

however, there is some encroachment on the 

eutrophic categories.
44 Malone

Figure 1. Malone and Pfeiffer (2005) identified 17 distinct

types of recirculating systems.

Table 1. Presumed water quality targets for warm water and

cold water marine hatchery systems.

Marine broodstock maturation systems are

generally designed to meet the highest water quality

expectations. These high standards reflect the high

value of the eggs produced by generally fecund

broodstock. Egg quality often reflects the health and

vigor of the broodstock.

Conditioning of broodstock often takes months

and few operations can risk loss of the time and

monetary investment associated with death of 

breeders 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), while 

secondary parameters (biochemical oxygen demand

[BOD] and total suspended solids [TSS]) are set at

levels 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 high

recirculation rates.

Fingerling production systems focus on the 

production of healthy fingerlings sized for release or

growout. The value of these fingerlings remains high

when measured on an individual weight basis and

thus justifies recirculating aquaculture systems (RAS) 

investment. Fingerlings are generally recognized as

sensitive to water quality, so toxic water quality

parameters are set at safe but manageable levels (<0.5

mg-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 the

detriment of nitrifiers (Ohashi et al. 1996, Ling and

Chen 2005).

Many marine fingerling production systems

approach densities and fingerling sizes that are more

typically associated with freshwater growout. Peak

TAN and nitrite levels are allowed to creep upwards

(<2.0 mg N/L), a strategy that benefits from pH

depression associated with rising carbon dioxide 

levels and the protective nature of chlorides on nitrite 

toxicity. Density is allowed to rise to 60 kg/m3while

the higher TAN targets lessen the importance of 

recirculation 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 with

elevated carbon dioxide can induce pH declines that

incapacitate nitrifying bacteria.

Current Treatment Strategies

Floating bead filters utilize a packed bed of

floating plastic beads. They display the inherent

ability to capture solids across a wide size spectrum.
Malone 45

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 bead

filters are able to provide for both fine and coarse

solids removal irrespective of the degree of water

reuse. 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 for 

simplification.

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 the 

application of pumped (circulation) floating bead

filters (clarification and biofiltration) with blown air 

(aeration and carbon dioxide stripping) to the 

categories 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 of 

complementing the bead filter with a large biofilter.

For example, a pumped (circulation) floating bead

filter (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 set

of designs are distinguished by the use of

microscreens for solids capture instead of a bead

filter. 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 cone 

with packed column carbon dioxide stripping

(Summerfelt and Sharrer 2004).

The unique demands of marine hatcheries are

stimulating the subtle shifts in system designs (Table

2). Central is the demand of pristine waters, a clear 

shift towards the oligotrophic compared to the 

demands for high carrying capacity emphasized in

the freshwater sector. Additionally, the heating and

cooling needs of the systems, environmental issues

associated with saltwater discharge, and, particularly,

biosecurity issues are encouraging more closure of 

systems. Biosecurity strategies lead to designs that

are more isolated and of smaller scale than

corresponding freshwater growout systems.

Table 2. Water treatment configurations must be adjusted to

meet the demands of marine hatchery systems.

It is becoming increasingly apparent that

biosecurity issues will force a rethinking of 

commercial marine hatchery configurations.

Biosecurity quickly renders liberal flushing of 

systems 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 of

biosecurity strategies seeking to provide protection of 

valuable broodstock and fingerlings from internal and

external disease vectors. Partitioning of large 

assemblies of tanks is dictated. Use of individual or 

small clusters of tanks facilitates treatment or, in the

worst case, limits losses when treatment is

impracticable. Partitioning also facilitates the 
46 Malone

manipulation of individual maturation tanks. Modern

biosecure hatcheries will consist of a large number of 

small isolated recirculating systems.

The engineering community must recognize the 

changing needs of the industry and focus on the 

development of treatment strategies appropriate for a

large number of dispersed systems. To that end, this

paper presents a bead filter/airlift combination that is

compatible 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 a

suitable 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 a 

fixed 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 the 

unit every few days after numerous backwashes have 

occurred. 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 a

triggering device that releases the captured air when

the charge chamber is filled. Then air bursts through

the bead bed, rapidly mixing it as it drops. Dirty

wash waters released by the bead bed rush into the 

charge chamber, filling it as the air escapes. The

bead bed reforms at the top as the filter refills and

water 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

Figure 3. The pneumatic charge chamber stores air to wash

the beads then acts as a settling basin recycling backwash

waters.

waters captured in the charge chamber have time to

settle out. A relatively clean stream of water is

slowly remixed with the circulating waters as the 

solids accumulate in the sludge basin. Sludge is

removed 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, and

thus, its biofiltration performance (Golz et al. 1999).

The PolyGeyser filter is normally packed with an

enhanced 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 a 

spherical bead bed without displaying excessive head

loss.

In the mid-1990s a number of studies confirmed

that 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 out

of his bubble-washed units at a backwash frequency

of five times a day. Finally, DeLosReyes et al.
Malone 47

(1997) found his unit performed the best with a 

backwash 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 core

of any recirculating system, three of them can be 

substantially achieved with the simple combination

of a piece of PVC pipe and an air blower. The

energy input that is normally reserved for the

circulating 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 efficient

in comparison to more traditional pumped

configurations.

More subtly, in contrast to centralized water 

pumps, use of airlift pumps allows the mechanical

source of power to be centralized without the 

corresponding 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 and

filter systems without the potential for cross

contamination. 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 the

industry 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 ratio

of the lift to the submergence, with flow delivery at a

fixed air input increasing dramatically as the lift

decreases. Given the constraints on depths of

injection, recirculating airlift pumps are most cost

effective 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 are 

controlled by changes in the rate of air injection (gas)

as it relates to the water flow (liquid). It is desirable 

from 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 both

oxygen 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 for 

water flow in recirculating applications. This work

concluded that airlifts should be designed to operate 

with submergence to lift ratios in the range of four to

five, 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 of

scour 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 screen
48 Malone

(or pipe) 25–28 cm below the tank water level. This

placement assures that there is sufficient water

pressure 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 a

variety of applications, the RAS airlifted PolyGeyser

tends to operate with a cumulative head loss from

tank to filter effluent in the range of 15–38 cm. This

low headloss range is compatible with airlifts that

have submergence depths in the range of 120–150

cm. Various engineering issues relating to the

PolyGeyser filter are presented in Table 3.

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 bead

filters. The table presents filter feed levels and

approximate 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 the 

lift tubes associated with the airlift recirculation are

provided. A dual lift tube option is also sized since

the use of two tubes facilitates energy conservation

during periods of light loading. Finally the air 

delivery pressure and flow are provided to allow for

selection of air pumps.

Table 4. These interim guidelines can be used as airlifted

PolyGeysers are introduced to support recirculating

aquaculture systems (metric version).

These criteria are applicable for a temperature 

range of 15–30°C. Application of these criteria under

peak loading outside that range should be undertaken

with caution. Below a temperature of about 10°C,

the overall nitrification capacity of the bioclarifier 

can be expected to decline. At these low

temperatures, systems failure will most probably be

indicated by chronic nitrite accumulation. Failure at

temperatures above 30°C are most likely to be first

evidenced by low dissolved oxygen levels in the

rearing 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 installation

levels to assure that the elevations for tank and filter

are properly selected and that secondary problems

such as filter flotation under high groundwater 

conditions are avoided (Table 4). The air delivery

requirements of an individual unit are 

straightforward. 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 to
Malone 49

assure that air distribution remains predictable.

Multiple airlifts serving a single PolyGeyser unit

should return discharge water just above the tank

waterline to avoid flow reversal between competing

airlifts.

There are a number of secondary processes that

can 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 be

enhanced by the addition of compatible technologies.

Addition of a temperature control loop facilitates

temperature manipulations used for condition in

broodstock. Moving bed reactors and rotating

biological filters are examples of two biological

filters 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.

Summary and Conclusions

The special needs of marine hatchery systems are

forcing engineers to reevaluate design strategies.

Although current design configurations are serving

the industry well, increasing biosecurity concerns

dictate greater isolation and encourage high reuse

designs that minimize exposure to external disease 

vectors. Internally, marine hatcheries benefit from

the 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 the 

operation of recirculating systems with high

hydraulic retention times. This feature also permits

the implementation of a high frequency backwashing

strategy that avoids headloss buildups in the bead

bed. 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

The authors acknowledge the contributions made

by commercial and other research and development

units toward the refinement of this strategy. Funding

for baseline studies was derived in past years from

the Louisiana and national Sea Grant College

programs. Commercial development was fostered by

a 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 for 

commercial 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 the

continued research efforts. Waddell Mariculture

Center in South Carolina formed an early test base
50 Malone

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 our

knowledge 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 with

application 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.

Loyless, J. C., and R. F. Malone. 1998. Evaluation of

airlift pump capabilities for water delivery, aeration, and

degasification for application to recirculating aquaculture

systems. Aquacult. Eng. 18:117–133.

Malone, R. F., and L. E. Beecher. 2000. Use of floating

bead filters to recondition recirculating waters in

warmwater aquaculture production systems. Aquacult.

Eng. 22:57–73.

Malone, R. F. 2002. Engineering for a responsible

aquaculture with focus on growout facilities for

commodity foodfish. In R. L. Creswell and R. Flos (eds.),

Perspectives on responsible aquaculture for the new

millennium, p. 94–111. World Aquaculture Society, Baton

Rouge, Louisiana, and European Aquaculture Society,

Oostende, Belgium.

Malone, R. F., and T. J. Pfeiffer. 2006. Design and

selection 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. Influence

of 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 and

biological 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 a

low-density media biofiltration unit for use in recirculating

fish culture systems. Master’s thesis. Louisiana State

University, Baton Rouge.
Uchida 51

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 enzymatic

saccharification 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 with 

iridovirus 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 production 

of cultured fish that are free of drugs.

In the case of Asian kelp (Undaria pinnatifida), the fronds are fragile and easily decomposed during the

saccharification 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 an

important role in agriculture and the livestock

industry 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 the 

approach of fermenting algae, which are plentiful in

marine 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 two

elements (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 starter 

microorganisms; usually lactic acid bacteria (LAB)

or yeast to initiate the process. This method can be

applied to any type of seaweed that contains cellulose 

(Table 1, Uchida and Murata 2004).

Figure 1. Categories of fermented food products.
52 Uchida

Table 1. Results of the fermentation test for various kinds of

seaweed with the addition of cellulase and the microbial

starter mixture (Uchida and Murata 2002).

The author is promoting the use of this fermented

material from seaweed as a fish dietary ingredient

with 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 by

cellulase during fermentation (Figure 2b). As a 

result, 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 of

seaweed and for preparing SCD-MS with high

efficiency. Furthermore, to demonstrate the potential

of 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, a 

feeding 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).

Figure 2. Fermentation process of seaweed. Lactic acid

fermentation (a) and lactic acid fermentation with single cell

detritus (SCD-MS) products (b).

Materials and Methods

Preparation of SCD-MS for determining

appropriate culture conditions. To determine the 

appropriate 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 NaCl

solution and cellulase (R-10, Yakult Pharmaceutical

Ind. Co.) (Table 1) and mixed well. The culture

bottles were capped and incubated at 20°C with

shaking several times a day to improve dispersion.

Initially, the preparation of SCD in cultures without

the addition of a microbial mixture was conducted

under 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 inoculation

of the microbial mixture. The microbial mixture was

composed 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 53

preincubated in MRS broth (Merck) and YM broth

(Difco), respectively, collected by centrifuge, washed

twice, 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 of

data were determined.

Mini preparation of SCD-MS. A sterile 50 mL

volume polypropylene centrifuge tube provided with

a screw cap (Iwaki) was used to prepare the 

fermentation cultures. To prepare NaCl-added

cultures (No. 1a–14a), 2.0 g of the Undaria product

were mixed with 40 mL of autoclaved 3.5% NaCl

solution, 40 mg of cellulase 12S (Yakult

Pharmaceutical), and 0.4 mL of bacterial cell

suspension 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, and

Leuconostoc mesenteroides IAM 13004T. The strains

were precultured with MRS medium, collected by

centrifuge, 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 used

instead of 3.5% NaCl solution. Cultures without

LAB inoculation were prepared as No LAB controls

(No. 15a and 15b). The triplicate cultures were

incubated 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 at

quantity of 5.0 × 1010 CFU (L. brevis), 5.5 × 107 CFU

(D. hansenii) and 1.2 × 108 CFU (Candida sp.). Each

culture bottle was capped, incubated at 20°C and

mixed several times a day to improve dispersion for 

the first eight days. The culture bottles were then

preserved at 20°C for 18 months.

Rearing trial for red sea bream challenged with

iridovirus. The Ecklonia-MS was prepared by

fermenting the Ecklonia maxima (a dried product

imported from South Africa) at 10°C for one month

after 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 the 

rearing trial, red sea bream (mean ±SD of body

weight = 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 cell

suspensions 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 was

used as a control diet (C-MS2). The SCD-MS2 and

C-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 a

temperature 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-filtered

seawater. 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 +
54 Uchida

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 the

spat 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 in

the 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 the

range 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.

Figure 3. Relationship between culture temperature and

production efficiency of SCD-MS.

Figure 4 shows a profile of the relationship

between cellulase concentration and the number of

SCD-MS in Undaria cultures prepared in the

presence of starter microbes. The number of SCDMS increased from 2.5 × 107

/mL (18.4% based on

volume proportion) at the start to 3.5–5.9 × 107/mL

(63.0–69.1% based on volume proportion) at 6 days

of 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 

odors and were judged to have rotted. In contrast, the 

culture media containing cellulase at 0.25–5.0% had

ester-like preferable odors and were judged to be

fermented.

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% of 

heterotrophic microorganisms) while the other 

contained 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 to

heterotrophic 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 that

the 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 and

production efficiency of SCD-MS.

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 55

D. hansenii and Candida sp., were isolated as

predominant microorganisms in the fermented Ulva.

We observed that the addition of the individually

cultured cell suspensions of these microorganisms as

a starter for fermentation of seaweed was effective in

the production of SCD-MS (Uchida and Murata

2004). However, we also found that lactic acid

bacteria alone can be used as a starter (Uchida et al.

2004b). Results of fermentation with NaCl-added

cultures are shown in Figure 5a. The predominance

of the inoculated species in the cultures was 100% for 

the cases of L. casei IFO 15883Tand 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, and

Leuconostoc 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 cultures

are shown in Figure 5b. The predominance of the

starter 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, and

Streptococcus Thermophilus. The LAB became

predominant (>90.0%) for 10 cases out of 14, while 

the inoculated species became predominant for the 6

cases out of 14. L. brevis, L. plantarum, L. casei, and

L. rhamnosus predominated in the MS cultures.

Promotion of survival rate of fish against viral

disease. Red sea bream challenged with iridovirus

and fed a diet containing the MS showed

significantly 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 the

supply of fish that are produced without the use of 

drugs, which is an important consideration for

consumers today.

SCD-MS as a replacement hatchery diet for

microalgae diet. Since the size of the SCD-MS is

about 5–10 μm in diameter, it has a potential to

replace microalgal diets in hatcheries. Table 3 shows

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 168

μm/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 prepared

from 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 partially

replace microalgal diets in hatcheries.

Table 3. Results of feeding trials of SCD-MS2 and C-MS2

with young pearl oyster (Uchida et al. 2004a).
56 Uchida

New Aquaculture Style Based on Marine

Silage

The development of the MS preparation

technology and its widespread use may change the 

way aquatic animals are fed in the future. Figure 6

presents the concept of aquaculture based on a

sustainable 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 such

as 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.

Figure 6. Concept of eco-friendly aquaculture based on a

marine silage diet.

References

Uchida, M., and M. Murata. 2002. Fermentative

preparation of single cell detritus from seaweed, Undaria

pinnatifida, suitable as a replacement hatchery diet for

unicellular 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. Mass

preparation of marine silage from Undaria pinnatifida and

its 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.
Smiley 57

Techniques for Live Capture of Deepwater Fishes with Special Emphasis 

on the Design and Application of a Low-cost Hyperbaric Chamber 

Jeffrey E. Smiley and Mark A. Drawbridge

Hubbs-SeaWorld Research Institute

2595 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 to

internal 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 the 

collection 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 and

two 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, and

brain; exophthalmia; internal and external

hemorrhage; everted stomachs; disoriented

swimming; altered behavior; and death (Gotshall

1964, Beyer et al. 1976, Feathers and Knable 1983,

Rogers et al. 1986, Gitschlag and Renaud 1994).

Specialized collection techniques are needed to

minimize barotrauma among deepwater fishes that

are 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 collected

using these methods are typically either moribund or 

dead. Special handling techniques include gradual

decompression and gas bladder deflation (Gotshall

1964, Lee 1992, Keniry 1996). Gas bladder deflation

has enabled collection of some specimens from the

sea, but mortality is highly variable and increases

with depth. To increase survivability, more time 

must be taken to decompress fish. In order to

accomplish this, specialized pressure-controlled

chambers 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 and

Gilchrist 1978, Wilson and Smith 1985, Koyama et

al. 2002).

Hyperbaric fish traps have made it possible to

collect 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, especially

for fish at shallower depths. To meet this need, we 

developed 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 species

of rockfish (Sebastes sp.) with the goal of holding the 

fish for breeding purposes.

Hyperbaric Chamber Design and

Construction

Design criteria. The hyperbaric chamber described

in this paper was designed based on the following

criteria:

1. Portability to allow manageable movement of 

fish from the field to the laboratory under

controlled conditions.
58 Smiley

2. Low cost using standardized parts that are

common 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 constant

conditions from the point of capture thereby

reducing stress on the fish.

5. Visual monitoring to ascertain fish health and

make decisions for safe decompression

schedules.

6. Water quality monitoring to ensure stable

seawater 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 and

secondary manifold and chamber. The primary

manifold was responsible for maintaining stable 

pressures for the secondary manifolds, the secondary

manifolds were responsible for maintaining stable 

pressures and flows in the chambers, and the 

chambers were responsible for specimen containment

and pressure isolation for greater than 30 minutes; the 

time allowed for transport from the boat back to the

lab.

Principle component: Primary manifold. Pressure 

in 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 the

sump and created stable backpressure in the system.

A portion of the backpressured seawater was diverted

before the pressure relief valve and served as the

source for the secondary manifolds. Four 1.9 cm ball

valves operating in parallel from the primary

manifold controlled the flow of seawater to four 

secondary manifolds through sections of 1.3 cm

flexible hose. The hose had a maximum operating

pressure of 20.4 atmospheres and ranged in length

from 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 was

bolted to a hand truck (Figure 1).

Principle component: Secondary manifold. A

secondary manifold was constructed for each of the

Figure 1. Portable primary manifold and booster pump

attached to a hand truck for ease of transport.

four chambers that allowed individual pressure and

flow control, safety, and bypass loops. Water from 

the primary manifold was diverted through a check

valve and a needle valve designed to control the 

amount 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 the 

secondary 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 when

system power failed. Seawater that had passed the

solenoid valve was routed through a rotary flow

indicator and two 0.6 cm labcock valves used to

divert outgoing flow to a cylindrical reservoir where 

water quality measurements could be taken. Two

other 1.3 cm ball valves were also used to bypass the 

solenoid and pressure relief valves in the event of 

component failure or operation at ambient pressure 

(Figure 2).

Figure 2. Schematic of secondary manifold and chamber

control valves.
Smiley 59

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 pressure

gauges 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 the

chamber. Connections from the chamber to the 

secondary 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 the

cylinder to complete the chamber. Pressure in the 

secondary manifold and chambers reached a

maximum of 9.5 atmospheres.

Figure 3. Hyperbaric chamber and secondary manifold (not

in view) attached to a hand truck for ease of operation and

portability.

Hyperbaric Apparatus Operation

Seawater supply. Seawater supply to the apparatus

depended on its location—either in the field or the 

laboratory. During field operation, the primary

manifold was supplied with chilled water from the

bait 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, and

chilled in a recirculating sump. The temperature

regulation systems at both locations were computer 

controlled, resulting in consistent supply

temperatures.

Pressurization sequence. Prior to receiving a fish,

each chamber was set on its end (open side up) and

filled with seawater (Figure 4). Water pressure was

regulated 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 a 

fish was caught and brought to the surface, it was

placed 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. Once

sealed, 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.

Monitoring environmental conditions and 

chamber 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 were 

recorded 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 in

pressure as the fish were being caught by hook and

line, recompressed, and decompressed in the

chamber. The depth recorder was programmed to log

data every 10 seconds when attached to the 

fisherman’s line, and every 30 seconds when placed
60 Smiley

in the chamber. The Reefnet logger measured depth

to the nearest 0.3 meters.

Water quality was monitored externally and

internally of the chamber. Externally water quality

was monitored downstream of each chamber using an

Oxyguard Handy Gamma probe. The probe was

factory fit with temperature (±0.1°C) and dissolved

oxygen (±0.1%). Measurements were recorded at

hourly 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 for

the chambers.

Decompression sequence. Fish were stabilized at

depth for 20 to 25.5 hours prior to beginning their 

decompression sequence. Four to six hours into the 

initial stabilization period the chambers were isolated

(valves closed and disconnected from the water

source on the boat) and transported back to the 

laboratory. Transportation times took no longer than

25 minutes, at which point water flow was

reestablished 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 rapid

change in pressure with an extended period of time to

decompress 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 the 

circulatory system.

Ascent rates used to calculate minimum time 

required 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 each

decompression sequence, the chamber was set

vertically (blind flange up) and the blind flange was

removed. The fish was then sluiced into a custombuilt, vinyl sling and placed into a 1.5 m diameter 

pool. The chambers were cleaned with freshwater 

and scrubbed as needed between uses. Maintenance

of the system was also conducted at this time.

Field Trials

Our apparatus was used to recompress and

decompress 14 cowcod. The fish were recompressed

to pressures equivalent to 84.7 m ±2.2 m in depth.

The average time taken to place the fish in the

chamber 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 were

allowed 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 ±0.6

m, 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 and

initial 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 water

temperature also shown.

Water temperature. Variation in water temperature

in the chamber was dependent on the operation of 

chillers 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 for

fish to be captured. During the recompression

sequence chilled water was circulated and the

temperature inside the chamber quickly decreased

from a maximum of 18°C to the target temperature of
Smiley 61

approximately 12°C. This usually occurred in the 

first 30 minutes of operation and thereafter the 

temperature stayed relatively constant.

Conclusion

The hyperbaric chamber was used successfully to

recompress, transport, and decompress a species

never before maintained in captivity. The apparatus

is portable and capable of stable operation under a

variety of field and laboratory conditions. The 

system is also relatively inexpensive to build,

operate, and maintain. Further modifications to the 

hatch design are being evaluated to expedite the

process of inserting the fish and initiating the 

recompression sequence. This modification may also

facilitate future attempts to use the chamber under 

water, 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 for

breeding purposes. We also plan to use the chambers

in 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 of

depressurization 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 red

snapper. 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 and

culture 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-sea

organisms. 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, with

observations 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 by

trawling 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 depth

of 5700 meters. Science 200:1056–1059.

Yoshimatsu 63

A Preliminary Report on the Use of Porphyra Protoplast as a Food Substitute 

for 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 enzymatic 

means. As protoplasts have no cell walls they are easily digestible when ingested by animals as food

components. In the present experiment we investigated the availability of Porphyra protoplasts (PP) as a

food 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 one 

of the most nutritious edible macroalgae (red algae)

and its thin sheet-like processed products are well

known as a wrapping material for a famous Japanese 

dish, sushi-rolls. Porphyra contains very high levels

of various kinds of minerals, and vitamins such as

vitamins A, B complex, C, and E. Laver is also a

good source of digestible protein (Resource Council

2000). In addition, it is notable that Porphyra

contains a high amount of taurine, an important

amino acid for larval fish (Kim et al. 2005).

Recently, efficient biotechnological techniques for 

producing protoplasts from macroalgae using purified

polysaccharases isolated from various bacteria were 

developed (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 called

something other than protoplasts because the cell

wall tends not to be destroyed completely by

enzymatic treatment and some parts of the outer cell

wall still remain intact. As protoplasts have no cell
64 Yoshimatsu

walls, they are easily digestible when ingested by

animals as a food ingredient. In the present

experiment we investigated the availability of 

Porphyra protoplasts (PP) as a live food substitute 

for culturing two types of aquatic animals: finfish and

bivalve molluscs.

Materials and Methods

The PP were prepared in the laboratory as

follows. As the cell wall of this alga is composed of

three kinds of polysaccharides (β-1, 4-mannan, β-1,

3-xylan, and porphyran), three enzymes (β-1, 4-

mannanase; β-1, 3-xylanase; and agarase) were 

produced from various types of bacteria which have

been isolated from marine environments (Araki et al.

1987, 1998, 2000; Aoki et al. 1990). Suitable

conditions for preparing a large amount of PP in the

laboratory were determined before the onset of

rearing experiments. Included were determinations

of the proper pH of the reaction mixture,

concentration of each enzyme, and time and

temperature of the reaction process, among others.

Since it is particularly difficult to distinguish and

separate protoplasts from spheroplasts in a whole 

reaction mixture, we use only the term protoplasts

here when referring to combined enzymatic-treated

product from Porphyra. After obtaining the 

protoplasts, 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 of

PP after grinding was varied from several μm to

several tens of μm depending on the level of

enzymatic reaction that had occurred.

One hundred liter polycarbonate aquaria were 

stocked with 13 fish each (initial average body

weight = 15.4 g ±0.2 g) that were fed the 

experimental diets for 6 weeks. Each aquarium was

aerated (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 during

biweekly measurement when all the experimental

fish were weighed individually after being

anesthetized with 2-phenoxyethanol (500 mg/L).

Table 1. Ingredient content and proximate analyses of the

experimental diets for red sea bream.

At the end of the feeding trials, in addition to the 

body weight and length measurements, hepato- and

viscero-somatic indices were calculated from the

weights of individual bodies, livers, and viscera.

Proximate analyses of liver and dorsal muscle were 

carried out from pooled samples of each group

obtained 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 and

initial 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 as

follows. 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 replicates

receiving each dietary treatment and reared in a

flowthrough system of temperature controlled

seawater (32–34 ppt, 20°C) for 6 weeks. Two diets
Yoshimatsu 65

(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. Adult

clams (mean shell length 35.3 mm) were reared under 

the same conditions as those used with spat and for 

the same period. Before feeding, the PP was mixed

with sea water using an electric mixer and offered to

the clams in liquid form. The dietary performance 

was evaluated by growth of the shells, body

composition and survival.

Results

Proximate compositions of dried Porphyra and

the 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 diets

with (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 was

more 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 two

test diets had no major differences in their proximate 

compositions (Table 2). There was no significant

difference in condition factor between the PP and

control groups. Nevertheless, higher values were

obtained 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 the

feeding trial are shown. After 6 weeks feeding, both

PP and control fish showed reduced moisture content

and increased crude protein and lipid content.

However, there were no significant differences in

proximate analyses data between them (p > 0.05).

Table 3. Effect of Porphyra protoplast supplementation on

the growth and feed utilization by red sea bream.

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 compared

with 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 was

observed in proximate carcass composition after the

feeding 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 as

obtained in red sea bream.
66 Yoshimatsu

Figure 1. Increase in shell length of spat fed PP as compared

with 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 by

supplementing PP as shown in Table 3. As a feed

ingredient, protein is the most important dietary

element and its efficient utilization must be thought

of 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 supplement

from 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 of 

macroalgal 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 

system or activating fat accumulation/mobilization as

well as improving absorption, assimilation, and

retention (Mustafa et al. 1995). Nevertheless, reports

of the supplemental effect of microalgal protoplasts

have apparently not been published to date.

Enhanced feed efficiency of red sea bream obtained

in the present experiment, when fed with diet

supplemented with 5% PP, may also be attributed to

the 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 the

feed 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 for 

culturing not only grown shellfish but also spat.

Under the experimental conditions employed, growth

in PP group of spat was inferior to that of spat fed

diatoms (Figure 1). Nevertheless, final survival was

high and almost same in the PP-fed group as in

natural food group. As there appeared to be no

difference in carcass composition between the two

groups, it should be possible in the future to improve 

the dietary effect by manipulating feeding techniques

and ingredients added to PP diet to enhance its

quality.

According to the experimental results obtained in

these 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 meet

the nutritional requirements of these aquatic animals

and develop the feeding techniques that might

enhance the performance of the PP.

Acknowledgment

This study was supported by a grant program of

the Agriculture, Forestry, and Fisheries Research

Council in Japan (Research project for utilizing

advanced technologies in agriculture, forestry, and

fisheries. No.1681, 2004-2006, Studies on the 

effective utilization of low-grade Porphyra by

enzymatic means).
Yoshimatsu 67

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 of

Porphyra yezoensis and green variant of P. tenera.

Nippon Suisan Gakk. 53:1623–1627.

Araki, T., Z. Lu, and T. Morishita. 1998. Optimization of

parameters 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.

Kim, S.K., T. Takeuchi, A. Akimoto, H. Furuita, T.

Yamamoto, M. Yokoyama, and Y. Murata. 2005. Effect

of taurine supplemented practical diet on growth

performance and taurine contents in whole body and

tissues 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 of

food composition in Japan (Fifth revised edition), p. 152-

159. Science and Technology Agency, Tokyo, Japan.

Jury 69

Aquaculture and Stock Enhancement Technologies Based 

on 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 in 

sensory 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 molecular 

cloning, cell biology, and physiological experiments show that CaRs play key roles in finfish osmoregulation 

and 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 to

fish 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 biological

changes 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), the 

SuperSmolt process reduces the deleterious effects of incomplete smoltification after the transfer of smolts to

seawater. This improves both the growth and feed conversion ratios while reducing mortalities during growout in

seawater. 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 and

preliminary 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 value 

marine 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 the

ionic and electric field environments that fish may encounter. While much of

this research is ongoing, we conclude that this CaR-based approach has considerable potential to improve

aquaculture and stock enhancement technologies in the U.S. and Japan.
70 Jury

Background. CaRs are part of a family of G-protein

coupled receptors that are closely related to

metabotropic glutamate and pheremone receptors and

are highly conserved between all species tested

(Breitwieser et al. 2004, Ingleton et al. 1999, Figure 

1). CaR proteins were first identified in human

parathyroid glands and their role in calcium 

homeostasis has been firmly established (Brown and

MacLeod 2001, Breitwieser et al.2004, Chang and

Shoback 2004). CaRs are responsive to

physiologically and environmentally relevant

changes 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), specific

amino acids (Conigrave et al. 2004), several heavy

metals; e.g., Pb2+ and Al

3+ (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 and

salinity 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 also

characterized a full-length CaR cDNA from dogfish

(Squalus acanthias) kidney (SKCaR) that is localized

in kidney, olfactory lamellae, epithelial cells and

vessels 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-length

transcripts or proteins. These species include the 

flounders, 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 Ca

2+

homeostasis in fish has been emphasized (Hubbard et

al. 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 propose 

that they are important in the integration of several

physiological and behavioral processes of concern to

the aquaculture and stock enhancement industries.

We have used the approach of studying receptor 

biology to address problems of concern in hatchery

production of anadromous and marine species. In

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 some 

research to develop additional technologies. These 

applications are: 1) SeaReady, 2) growing marine 

fish in near freshwater, and 3) the role of CaR in

sensory biology.

SeaReady. Anadromous species, including

salmonids, are able to survive in either freshwater or

seawater due to osmoregulatory adjustments in

tissues including gill, skin, kidney, and

gastrointestinal 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 in

salmonids raised under hatchery production

conditions prepares salmon for the remodeling of 

their tissues after seawater transfer. These 

physiological changes must be accomplished within a 

limited 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 during

smolting determine how ready salmonids are for a

seawater existence. Smoltification has traditionally

been manipulated under hatchery rearing conditions

with varying degrees of success by changes in

photoperiod, temperature, and feeding (McCormick

et al. 1998). While the factors that initiate and

control smoltification continue to be the subject of
Jury 71

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 smoltification

and subsequent osmotically induced lethal and

sublethal effects upon transfer of fish to seawater.

These effects can include significant elevations in

plasma ionic content resulting in the dysfunction of

various organ systems, impaired ion homeostasis,

abnormal swimming behavior, morphological

changes, impaired feeding behavior, and often

increased mortality.

Based on our understanding of the distribution

and function of calcium-sensing receptors relative to

osmoregulation, as well as the logistic and

socioeconomic 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 enhance 

smoltification in salmonid fishes while they are still

in the hatchery prior to release into rivers or transfer

to seawater for growout. This process consists of 

manipulating the ionic environment (e.g., Ca2+ and

Mg2+) as well as application of feed addititives (NaCl

and CaR reactive amino acids) over a period of 4–6

weeks (Figure 2).

Figure 2. SeaReady process overview.

SeaReady serves to preadapt salmon to seawater

and 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 to

seawater 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

growth 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 this

preadaptation 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 or

near 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, then

transfer them to marine sites for growout. This

would allow for raising fish to a larger size under 

controlled hatchery conditions, potentially at a lower

cost. The reasons for using freshwater systems

include: 1) coastal property is very expensive for

shore based tank farms and political/environmental

pressures are restrictive along the coast in many

areas, 2) environmental regulations limit the amount

of saline water discharge and sludge disposal from 

recirculating 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 freshwater 

by 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 decrease 

in salinity for a period of time prior to transfer to

freshwater, 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 maintain

expression 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 from 

these conditions back to 35 ppt seawater. MariCal

plans to grow cobia to market size in future large 

scale trials.
72 Jury

Role of CaRs in sensory biology. CaRs are 

expressed in most osmoregulatory tissues in salmon,

but they are also found throughout the peripheral and

central nervous systems (Nearing et al. 2002, Nearing

et al. submitted). In particular CaRs have been found

in the olfactory system (olfactory epithelia, olfactory

bulb, 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 have 

determined that Atlantic salmon are responsive to

calcium, magnesium, gadolinium, and strontium at

concentrations that are consistent with CaR activation

or modulation (Nearing et al. 2002, Nearing et al.

submitted). On this basis, we propose that CaRs play

a role in multiple osmoregulatory, and sensory

functions in salmon which experience large changes

in the concentration and composition of the ionic 

environments encountered during their normal life

history. This has many implications for stock

enhancement 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 stock

enhancement and aquaculture industries.

Conclusions. The identification of CaRs in multiple 

tissues of many species of finfish is an important

discovery opening an area of research on the

integration of environmental and extracellular 

signalling of cations. Exposure of finfish to variable 

ionic environments under naturally occurring

conditions (e.g., the typical life history of 

anadromous species) or under hatchery conditions

(e.g., SeaReady) influences CaR signaling in specific

tissues (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 the 

stock enhancement and aquaculture industries in the 

U.S. and Japan.

References

Breitweiser, G. E., S. U. Medlich, and M. Zhang. 2004.

Calcium sensing receptors as integrators of multiple

metabolic signals. Cell Calcium 35:209–216.

Brown, E. M., and R.J. MacLeod. 2001. Extracellular

calcium sensing and extracellular calcium signaling.

Physiol. Rev. 81:239–297.

Chang, W., and D. Shoback. 2004. Extracellular Ca

2+

-

sensing receptors-an overview. Cell Calcium 35:183–196.

Conigrave, A. D., H. Mun, L. Delbridge, S. J. Quinn, M.

Wilkinson, and E. M. Brown. 2004. L-amino acids

regulate parathyroid hormone secretion. J. Biol. Chem.

279:38151–38159.

Doroszewicz, J., P. Waldegger, N. Jeck, H. Seyberth, and

S. Waldegger. 2005. pH dependence of extracellular

calcium sensing receptor activity determined by a novel

technique. Kidney Int. 67:187–192.

Fellner, S. K., and L. Parker. 2002. A Ca

(2+)

-sensing

receptor modulates shark rectal gland function. J. Exp.

Biol. 205:1889–1897.

Flanagan, J. A., L. A. Bendell, P. M. Guerreiro, M. S.

Clark, D. M. Power, A. V. M. Canario, B. L. Brown, and

P. M. Ingleton. 2002. Cloning of the cDNA for the

putative calcium-sensing receptor and its tissue

distribution in sea bream (Sparus aurata). Gen. Comp.

Endocr. 127:117–127.

Handlogten, M. E., C. Huang, N. Shiraishi, H. Awata, and

R.T. Miller. 2000. Extracellular Ca

2+

-sensing receptor is a

promiscuous divalent cation sensor that responds to lead.

Am. J. Physiol.-Renal. 279:F1083–1091.

Hentschel, H., J. Nearing, H. W. Harris, M. Betka, M.

Baum, S. C. Hebert, and M. Elger. 2003. Localization of

Mg2+-sensing shark kidney calcium receptor SKCaR in

kidney of dogfish, Squalus acanthias. Am. J. Physiol.-

Renal. 285:F430–439.

Hubbard, P. C., E. N. Barata, and A. V. Canario. 2000.

Olfactory sensitivity to changes in environmental [Ca

(2+)

]

in the marine teleost Sparus aurata. J. Exp. Biol.

203:3821–3829.

Hubbard, P. C., P. M. Ingleton, L. A. Bendell, E. N.

Barata, and A. V. Canario. 2002. Olfactory sensitivity to

changes in environmental [Ca

(2+)

] in the freshwater teleost

Carassius auratus: An olfactory role for the Ca

(2+)

-sensing

receptor? J. Exp. Biol. 205:2755–2764.
Jury 73

Ingleton, P. M., P. C. Hubbard, J. A. Danks, G. Elgar, R.

A. Sandford, and R. J. Balment. 1999. The calciumsensing receptor in fishes. In J. Danks, C. Dacke, G. Flik,

and C. Gay (eds.), Calcium metabolism: Comparative

endocrinology, p. 45–48. BioScientifica Ltd., Bristol, UK.

Linley, T. 2000. Influence of short-term estuarine rearing

on the ocean survival and size-at-return of coho salmon in

southeast Alaska. N. Am. J. Aquacult. 63:306–311.

Loretz, C. A, C. Pollina, S. Hyodo, Y. Takei, W. Chang,

and D. Shoback. 2004. cDNA cloning and functional

expression of a Ca

2+-

sensing receptor with truncated Cterminal tail from the Mozambique tilapia (Oreochromis

mossambicus). J. Biol. Chem. 279:53288–53297.

McCormick, S. D., L. P. Hansen, T. P. Quinn and R. L.

Saunders. 1998. Movement, migration and smolting of

Atlantic salmon (Salmo salar). Can. J. Fish. Aquat. Sci.

55:77–92.

Naito, T., Y. Saito, J. Yamamoto, Y. Nozaki, K. Tomura,

M. Hazama, S. Nakanishi, and S. Brenner. 1998. Putative

pheromone receptors related to the Ca

2+

-sensing receptor

in Fugu. Proc. Natl. Acad. Sci. 95:5178–5181.

Nearing, J., M. Betka, S. Quinn, H. Hentschel, M. Elger,

M. Baum, M. Bai, N. Chattopadyhay, E. M. Brown, S. C.

Hebert, and H. W. Harris. 2002. Polyvalent cation

receptor proteins (CaRs) are salinity sensors in fish. Proc.

Natl. Acad. Sci. 99:9231–9236.

Nearing, J., M. Betka, S. Jury and H.W. Harris.

Submitted. Atlantic salmon (Salmo salar) express

calcium-sensing receptors (CaRs) in various tissues. J.

Exp. Biol.

Quinn, S. J., O. Kifor, S. Trivedi, R. Diaz, P. Vassilev, and

E. Brown. 1998. Sodium and ionic strength sensing by

the calcium receptor. J. Biol. Chem. 273:19579–19586.

Quinn, S. J., M. Bai, and E. M. Brown. 2004. pH sensing

by the calcium-sensing recptor. J. Biol. Chem.

279:37241–37249.

Radman, D. P., C. McCudden, K. James, E. M. Nemeth,

and G. F. Wagner. 2002. Evidence for calcium-sensing

receptor mediated stanniocalcin secretion in fish. Mol.

Cell. Endocrinol. 186:111–119.

Speca, D. J., D. M. Lin, P. W. Sorensen, E. Y. Isacoff, J.

Ngai, and A. H.Dittman. 1999. Functional identification

of a goldfish odorant receptor. Neuron 23:487–498.

Niwa 75

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, the 

process by which the bacterium colonizes gut of abalone is unknown. Therefore, our goal is to clarify when

colonization 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 the 

world 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 and

aquaculture. Abalone ingest mainly algae, with a

preference for brown algae. Abalone are distributed

along the coastal reefs in Japan, where many kinds of 

kelp in the order Laminariales (brown algae) grow

abundantly. Laminaria sp. is known to be the 

original source of alginate. Alginate accounts for 

more than 30% of the dry weight of the thallus in

some algae. It is known that the cell walls of brown

algae 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 those 

polysaccharides. Many researchers have reported the 

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 Shepherd

1992) investigated the nutritional requirements for

the development of artificial feeds for abalone and

suggested that abalone can efficiently use alginic acid

as an energy source. It has been reported that

alginate 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 from 

the digestive diverticulum of H. discus hannai

(Shimizu et al. 2003). Recently, we succeeded in

cloning of the equivalent gene from H. discus discus

(in preparation). Furthermore, 68% of all alginolytic 

bacteria isolated from H. discus hannai showed a 

strong preference for the polyG block (Sawabe et al.

1998). Therefore, it is reasonable to speculate that

the polyM part of ingested alginate is degraded by the 

digestive 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 et
76 Niwa

al. 1995). The bacterium produced at least six types

of alginate lyases, two of which were observed to be

specific for polyM and four of which were specific

for 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 those 

microfloras (Sawabe et al. 2003). The bacterium was

also found in the gut of a turban shell, Turbo

cornutus, although the occupation rate was

significantly smaller than that found in Haliotis spp.

Furthermore, acetic acid and formic acid were

detected as major fermentation products of alginate in

the V. halioticoli strains. The acetate is absorbed

from the gut tissue of abalone and metabolized as an

oxidative energy source (Breznak 1982). It is

suggested that the abundant populations of V.

halioticoli in the gut of Haliotis abalone may have a 

great 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 that

the main source of the bacterium is thalli of

laminaria, V. halioticoli has never been isolated from 

that 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 the

abalone and to what extent the bacterium contributes

to digestion of polysaccharides.

References

Boyen, C., B. Kloareg, M. Polne-Fuller, and A. Gibor.

1990. Preparation of alginate lyase from marine mollusks

for protoplast isolation in brown algae. Phycologia

29:173–181.

Breznak, J. A. 1982. Intestinal microbiota of termites and

other xylophagous insects. Annu. Rev. Microbiol. 36:323–

343.

Kloareg, B., and R. S. Quatrano. 1988. Structure of the

cell walls of marine algae and ecophysiological functions

of the matrix polysaccharides. Oceanogr. Mar. Biol. Ann.

Rev. 26:259–315.

Nakada, H. I., and P. C. Sweeny. 1967. Alginic acid

degradation by eliminases from abalone hepatopancrease.

J. Biol. Chem. 242:845–851.

Oakes, F. R., and R. D. Ponte. 1996. The abalone market:

Opportunities for cultured abalone. Aquaculture 140:187-

195.

Ohnishi, T., M. Suzuki, and R. Kikuchi. 1985. The

distribution of polysaccharide hydrolase activity in

gastropods and bivalves. Bull. Jpn. Soc. Sci. Fish. 51:301–

308.

Sawabe, T., Y. Oda, Y. Shiomi, and Y. Ezura. 1995.

Alginate degration by bacteria isolated from the gut of sea

urchins and Abalones. Microbial. Ecol. 30:193–202.

Sawabe, T., I. Sugimura, M. Ohtsuka, K. Nakano, K.

Tajima, Y. Ezura, and R. Christen. 1998. Vibrio

halioticoli sp.nov., a non-motile alginolytic marine

bacterium isolated from the gut of the abalone Haliotis

discus hannai. Int. J. Syst. Bacteriol. 48:573–580.

Sawabe, T., N. Setoguchi, S. Inoue, R. Tanaka, M.

Ootsubo, M. Yoshimizu, and Y. Ezura. 2003. Acetic acid

production of Vibrio halioticoli from alginate: a possible

role for establishment of abalone-V.halioticoli association.

Aquaculture 219:671–679.

Shepherd, S. A. 1992. Abalone of the World, p. 504-517.

Fishing News Books, Oxford, UK.

Shimizu, E., T. Ojima, and K. Nishita. 2003. cDNA

cloning of an alginate lyase from abalone, Haliotis discus

hannai. Carbohydr. Res. 338:2841–2852.

Tanaka, R., I. Sugimura I., T. Sawabe, M. Yoshimizu, and

Y.Ezura. 2003. Gut microflora of abalone Haliotis discus

hannai in culture changes coincident with a change in diet.

Fish. Sci. 68:227–229.

Tsujino, I., and T. Saito. 1962. Studies on alginase. Part

II. A new unsaturated uronide isolated from alginase

hydrolysate. Agr. Biol. Chem. Tokyo 26:115–118.