NOAA Technical Report NMFS 70

November 1988

New and Innovative Advances in
Biology/Engineering withPotential for Use in Aquaculture
Proceedings of the Fourteenth
U.S.-Japan Meeting on Aquaculture
Woods Hole, Massachusetts
October 16-17, 1985
Albert K. Sparks (editor)

U.S. Department of CommerceNOAA TECHNICAL REPORT NMFS
The major responsibilities of the National Marine Fisheries Service (NMFS) are to monitor and assess the abundance and geographic distribution of fishery resources, tounderstand and predict fluctuations in the quantity and distribution of these resources, and to establish levels for their optimum use. NMFS is also charged with the development
and implementation of policies for managing national fishing grounds, development and enforcement of domestic fisheries regulations, surveillance of foreign fishing off UnitedStates coastal waters, and the development and enforcement of international fishery agreements and policies. NMFS also assists the fishing industry through marketing service
and economic analysis programs, and mortgage insurance and vessel construction subsidies. It collects, analyzes, and publishes statistics on various phases of the industry.
The NOAA Technical Report NMFS series was established in 1983 to replace two subcategories of the Technical Reports series: "Special Scientific Report-Fisheries" and"Circular." The series contains the following types of reports: Scientific investigations that document long-term continuing programs of NMFS; intensive scientific reports on
studies of restricted scope; papers on applied fishery problems; technical reports of general interest intended to aid conservation and management; reports that review in considerable detail and at a high technical level certain broad areas of research; and technical papers originating in economics studies and from management investigations. Since
this is a formal series, all submitted papers receive peer review and those accepted receive professional editing before publication.
Copies of NOAA Technical Reports NMFS are available free in limited numbers to governmental agencies, both Federal and State. They are also available in exchange forother scientific and technical publications in the marine sciences. Individual copies may be obtained from: U.S. Department of Commerce, National Technical Information Service,5285 Port Royal Road, Springfield, VA 22161. Although the contents have not been copyrighted and may be reprinted entirely, reference to source is appreciated.
34. Additions to a revision of the shark genus Carcharhinus: Synonymy of Aprionodon and Hypoprion, and description of a new species of Carcharhinus (Carcharhinidae),by J. A. F. Garrick. November 1985, 26 p.
35. Synoptic review of the literature on the Southern oyster drill Thais haemastoma
floridana, by Philip A. Butler. November 1985, 9 p.
36. An egg production method for estimating spawning biomass of pelagic fish:
Application to the northern anchovy, Engraulis mordax, by Reuben Lasker (editor).
December 1985, 99 p.
37. A histopathologic evaluation of gross lesions excised from commercially important North Atlantic marine fishes, by Robert A. Murchelano, Linda DespresPatanjo, and John Ziskowski. March 1986, 14 p.

50. Preparation of acetate peels of valves from the ocean quahog, Arctica islandica,
for age determinations, by John W. Ropes. March 1987, 5 p.
51. Status, biology, and ecology of fur seals. Proceedings of an international
workshop, Cambridge, England, 23-27 April 1984, by John P. Croxall and Roger
L. Gentry (editors). June 1987, 212 p.
52. Limited access alternatives for the Pacific groundfish fishery, by Daniel D.
Huppert (editor). May 1987, 45 p.
53. Ecology of east Florida sea turtles: Proceedings of the Cape Canaveral, Florida,
sea turtle workshop, Miami, Florida, February 26-27, 1985, by Wayne N. Witzell(convener and editor). May 1987, 80 p.

38. Fishery atlas of the northwestern Hawaiian Islands, by Richard N. Uchida andJames H. Uchiyama (editors). September 1986, 142 p.

54. Proximate and fany acid composition of 40 southeastern U.S. finfish species,
by Janet A. Gooch, Malcolm B. Hale, Thomas Brown, Jr., James C. Bonnet, Cheryl
G. Brand, and Lloyd W. Reiger. June 1987, 23 p.

39. Survey of fish protective facilities at water withdrawal sites on the Snake andColumbia Rivers, by George A. Swan, Tommy G. Withrow, and Donn L. Park. April
1986, 34 p.

55. Proximate composition, energy, fany acid, sodium, and cholesterol content of
finfish, shellfish, and their products, by Judith Krzynowek and Jenny Murphy. July
1987, 53 p.

40.

56. Some aspects of the ecology of the leatherback turtle Dermochelys coriacea at
Laguna Jolova, Costa Rica, by Harold F. Hirth and Larry H. Ogren. July 1987, 14 p.

Potential impact of ocean thermal energy conversion (OTEC) on fisheries, by
Edward P. Myers, Donald E. Hoss, Walter M. Matsumoto, David S. Peters, Michael
P. Seki, Richard N. Uchida, John D. Ditmars, and Robert A. Paddock. June 1986,
33 p.

41. A stationary visual census technique for quantitatively assessing community structure of coral reef fishes, by James A. Bohnsack and Scott P. Bannerol. July 1986, 15 p.
42. Effects of temperature on the biology of the northern shrimp, Pandalus borealis,in the Gulf of Maine, by Spencer Apollonio, David K. Stevenson, and Earl E. Dunton,
Jr. September 1986, 22 p.
43. Environment and resources of seamounts in the North Pacific, by Richard N.
Uchida, Sigeiti Hayasi, and George W. Boehlert (editors). September 1986, 105 p.

44. Synopsis of biological data on the porgies, Calamus arctifrons and C. proridens(Pisces: Sparidae), by George H. Darcy. September 1986, 19 p.
45. Meristic variation in Sebastes (Scorpaenidae), with an analysis of character
association and bilateral pattern and their significance in species separation, by Lochai Chen. September 1986, 17 p.
46. Distribution and relative abundance of pelagic nonsaimonid nekton off Oregon
and Washington 1979-84, by Richard D. Brodeur and William G. Pearcy. December
1986, 85 p.
47. Reproduction, maturation, and seed production of cultured species: Proceedingsof the twelfth U. S. -Japan meeting on aquaculture, Baton Rouge, Louisiana, October
25-29, 1983, by Carl J. Sindermann (editor). February 1987,73 p.
48. Widow rockfish: Proceedings of a workshop, Tiburon, California, December
11-12, 1980, by William H. Lenan and Donald R. Gunderson (editors). January 1987,
57 p.
49. Reproduction, movements, and population dynamics of the southern kingfish,
Menticirrhus americunus, in the northwestern Gulf of Mexico, by Stephen M. Harding
and Mark E. Chittenden, Jr. March 1987, 21 p.

57. Food habits and dietary variability of pelagic nekton off Oregon and Washington,
1979-1984, by Richard D. Brodeur, Harriet V. Lorz, and William G. Pearcy. July
1987, 32 p.
58. Stock assessment of the Gulf of Menhaden, Brevoonia patronus, fishery, by
Douglas S. Vaughan. September 1987, 18 p.
59. Atlantic Menhaden, Brevoortia tyrannus, purse seine fishery, 1972-84, with abrief discussion of age and size composition of the landings, by Joseph W. Smith,William R. Nicholson, Douglas S. Vaughan, Donnie L. Dudley, and Ethel A. Hall.
September 1987, 23 p.
60. Gulf Menhaden, Brevoortia palronus, purse seine fishery, 1974-85, with a brief
discussion of age and size composition of the landings, by Joseph W. Smith, Eldon
J. Levi, Douglas S. Vaughan, and Ethen A. Hall. December 1987, 8 p.
61. Manual for starch gel electrophoresis: A method for the detection of genetic variation, by Paul B. Aebersold, Gary A. Winans, David J. Tee1, George B. Milner, andFred M. Utter. December 1987, 19 p.
62. Fishery publication index, 1980-85; Technical memoradum index, 1972-85, byCynthia S. Martin, Shelley E. Arenas, Jacki A. Guffey, and Joni M. Packard.
December 1987, 149 p.
63. Stock assessment of the Atlantic Menhaden, Brevoonia tyrannus, fishery, by
Douglas S. Vaughan and Joseph W. Smith. January 1988, 18 p.
64. illustrated key to penaeoid shrimps of commerce in the Americas, by Isabel Perez
Farfante. April 1988, 32 p.
65. History of whaling in and near North Carolina, by Randall R. Reeves and Edward
Mitchell. March 1988, 28 p.
66. Atlas and zoogeography of common fishes in the Bering Sea and Northeastern
Pacific, by M. James Allen and Gary B. Smith. April 1988, 151 p.

NOAA Technical Report NMFS 70

New and Innovative Advances in
Biology/Engineering withPotential for Use in Aquaculture
Proceedings of the Fourteenth
U.S.-Japan Meeting on Aquaculture,
Woods Hole, Massachusetts
October 16-17, 1985

Albert K. Sparks (editor)
Panel Chairmen:
Conrad Mahnken, United States
Shigekatu Sato, Japan
Under the U.S.-Japan Cooperative Programin Natural Resources (UJNR)

November 1988

U.S. DEPARTMENT OF COMMERCE
C. William Verity, Jr., Secretary

National Oceanic and Atmospheric Administration
William E. Evans, Under Secretary for Oceans and Atmosphere

National Marine Fisheries Service
James Brennan, Assistant Administrator for Fisheries

PREFACE_

The United States and Japanese counterpart panels on aquaculture were formed in 1969 under the United StatesJapan Cooperative Program in Natural Resources (UJNR). The panels currently include specialists drawn from thefederal departments most concerned with aquaculture. Charged with exploring and developing bilateral cooperation, the panels have focused their efforts on exchanging information related to aquaculture which could be of bene til
to both countries.
The UJNR was begun during the Third Cabinet-Level Meeting of the Joint United States-Japan Committee on
Trade and Economic Affairs in January 1964. In addition to aquaculture, current subjects in the program include
desalination of seawater, toxic microorganisms, air pollution, energy, forage crops, national park management, myel>plasmosis, wind and seismic effects, protein resources, forestry, and several joint panels and committees in marine
resources research, development, and utilization.
Accomplishments include: Increased communication and cooperation among technical specialists; exchanges of
information, data, and research findings; annual meetings of the panels, a policy-coordinative body; administrative
staff meetings; exchanges of equipment, materials, and samples; several major technical conferences; and beneficial
effects on international relations.

Conrad Mahnken - United States
Shigekatu Sato - JapanThe National Marine Fisheries Service (NMFS) does not approve, recommend or endorse any proprietary product or proprietary material mentioned
in this publication. No reference shall be made to NMFS, or to this publication furnished by NMFS, in any advertising or sales promotion which would
indicate or imply that NMFS approves, recommends or endorses any proprietary product or proprietary material mentioned herein, or which has asits purpose an intent to cause directly or indirectly the advertised product tobe used or purchased because of this NMFS publication.

ii

CONTENTS
Kawauchi, Hiroshi, and Syunsuke Moriyama
Chum salmon growth hormone: Isolation and effects on growth of juvenile rainbow trout 1
Itoh, Seiga, Susumu Sekine, and Hiroshi Kawauchi
Cloning and expression of cDNA for salmon growth hormone in Escherichia coli 7
Chen, Thomas, T., Rebecca J. Van Beneden, Luis B. Agellon, Duane A. Howard, and Ronald A. Sonstegard
Molecular toxicology: A new frontier 9
Teshima, Shin-ichi, and Akio Kanazawa
Necessity of dietary sterols and phospholipids for growth of the prawn, Penaeus japonicus Bate 15
Dickhoff, Walton W.
Applications of endocrinology to salmon culture: Hormonal induction of spawning of adults andhormone patterns during development of juveniles 21
Kito, Hitoshi
Isolation and development of protoplast in Porphyra 23
Cheney, Donald P.
Genetic engineering and biotechnology of economically important seaweeds 27
Takahashi, Kanji, and Akimitsu Koganezawa
Mass culture of Ulvella lens as a feed for abalone, Haliotis discus hannai 29
Weiner, Ronald M., Levent Dagasan, Michael P. Labare, Rita R. Colwell, Dale B. Bonar, andSteven L. Coon
Bacterial products and polysaccharide films as cues and enhancers of oyster set 37
Laufer, Hans, David Borst, Fred C. Baker, and David A. Schooley
Juvenile hormone in crustacea 39
Manzi, John J., and Nancy H. Hadley
Recent advances in nursery culture of bivalve mollusks in North America 41
Saga, Naotsune, and Yoshiaki Sanbonsuga
Tissue culture and genetic engineering for seaweed aquaculture 47
Ellis, Lehman L.
Preliminary investigations on cryopreservation of marine bivalve gametes and larvae 55
Kanazawa, Akio, and Shin-ichi Teshima
Microparticulate diets for fish larvae 57
Ito, Hiroshi
Sowing culture of scallop in Japan 63iii

Chum salmon growth
hormone: Isolation andeffects on growth ofjuvenile rainbow trout
HIROSHI KAWAUCHISYUNSUKE MORIYAMA
Laboratory of Molecular Endocrinology
School of Fisheries Sciences
Kitasato University
Sanriku-cho Kesen-gun
Iwate Prefecture. 022-01 Japan

Growth hormone (GH) stimulates animal growth throughout thevertebrates. In view of the potential applications in fish culture,interest is increasing in the study of fish pituitary hormone ingeneral, and the growth hormone in particular. However, until
recently chemical knowledge of GH has been mainly confined tomammals, although many attempts have been made to purify these
hormones in lower vertebrates such as shark (Lewis et al. 1972),
tilapia (Farmer et al. 1976), salmon (Higgs et al. 1978; Komourdjian and Idler 1979, Wagner et al. 1985), sturgeon (Farmer et al.
1981), and carp (Cook et al. 1983). Our recent studies on the isolation and characterization of chum salmon GH (Kawauchi et al.
1986a) and the gene cloning (Sekine et al. 1985) will promote a
better understanding of fish physiology and application to fishculture.
The present paper describes isolation and characterization of two
genetic variants of chum salmon GHs (I and II) and examines effectsof salmon GH on growth of juvenile rainbow trout.

Materials and methods_

ABSTRACT

Materials
Two molecular forms of salmon growth hormone, GH I andII, have been isolated from the pituitary glands of the chumsalmon, Oncorhynchus keta. A two-step procedure was employed
for extraction of the salmon GHs under an alkaline (pH 9-10)
condition, subsequent to acid acetone extraction. The GHs werethen purified by isoelectric precipitation at pH 5.6, gel filtration on Sephadex G 100, and high-performance liquid chromatography on ODS. A molecular weight of 22,000 was estimatedfor both forms of salmon GH by gel electrophoresis in sodium
dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6
and 6.0 were estimated for salmon GH I and II, respectively,
with differences present in the amino acid composition and the
N-terminal residue, suggesting that they may be genetic variants
coded on two separate genes. The partial amino acid sequenceof salmon GH I at both terminal regions has been determined.
Intraperitoneal injection of salmon GH I at doses of 0.01
Jig and 0.1 Jig/g body weight resulted in a significant increasein body weight and length ofjuvenile rainbow trout. The growthpromoting effect lasted longer even after cessation of GH treatment. The long-lasting effect of GH treatment was also induced
by a single injection at higher doses of 5 to 50 Jig/fish (30 g bodyweight).

Mature female chum salmon, Oncorhynchus keta, 3 to 5 years old
were caught by a fishweir 300 m from the mouth of the Tsugaruishi
River in Iwate Prefecture, Japan, and kept in a live well overnight.
Whole brain and pituitary were dissected from the side of thepharynx using a cylindrical borer (2.5 cm in diameter, 20 cm inlength) made of stainless steel within 20 min after transfer fromthe live well. Pituitary glands were frozen immediately in liquid
nitrogen and maintained until use. Escherichia coli-sGH has been
expressed by the cDNA encoding salmon GH in E. coli (Sekine
et al. 1985).

Isolation
The pituitary glands (50 g) were initially extracted with acid-acetone
as described for sPRL (Kawauchi et al. 1983a). The residue oftheacid acetone extract was re-extracted with 250 mL of water adjusted
to pH 9-10 with Ca(OHh at 4°C for I h. The supernatant was adjusted to pH 5.6 with 0.1 N HCl, and the resulting precipitate
redissolved in 200 mL of water at pH 9.0, followed by reprecipitation at pH 5.6. The precipitate was dissolved in 30 mL of 0.05 M
ammonium acetate, pH 10, and the supernatant subjected to gel
filtration on a Sephadex G-l00 (5 x 70 cm) column, equilibrated
with the same buffer. Growth-promoting activity of the putative
GH fraction was examined using juvenile rainbow trout as described
below. The biologically active fraction was dissolved in 0.1 %trifluoroacetic acid containing urea (about I M) and subjected tohigh-performance liquid chromatography on a reverse phase (TSKGel ODS-l20T, 5 Jim) column (0.4 x 25 cm), at a column temperature of 40°C and a flow rate of 1 mLlmin. Linear gradientelution was performed with acetonitrile containingO.1 % trifluoroacetic acid. The eluate was monitored by measuring the absorbanceat 220 nm, and each GH peak was collected in one tube.

Chemical and physicochemical characterization
The purified GHs were examined by slab gel electrophoresis at pH
4.3 in 7.4% polyacrylamide gel, and by gel electrofocusing in 5%

EP

50S

(%)

100

sGH-'

(J

/
sGH-II:

~

:'

m.wt

80

pi

60

40

1- -

5.e

11-

e.o

-

"

. . . _....

42,900-28,600

22,000

20

•

I II

o

10
Time

20

30

-

14,300

ref

40

(min)

Figure 1
HPLC: High-performance liquid chromatography of fraction 2 on TSK gel ODS-l20T column (O.4x25 em, 51ffi1). The fraction was dissolvedin 1 M urea containing 0.1 % trifluoroacetic acid. Elution was performed by a linear gradient of acetonitrile containing 0.1 % trifluoroacetic
acid. The now rate was 1 mL/min. Two molecular forms were designated as salmon GH I and II according to the order of elution. EP: Gelisoelectric focusing patterns of salmon GH I and II. SDS: SDS-gel electrophoresis patterns of salmon GH I and II.

polyacrylamide gel at 2% ampholine (pH 3.7-10). The molecularweight was estimated by sodium dodecyl sulfate (SDS) slab gelelectrophoresis.
For amino acid analysis, the proteins were hydrolyzed for 22 h
at 110°C with constant boiling HC!. The analysis was carried out
in an LKB automated amino acid analyzer (Type 44(0). N-terminal
analysis was performed by the DNS method (Gray 1967).
Cyanogen bromide cleavage of salmon GH I was performed in
70% formic acid, followed by performic acid oxidation. The resulting fragment peptides were subjected to HPLC on a TSK-Gel
ODS-l20T column using a linear gradient with 0.1 % tritluoroacetic
acid and acetonitrile.
Automatic microsequencing was performed with a gas-liquid
sequencer (Applied Biosystems, model 470A) (Hewick et al. 1981).
Identification of phenylthiohydantoin amino acid was carried out
by HPLC (Spectro Physics, SP8100 system) on a C8 reverse phasecolumn (Senshu Kagaku SEQ-4, 7 JA, 0.46 x 30 cm) using a gradient elution with 40 mM sodium acetate (pH 4.9) and acetonitrile.

once. Doses of the hormones were 0.01 and 0.1 JAg/g body weightin repeated injections and 5, 10, and 50 JAg/fish of "'30 g bodyweight in a single injection. Fish were fed to satiation with a commercially prepared diet (Masu No. 4P, Nihonhaigo Shiryo Co.)
twice daily. Growth-promoting activity was evaluated by bodylength increase and weight gain.

Results_

Isolation of two salmon GHs
Chum salmon GH was extracted from the residue of the acid acetone
extract of the pituitary glands at pH 9, and purified by repeating
the isoelectric precipitation at pH 5.6. The wet precipitate wasimmediately dissolved in 0.05 M ammonium acetate, pH 9.0, andsubjected to gel filtration on Sephadex G 100. The salmon GH
fraction produced a single band on SDS and polyacrylamide gelelectrophoresis at pH 4.3, but produced two bands in gel electrofocusing (pH 3.5-10). These two components were separated by
HPLC on an ODS column and designated as salmon GH I and II,
according to the order of elution (Fig. I).

Growth-promoting activity
Growth-promoting activity of the purified salmon GH I and E. colisGH was examined using juvenile rainbow trout, Salrno gairdneri.
All animals were maintained in running water under a natural
photoperiod from August to December. The water temperature
varied between 13 and 15°C during the period of treatment, andgradually decreased to 4°C by the end of December. The hormones
were dissolved in a small volume of I M urea and then diluted insaline (0.9% NaCl), or in a minimum volume of water at pH 10
and then diluted in saline containing I % bovine serum albumin,
and injected intraperitoneally at 4-day or I-week intervals, or only

Chemical and physicochemical properties
Salmon GHs were hardly soluble in an aqueous buffer. The isoelectric points of salmon GH I and II were estimated to be 5.6 and 6.0,
respectively, by gel electrofocusing. Both salmon GH I and II exhibited an identical molecular weight of 22,000 daltons, estimatedby SDS gel electrophoresis (Fig. I). However, they differed inamino acid compositions as presented in Table I, suggesting that2

they are not electroisomers generated by deamidation. The total
number of basic and acidic amino acids is comparable with thatof human GH. A relatively high value of Val and lIe, and a low
value of Phe, may be characteristic of salmon GHs.

Table 1
Amino acid compositions of salmon growth hormones.
Amino acidsGH I

sGH IIcDNA:j:Human GH:j::j:

Asp
Thr
Ser
Glu
Pro
Gly
Ala
Cys#
Val
Met
lIe
Leu
Tyr
Phe
Trp##
His
Lys
Arg

28.0
6.8
11.7
22.5
3.7
7.3
6.6
3.3
10.7
2.7
10.6
28.5
6.8
6.8
1.0
4.2
12.6
8.6

25.1
5.7
10.1
25.6
3.9
9.3
5.0
3.4
9.6
4.0
10.3
28.3
5.5
6.7
1.0
4.3
14.7
8.5

28
7
17
20
5
8
6
4
10
3
12
28
8
7

20
10
18
27
8
8
7
4
7
3
8
26
8
13

I

I

3
12
9

3
9
II

Total
N-terminus

182.3
lIe

181.0
ND

188
lIe

191
Phe

Partial amino acid sequence of salmon GH I
Isoleucine was identified as a sole amino-terminal residue of salmon
GH I, but no N-terminal amino acid was found for salmon GH II
by the dansyl method. The amino acid sequence of the N-terminal
33 residues of salmon GH I was identified, by an automatic
microsequencer with 40 J1g of salmon GH I, as follows: H-lle-GluAsn-Glu-Arg-Leu-Phe-Asn-IIe-A1a-Val-Ser-Arg-Val-Gln-His-LeuHis-Leu-Leu-Ala-Gln-Lys-Met-Phe-Asn-Asp-Phe-Asp-Gly-ThrLeu-Leu-. Cyanogen bromide cleavage followed by performic acidoxidation after fractionation by HPLC resulted in three fractions
from salmon GH I.
The first fraction gave a sole amino-terminal residue His. The
sequence was determined as follows: H-His-Lys-Val-Gln-Thr-TyrLeu-Thr-Val-Ala-Lys-(Cys)-Arg-Lys-Ser-Leu-Glu-Ala-Asn-CysThr-Leu-. The second fraction gave a sole N-terminal residue of
TIe. The amino acid composition is consistent with that ofGH (1-24).
The third fraction gave three amino-terminal residues, lIe, Phe andVal, suggesting contamination of three fragment peptides.

:j:Based on the nucleotide sequence of the cDNA cloned sGHmRNA (Sekine
et al. 1985).
:j::j:Taken from Martial et al. 1979.
#Determined after performic acid oxidation.
##Detected by UV absorption.
ND Not detected by the dansyl method.

Biological activity
The effects of salmon GH I on growth of intact juvenile rainbow
trout are shown in Figure 2A. Animals received a total of sevenIS

LENGTHWEIGHT0

0.1 ug/gBW•

0.01 Ug/gBW

0

0.01

x saline
10

ug/g~

%

Injection]%

Length

200

900

150

500

Weight

2

-c

.~

"3,

~

c

~ 5

A

0-1 lJ')/gBW•x saline

....

D,y

8

D,y

100 l~0==~4~0-----'70~---:,oT.<10Day

100 L~0~=::::;4~0;--'7,;,0---,i1nlo
Day

Figure 2
Effects of salmon GO I on growth of juvenile rainbow trout. (A) During treatment: Fish received a total of 7 intraperitoneal injections at a dose of either 0.01 lAg or
O.llAg/g body weight every 4 days. Initial body weight and fork length <X±SE, n=15) for control group (_), 9.70±1.33 g and 9.16±0.15 cm; for 0.01 lAg group(.), 10.26± 1.15 g and 9.06±0.63 cm; for 0.1 lAg group (0), 10.86± 1.93 g and 9.36±0.53 cm, respectively. (B) After treatment: Growth of these groups was followed
for 110 days. Vertical lines represent one standard error of mean. Arrows represent time of injection.

3

(em)

(9)

55

0

•

Natural sGH
~ sGH

14

l/Jg/f Ish
lpg/fl sh1;.

• sal ine

45

0

••

Natural sGH
E. call sGH
salineo

14

lpg/fish
lpg/fish

13
.t::

'Cl
c

:E
Ol

~

~

35

oX:

0

12

u.

25

11

15

o

14

•

•

!

•

28

42

56

70

8491

28

42

56

70

84 91

Days

Days

Figure 3
Effects of the natural salmon GHs and E. coli-sGH of growth of juvenile rainbow trout. Fish received intraperitoneal injections at a dose of 0.1 ",gig body weight once a week. Saline group (.); natural salmon GHs (a mixture of I and II) group(0); and E. coli group (.). Vertical lines represent one standard error of mean. t Significantly different from saline-injected
control at 0.05.

injections of either O.OIj.lg or O.Ij.lg/g body weight at 4-day intervals. Salmon GH-injected groups had greater increases in weight
and length than the control group. Furthermore, there is a clear
dose-response relationship in both weight gain and length increase.
Figure 2B represents the growth after cessation of the treatment.
The GH-treated groups maintained higher growth rate than the control group. The growth-promoting effects of E. coli-sGH and a combination of natural salmon GH I and II at a dose 0.1 j.lg/g of bodyweight once a week are summarized in Figure 3. It is evident thatboth GHs are equipotent in growth-promoting activity over a
13-week period. Figure 4 shows the growth-promoting effect ofa single injection of salmon GH. The single injection of doses of
5, 10, and 50 j.lg salmon GH also significantly stimulated growthof juvenile rainbow trout (30 g), whereas they did not show any
dose dependence.

Discussion

et al. 1983). Nevertheless, alkaline extraction resulted in only limited
success for the isolation of salmon GH. This may be due, in part,to contamination with gonadotropin (Higgs et a1. 1978) which isthe predominant component in the pituitary of the mature fish
(Kawauchi et a1. 1986b). Hence the residue of the acid acetone
extract was subjected to re-extraction in alkaline pH. HPLC revealed
that the major components in the alkaline extract were putative
salmon GH and various glycoproteins, which were assigned as
SUbunits of glycoprotein hormone. It was also confirmed that noneof these glycoproteins in this alkaline extract exhibited gonadotropic
activity in rainbow trout, but possessed potent antigenicity in rabbits. Thus the two-step extraction procedure adapted for isolationof salmon GH is superior to the direct extraction with alkaline pH
in preventing contamination by biologically active hormones. An
isoelectric fractionation procedure was employed to remove theglycoproteins. The preparation, however, still had two bands ongel electrofocusing. The two components, salmon GH I and II, could
be separated by HPLC on a reverse phase column.
There was no apparent size heterogeneity between salmon GH
I and II (Fig. I). Although it appeared that they were electroisomers
derived from deamidation, their amino acid composition andanalyses of N-terminal residues strongly suggest that they are genetic
variants. Indeed, it has been noted that the salmon pituitary glandcharacteristically secretes two isoforms of a-, (3-melanotropins andendorphins, and proposed that they may be derived from two distinct
proopiocortins, coded on two separate genes (Kawauchi 1983). This
unusual multiplicity of molecular forms has also been found insalmon PRL preparations, which differ from each other by
replacements of only 4 of 187 residues (Yasuda et al. 1986). The
genetic variant forms observed in the chum salmon pituitary hor-_

Prolactin (Kawauchi et a1. 1983a) together with proopio,::ortinrelated hormones (Kawauchi et a1. 1980ab. Kawauchi 1983) andMCH (Kawauchi et a1. 1983b) were successfully isolated from anacid acetone extract of chum salmon pituitaries. However, no
presumptive GH fraction was found in the extract, suggesting thatthe salmon GH might not be extractable with acid acetone. In fact,
an alkaline extraction procedure described for mammalian GH by
Papkoff and Li (1958) has been exclusively employed for the isola·tion of fish GHs such as shark (Lewis et a1. 1972), tilapia (Farmer
et aI. 1976), salmon (Higgs et al. 1978, Komourdjian and Idler 1979,
Wagner et aI. 1985), sturgeon (Farmer et al. 1981). and carp (Cook

4

%

%

length

300

140

~
~

4>

-;;...

-=.

-=~
~

~

Weight

~

0

~

120

200

100

100

o

14

28

42

56

o

70

14

28

42

56

70

Day

Day

Figure 4
Effects of salmon GH (a mixture of I and II) on growth of juvenile rainbow trout. Fish (N= 10) received a single intraperitoneal
injection at a dose of either 5 f'g or 10 f'g/g or 50 f'g/fish ("-'30 g). Control group (_); 5 ~ group (0); 10 f'g group (.); SO ~ group(0). Body weight and fork length were measured once a week. Vertical lines represent one standard error of mean.

Acknowledgments

mones may be accounted for by the tetraploid hypothesis of
salmonidae (Allendorf and Thorgaard 1984).
cDNA of salmon GH has been cloned from a cDNA library
prepared from chum salmon pituitary gland poly(A)+RNA withsynthetic DNA probes corresponding to the residues 23-28 of salmon
GH I. The sequence analysis of nucleotide of the cDNA revealed
that the cDNA codes for a polypeptide of21O amino acids (Sekine
et al. 1985). The N-terminal 38 residues of salmon GH I and the
C-terminal 22 residues are identical to the segments between 23
and 61 and between 182 and 210, respectively, in the predicted
amino acid sequences. Thus, it is clear that a putative signal peptide of 22 amino acids is cleaved from a pre-salmon GH of 210
residues, to produce the I88-amino-acid salmon GH I. The aminoacid composition of salmon GH I is consistent with the values basedon the structure of 188 residues. Moreover, the recombinant salmon
GH corresponding to the nucleotide sequence has been empressedin E. coli, and found to be identical to salmon GH I on HPLC.
The highly purified salmon GH, as well as E. coli-sGH, elicitedprofound growth-stimulating effects on juvenile rainbow trout at
doses of O. aI and O. I JAgl g body weight, the natural and the recombinant salmon GHs being equipotent. The effects of salmon GH
were more marked on weight gain than on length increase, andsimilar effects of GH have been shown in the carp (Cook et aI.
1983) and coho salmon (Higgs et al. 1976). Even after cessationof treatment,salmon GH-treated groups maintained higher growth
rates than the control group (Fig. 2B). This long-lasting effect has
also been observed by a single injection of salmon GH. It should
also be noted that the growth rates during treatment were lower
than post-treatment, particularly in the control group (Fig. 2B),
which may be due to handling stress during injections and measurements of body weight and length under anesthesia. In fact, whenfish received frequent injections at 2-day intervals, they lost weightand showed high mortality.

_

We thank Mr. Yamaguchi and Tokyo Research Laboratories,
Kyowa Hakkou Kogyo Co., for their assistance in the sequence
determination. This work was supported in part by grants-in-aid
for scientific research from the Japan Ministry of Education, Science
and Culture, and from the Nihon Kagaku-Kenkyukai Foundation
to H.K.

Citations_

Allendorf, F.W., and G.H. Thorgaard.
1984. Tetraploidy and the evolution of salmonid fishes. In Turner. B.J. (ed.).
Evolutionary genetics of fishes. p. 1-53. Plenum. New York.

Cook, A.N., A.W. Wilson, and R. Peter.
1983. Development and validation of a salmon growth hormone radioimmunoassay. Gen. Compo Endocrinol. 50:335-347.

Farmer, S.W., H. Papkoff, T.A. Bewley, T. Hayashida, R.S. Nishioka, H.A.
Bern, and C.H. Li.
1976. Isolation and properties of teleostean growth hormone. Gen. Compo
Endocrinol. 30:91-100.

Farmer, S.W., T. Hayashida, H. Papkoff, and A.L. Polenov.
1981. Characteristics of growth hormone isolated from sturgeon (Acipenser
guldens/ad/i) pituitaries. Endocrinology 108:377-381.

Gray, W.R.
1967. Sequential degradation plus dansylation. In Hirs. C.H.W. (ed.), Methods
in enzymology. Vol. 11, p. 469-475. Academic Press. New York.

Hewick, R.M., M.W. Hunkapiller, L.E. Hood, and W.J. Dreyer.
1981. A gaS-liquid solid phase peptide and protein sequenator. J. BioI. Chern.
256:7990-7997.

Higgs, D.A., E.M. Donaldson, H.M. Dye, and J.R. McBride.
1976. Influence of bovine growth hormone and L-thyroxine on growth, muscle
composition. and histochemical structure of the gonads. thyroid, pancreas. andpituitary of coho salmon (Oncorhynchus kisutch). J. Fish. Res. Board Can.
33: 1585-1603.

5

Higgs, D.A., E.M. Donaldson, J.R. McBride, and H.M. Dye.
1978. Evaluation of the potential for using a chinook salmon (Oncorhynchus/shawy/scha) pituitary extract versus bovine growth of coho salmon (Oncorhynchus kisu/ch). Can. J. Zoo!. 56:1226-1231.
Kawauchi, H.
1983. Chemistry ofproopiocortin-related peptides in the salmon pituitary. Arch.
Biochem. Biophys. 227:343-350.
Kawauchi, H., M. Tsubokawa, A. Kanezawa, and H. Kitagawa.
198Oa. Occurrence of two different endorphins in the salmon pituitary glands.
Biochem. Biophys. Res. Commun. 92:1278-1288.
Kawauchi, H., Y. Adachi, and B. Ishizuka.
198Ob. Isolation of another fJ-melanocyte stimulating hormone in the salmon
pituitary glands. Int. J. Pept. Protein Res. 16:79-82.
Kawauchi, H., K. Abe, A. Takahashi, T. Hirano, S. Hasegawa, N. Naito, and
Y. Nakai.
1983a. Isolation and properties of chum salmon prolactin. Gen. Compo
Endocrino!. 49:446-458.
Kawauchi, H., I. Kawazoe, M. Tsubokawa, M. Kishida, and B.I. Baker.
1983b. Characterization of melanin-concentrating hormone in the chum salmon
pituitaries. Nature 305:321-323.
Kawauchi, H., S. Moriyama, A. Yasuda, K. Yamaguchi, K. Shirahata, J. Kubota,
and T. Hirano.
1986a. Isolation and characterization of chum salmon growth hormone. Arch.
Biochem. Biophys. 244:542-552.
Kawauchi, H., Suzuki, K., Y. Nagahama, S. Adachi, N. Naito, and Y. Nakai.
1986b. Occurrence of two distinct gonadotropins in chum salmon pituitary. In
Yoshimura, F., and A. Gorbman (eds.), Pars Distalis of the pituitary gland,p. 383-390. Excerpta Medica, Amsterdam-New York-Oxford.
Komourdjian, M.P., and D.R. Idler.
1979. Chum salmon pituitary fractions: Somatotropic activity and cytoimmunofluorescence studies, Gen. Compo Endocrino!. 37:343-349.
Lewis, U.J., R.N.P. Singh, B.K. Seavey, R. Lasker, and G.E. Pickford.
1972. Growth hormone- and prolactin-like proteins of the blue shark (Prionilce
glauco). Fish. Bull., U.S. 70:933-939.
Martial, J.A., R.A. Hallewell, J.D. Baxter, and H.M. Goodman.
1979. Human growth hormone: Complementary DNA cloning and expression
in bacteria. Science (Wash., DC) 205:602-607.
Papkoff, H., and C.H. Li.
1958. The isolation and characterization of growth hormone fron anterior lobesof whale pituitaries. J. Bio!. Chern. 231 :367-377.
Sekine, S., T. Mizukami, T. Nishi, Y. Kuwana, A. Saito, M. Sato, S. Itoh, and
H. Kawauchi.
1985. Cloning and expression of cDNA for salmon growth hornonc in
Escherichia coli. Proc. Nat!. Acad. Sci. (USA) 82:4306-4310.
Wagner, G.F., R.e. Fargher, J.e. Brown, and B.A. McKeown.
1985. Further characterization of growth hormone from the chum salmon(Oncorhynchus ketal. Gen. Compo Endocrino!. 60:27-34.
Yasuda, A., H. Itoh, and H. Kawauchi.
1986. Primary structure of chum salmon prolactins: Occurrence of highly conserved regions. Arch. Biochem. Biophys. 244:528-541.

6

Human growth hormone (hGH) and other mammalian hormones(GHs) can now be produced by recombinant DNA technology, butthere is no report on cloning and expression of salmon growth hormone (sGH). Massive supplies of sGH produced in Escherichia
coli will provide new means to fish husbandry. Recently, sGH was
purified from chum salmon (Oncorhynchus keta) pituitaries(Kawauchi et al. 1986) To clone sGH cDNA, total RNA was extracted from salmon pituitaries and purified by oligo(dT) cellulose
column.
Using this poly(A)+ RNA, a cDNA library was constructed by
the Okayama and Berg (1983) method. Two types of DNA probes(A and B) were synthesized based on the partial amino acid sequenceof sGH. First, probe A was used to screen this cDNA library andthe candidate clones were also confirmed by hybridization withprobe B. Finally, eight clones were found to hybridize with thesetwo probes and possessed 1-1.3 kb cDNA insert.
One of the cDNA from the candidate clone was sequenced? andresults showed that the deduced amino acid sequences (1-40 and
167-188) were completely identical with the partial amino acid sequence for sGH. This sequence predicts that sGH will be synthesized as a precursor consisting of 210 amino acids which includes
22 NHz-terminal amino acids as a signal peptide. Consequently,
mature sGH consists of 188 amino acids, whereas hGH and bovine
GH consist of 191 and 190 amino acids, respectively. To calculateamino acid homologies between sGH ahd hGH, the sequences arealigned by introducing arbitrary gaps to maximize their homologies.
sGH has 35% amino acid and 50% nucleotide homology with hGH.
For the expression of mature sGH, a plasmid vector which contains tandem trp promoter and lpp terminator was chosen, because
IFN-y was expressed in a large amount. Purified Mbo II-Sal I fragment and Sal I-Bam HI fragment from this cDNA were inserted
into trp-vector together with a synthetic linker which contains initiation codon just in front of mature sGH. This approach allows
direct expression of mature sGH in E. coli, and the product waschecked by NaDodS04 /PAGE.
The amount of sGH synthesized in E. coli carrying psGHIB-2
and its improved vector was estimated to comprise about 15% and
25 % of total cellular protein, respectively. In addition, inclusion
bodies were observed in the E. coli cells containing psSGIB-2, andthe particles were easily recovered and purified from sonicated cellsby centrifugation. The particles collected by centrifugation weresolubilized in 8M urea solution and renatured by dilution and dialysis
as described by Marston et al. (1984) with a slight modification.
The partially purified sGH sample was tested for its growthpromoting activity on rainbow trout. sGH was injected intraperitoneally into rainbow trout less than I year old (9-9.5 cm fork lengthand 10-12 g body weight), and weight and length were measured
every week. The results clearly show that sGH synthesized in E.
coli is equipotent to the natural sGH in promoting increases in weight
and length of rainbow trout.
Since the cloning and expression of sGH has made possible theproduction of this hormone in large quantities, we would like to
study the use of this hormone on a variety of fish. If sGH is effective on many fish by a practical method such as dipping or feeding,this hormone may have great potential value in fish culture.

Cloning and expression of
eDNA for salmon growth
hormone in Escherichia coli!
SEIGA ITOH
SUSUMU SEKINETokyo Research Laboratory
Kyowa Hakko Kogyo Co., Ltd.
3-6-6 Asahicho, Machidashi
Tokyo, 194 JapanHIROSHI KAWAUCHI
School of Fisheries Sciences
Kitasato University
Sanriku, [wate 022-01, Japan'The complete paper has been published in Proc. Nat!. Acad. Sci. U.S.A. 82:43064310 (July 1985).

7

Citations_

Kawauchi, H., S. Moriyama, A. Yasuda, K. Yamaguchi, K. Shirahata, J. Kubota,
and T. Hirano.
1986. Isolation and characterization of chum salmon growth hormone. Arch.
Biochem. Biophys. 244:542-552.
Marston, F.A.O., P.A. Lowe, M.T. Doel, J.M. Schoemaker, S. White, and
S. Angal.
1984. Purification of calf prochymosin (prorennin) synthesized in Escherichia
coli. Bio-Teehnology (N.Y.) 2:800-804.
Okayama, H., and P. Berg.
1983. A eDNA cloning vector that permits expression of eDNA inserts in mammalian cells. Mol. Cell. BioI. 3:280-289.

8

The widespread use of polyhalogenated and/or polynuclear aromatichydrocarbons and other organic compounds for various industrial
purposes in recent years has resulted in serious accumulation ofthese compounds or their metabolites in aquatic ecosystems. There
are numerous reports documenting detrimental effects caused bythese compounds on humans, birds, and fish (for review see Safe
1984). In humans, for instance, polychlorinated biphenyls (PCB's)
can cause nausea, anorexia, dermal cysts, comedos, and a vast arrayof other symptoms (Safe 1984). Recent reports by Hickey andAnderson (1968), Peakall (1975), and Eroschenko and Wilson(1975) have shown independently that birds eating fish with highaccumulated levels of halogenated hydrocarbons produced eggs withunusually thin shells, suggesting possible dysfunction in calcium
homeostasis. These eggs also resulted in low hatch. Furthermore,
there are positive correlations between levels of halogenatedhydrocarbon contamination in fish and reproductive dysfunction(Wasserman et al. 1979). Unfortunately, the biochemical mechanisms of the toxic effects of these compounds in fish reproduction await elucidation.
Large populations of fish in the Great Lakes bear a wide rangeof tumors, such as papilloma in white sucker (near 50%), thyroid
hyperplasia in coho salmon (about 80% in Lake Ontario), lymphosarcoma in northern pike or muskellunge (about 20%), and hepatoma in rainbow trout (Sonstegard 1977). In recent years there has
been increasing concern about the effects on oncogenesis in fishfrom nonvolatile halogenated hydrocarbons and other organopollutants in the aquatic environment and the potential health effects
to humans from consuming fish living in such an environment.
Therefore, molecular biological and biochemical studies should be
conducted to investigate the cause of neoplasia formation in fish.
In addition, it is also essential to devise a reliable system for theassessment of potential carcinogenic effects of chemical pollutantsin the aquatic environment.
Although there are methods available for detecting the presenceof polyhalogenated and/or polynuclear aromatic hydrocarbons in
the aquatic environment, these methods are unsuitable for assessing the earliest biological effects caused by these chemical pollutants
at sublethal levels. There is an urgent need to establish rapid,
reliable, and sensitive bioassays for detecting the adverse effectsof these organic pollutants in our aquatic environment at these levels.
Such methods are essential in generating needed information forassessment and management of polluted lakes and rivers because
the effects to humans from consumption of contaminated fish
(especially by pregnant women and young children) have yet to be
determined.
Our laboratory is addressing a number of these issues using rainbow trout as a model fish species. In this paper we shall discuss
some examples to illustrate our approaches.

Molecular toxicology:
A new frontier
THOMAS T. CHEN
Center of Marine Biotechnology
University of Maryland
600 E. Lombard Street
Baltimore, Maryland 21202

REBECCA J. VAN BENEDEN 1
Chesapeake Bay Institute
The Johns Hopkins University
Baltimore, Maryland 21218

LUIS B. AGELWN2
Department of Biology
The Johns Hopkins University
Baltimore, Maryland 21218

DUANE A. HOWARD3RONALD A. SONSTEGARD4
Department of Biology
McMaster University
Hamilton, Ontario LSS 4Kl Canada

ABSTRACT
We are investigating sensitive biochemical and molecular biologicalparameters for assessing pathobiological effects of chemical toxicants
to fish at sublethal levels in the aquatic environment. In rainbow trout,the production of vitellogenin (VG) in the liver can be induced by treatment of fish with 17B-estradiol. The levels of VG in sera of induced
fish can be measured quantitatively by rocket immunoelectrophoresis.
In a series of feeding experiments, it was found that Aroclor and/or
Mirex suppressed the production of VG induced by 17B-estradiol. These
results suggest that the production of VG induced by estradiol can beused as a biocheInical parameter for evaluating the effects of halogenatedhydrocarbons on fish reproduction at sublethal levels. Via molecularcloning, complementary DNA (cDNA) genes to VG and other estrogenresponsive mRNA have been prepared. These cDNA genes have beenused as probes in a quantitative dot blot hybridization analysis to monitor
the expression of estrogen-responsive genes in vivo and in vitro in respouseto the stimulation by 17B-estradiol. By employing the quantitative immunochemical and molecular hybridization assays, we are studying theeffects of halogenated hydrocarbons on fish reproduction at sublethallevels.
The genetic element responsible for the oncogenic activity of avian
myelocytomatosis virus (MC29) has been determined to be the v-mye
gene. Genomic DNA of chicken, human and mouse are found to contain sequences homologous to v-mye, and are termed e-mye. Using v-mye
and chicken e-mye as probes, e-mye gene sequence has also been isolated
from a rainbow trout gene bank. The gene was characterized by restriction enzyme analysis and DNA sequence deterInination. This is the firste-mye gene identified from a lower vertebrate. Using rainbow trout e-mye
as a probe, we have screened northern pike lymphosarcoma DNA for
amplification or rearrangement of e-mye gene. Differences were detectedon Southern blots between endonuclease restriction patterns of DNA
of normal and lymphosarcoma tissues. These results suggest that lymphosarcoma in northern pike may involve rearrangement of e-mye gene.
Work is underway to develop in vitro systems for assessing effects ofcheInical pollutants on oncogene activation in the aquatic enviromnent.

Vitellogenin_

The egg-yolk precursor protein, vitellogenin (VG), in all oviparous
vertebrates is synthesized in the liver under the control of estrogen.
Current addresses:'Duke University Marine Lab., Beaufort, NC 28516.
'College of Physicians and Surgeons, Columbia University, New York, NY 10032.
JSchering Canada, 3535 Trans-Canada Hwy, Pointe Claire, Quebec H9R 184,
Canada.
'Department of Comparative Medicine, School of Veterinary Medicine, Tufts
University, Grafton, MA 01536.

9

The dose-response relationship of VG accumulation in the serumof juvenile rainbow trout following 17B-estradiol treatment has
recently been studied. Estradiol at the concentration of 2 J.lg/loo
g body weight induces detectable levels of VG in the serum. Maximum induction of VG accumulation can be achieved at the doseof 200 J.lg/loo g body weight of the hormone (Chen et al. 1986).
We have begun to investigate the effect of polyhalogenatedhydrocarbons on the induction of VG synthesis in rainbow trout.
Groups of juvenile fish were exposed to Aroclor 1254 and/or Mirex
in the diet. After feeding for 6 months, both control and experimental
animals were treated with 17B-estradiol at the concentration of 20
or 80 J.lg/loo g body weight, and the levels of serum VG were determined by quantitative rocket immunoelectrophoresis. As shown in
Table I, Aroclor 1254, Mirex, and a mixture of Aroclor 1254 andMirex significantly suppressed levels of VG induced by 17Bestradiol (Chen et al. 1986). Although the mechanisms by which
halogenated hydrocarbons exert reproductive toxicity are unknown,
they may be related to altered steroid metabolism (Safe 1984). It
is conceivable that mixed-function oxidase induction by halogenatedhydrocarbons may elevate estradiol catabolism, thus decreasing VG
levels. Similarly, PCB's and Mirex are hepatic toxins in fish as
evidenced by hepatomegaly and histopathological effects (Leatherland et al. 1979). This may lower VG synthesis at the organ level.
Although more in-depth analyses are required, the above results
strongly suggest that estradiol-induced VG synthesis can be used
as a sensitive biochemical parameter for assessing or screening
chemicals that may be hepatotoxins or may disturb reproductive
physiology.
There are some problems inherent in the bioassay describedabove: (I) the relatively long time required for the test chemicals
or hormones to reach the target organ; (2) higher levels of thechemicals and hormones required to show adverse effects; (3) highmortality of test animals due to injury caused by injection of hormones; (4) fluctuation of hormone levels retained in individual
animals due to leakage of hormones. Development of an in vitro
biossay using rainbow trout hepatoma cell line or primary hepatocytes as target cells would help to overcome these problems.

A

B

e

deb

4

8

a

40

E

§.
~

Cl

'Qj
::I:

20

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U

0

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12

Lipovitellin (jtg/10 ILl>

Figure 1
Quantitative measurement of Iipovitellin by rocket immunoelectrophoresis
carried out in a 1.5-mm-thick slab agarose gel (1.0%) containing 0.05%
anti- VG serum of rainbow trout. (A) Increasing amounts of lipovitellinin each sample well was electrophoresed at 40 V overnight at 4°C. (B)Linear relationship between peak height and amount of Iipovitellin in eachwell (from Chen et al. 1986 with permission).

After synthesis, it is secreted into the vascular system and depositedin the oocytes as lipovitellin and phosvitin (Tata and Smith 1979).
In rainbow trout, VG is synthesized as a polypeptide of 160,000
daltons, which is post-translationally processed into its mature formof 170,000 daltons (Chen 1983). Upon treatment of juvenile maleand female or mature male rainbow trout with 17B-estradiol, theproduction of VG can be determined quantitatively by rocket immunoelectrophoresis (Fig. 1; Chen et al. 1986). This method can
detect VG levels as low as 0.05 mg/mL of serum and gives reliable
quantification of VG in both hormone-induced and non-induced fish.

Estrogen-responsive
eDNA genes_

Recently Chen (1983) reported that treatment of mature male orjuvenile rainbow trout of both sexes with 17B-estradiol resulted in
the induction of VG mRNA and another mRNA (ULER2) with asize smaller than that of the 18S rRNA. The cDNA genes of these

Table 1
Effect of Aroclor and/or Mirex on serum vitellogenin levels.
Vitellogenin present in the serum (mg/mL ± SD)
Aroclor 1254

Estradiol(lAg/lOO gbody weight)

Control80205.06±0.45
2.72±0.54

Mirex

Aroclor 1254/Mirex
30/5 IAg/g

2.00±0.86
1.95±0.40

1.12±0.27
1.50±0.23

1.41 ±0.17
1.27±0.23

3.96±0.62
2.51 ±0.56*

2.20±0.5l
1.71 ±0.35

1.1O±0.48
1.54±0.28

1.82±0.30
1.24±0.42

Groups of fish were fed diets contaminated with known quantities of chemicals indicated for a period of 6 months. Serum samples were collected 6 days following treatment
with 17B-estradiol. A group of 20 fish was used per treatment (from Chen et aI. 1986 with permission).
*Not significant using student T-test (P<0.05).

10

I

A a b

c

d

e

h

9

k

m

n

o

B-------'4285

Figure 2
Appearance of estrogen·responsive mRNA in fish treated with 17B·estradiol in vivo. Juvenile rainbow trout each
received 100 I'g/l00 g body weight of 17B-estradiol, and RNA was prepared from livers of the treated fISh at
different time intervals. Following electrophoresis 00 1.4% agarose gels, the RNA was transferred to nitrocellulose
ftIter, and probed with radiolabeled pTRC2 and pRTCS DNA. (A) RNA samples probed with radiolabeled pRTC2.
(8) RNA samples probed with radio-labeled pRTCS. (a) control; (b--g) 12, 24, 48, 72, 96 h after 17B-estradiol
treatment. A second dose of 17B-estradiol (100 I'g/l00 g body weight) was injected into animals 2 weeks after
primary induction. (h--o) 12, 24, 36, 48, 72, 96, 120, 144 h after hormone injection.

protein products and by dot blot hybridization of the mRNA. The
effect of mammalian S-9 activation on the toxicity of the test
chemicals can also be investigated. There are several unique featuresassociated with this testing method: (1) It detects the adverse effects of testing chemicals at the levels of transcription and translation; (2) shorter exposure time is required for the test; (3) every
cell receives homogeneous levels of the test chemicals or hormones;and (4) compared with the in vivo bioassay, lower levels of the test
chemicals and hormones are required.

two estrogen-responsive mRNA sequences have been isolated bymolecular cloning (Chen et al. 1988) and are named as pRTC2 andpRTC5, respectively. Using these two cDNA genes as probes, theexpression of estrogen-responsive genes induced in vivo by 17Bestradiol has been monitored by RNA northern blot analysis (Fig.
2). In order to measure the levels of estrogen-responsive mRNA
sequences in the target tissue accurately, a quantitative molecularhybridization method (dot blot hybridization method) has beendeveloped, using pRTC2 and pRTC5 as probes (Fig. 3; Chen et al.
1988). By employing this method, the dose-response relationshipand the time course of accumulation of estrogen-responsive mRNAin juvenile rainbowtrout following 17B-estradiol treatment have beenstudied (Chen et al. 1988).
Estradiol at a concentration of 1 x 10- 9 was found to induce expression of estrogen-responsive genes in a rainbow trout hepatoma
cell line. This finding has opened the possibility of developing a
rapid in vitro bioassay for screening chemicals that may disrupt the
reproductive physiology of fish. In addition, it also provides an ideal
system for elucidating the actions of chemical pollutants on fish
reproduction at the molecular level. Figure 4 summarizes such a
proposed in vitro testing system, in which rainbow trout hepatocytes
or hepatoma cells are exposed to the test chemicals for various
lengths of time. Following exposure, the cells are treated with l7Bestradiol, and the expression of the estrogen-responsive genes ismeasured quantitativeiy by radioimmunochemical analysis of the

Cellular myc gene(c-myc) in fish_

Avian myelocytomatosis virus (MC29) is a replication-defective
retrovirus that induces a broad spectrum of malignant diseases, including myelocytomas, renal and liver tumors, carcinomas, sarcomas, and erythroblastosis (Bishop 1983). The genetic element
responsible for the oncogenic transformation activity of the virus
has been identified as the v-myc gene of about 1568 base-pairs (bp)
(Lautenberger et al. 1981). Genomic DNA from a variety of species,
from mammals to insects, has been shown to contain sequences
homologous to v-myc by Southern blot analysis (Shilo and Weinberg
1981). However, these sequences have been isolated only bymolecular cloning from chicken, mouse, and human, and are known11

Test chemicalAaPrimary hepatocyte or
hepatoma cell line

b

I

I

c

Primary hepatocyte or
hepatoma cell line
+ 8-9 fraction

I

mRNA

mRNA

I

d

I

dot
hybridization

e

I

dot
hybridization
Immunochemical
analysis ofprotein products

lmmunochemical
analysis ofprotein products

9
Figure 4
Summary of proposed in vitro testing system.

hfunction of the gene. The most primitive class of vertebrates foundto date by Southern blot analysis to contain myc gene sequences
are the bony fishes. Hence, we have initiated studies to isolate andcharacterize the c-myc gene from rainbow trout. Several recombinant phage clones containing c-myc sequences have been isolated
from a rainbow trout gene bank, using v-myc gene as a probe underrelaxed conditions (30% fonnamide, 5 xSSC, 37°C) (Van Beneden
et al. 1986). Further characterization of these clones involved restriction enzyme analysis and hybridization using chicken c-myc exon
II (190 bp Pst-l - Hidf-l fragment) and exon III (849 bp Cia-l EcoRl fragment) as probes. A 3.7 Kb EcoRl DNA fragment fromone ofthe positive clones (RTC-l) was found to contain sequences
homologous to both chicken c-myc exon II and III. This DNA fragment was subcloned into plasmid pBR325, and was tenned Cl-8!.
The nucleotide sequence of this DNA insert was determined by themethod of Maxam and Gilbert (1977). Examination of the nucleotide
sequence of rainbow trout reveals the presence of two exons (IIand III). Trout exon II is shorter (664 bp) than those of chicken(702 bp), mouse (757 bp), and human (771 bp) (Watson et al.
1983a,b; Stanton et al. 1984; Van Beneden 1986). On the other
hand, trout exon III is longer (578 bp) than those of chicken, mouse,and human, all of which have 561 nucleotides. Trout exons II and
III are interrupted by an intron (332 bp) which is considerably
shorter than those of chicken (971 bp), mouse (1.2 kb), and human(1.38 kb). These observations strongly suggest that more primitive
organisms have smaller introns.
The c-myc exon I has been localized for chicken (Linial and
Groudine 1985), mouse (Stanton et al. 1984) and human (Hamlyn
and Rabbits 1983) genes. However, using a DNA probe derived
from either chicken or human exon I, it has not been possible todetect homologous sequences in rainbow trout (Van Beneden et al.
1986). This result suggests that c-myc gene exon I has diverged
more rapidly relative to exons II and III.
The degree of homology in nucleotide and predicted amino acidsequence among c-myc genes of rainbow trout, chicken and humanhas been determined (Van Beneden et al. 1986). Overall, the third
exon is more conserved than the second exon at both nucleotide
and amino acid levels. However, several segments of the second
exon are highly conserved among the three organisms, including
the two myc box regions (A and B) homologous to N-myc (Schwab
et al. 1983). In addition, it is rather interesting to note that these

.

B
12
N

E

E
N

...><

o

:;;'"

8

~

4

o

o

2
standard induced RNA

3
(JA9)

Figure 3
A representative standard curve of the quantitative RNA dot blot hybridization.
Different amounts of total induced RNA in 50 flL was spotted on nitrocellulose
tilters, and then hybridized to rJ2Pl - pRTC2 DNA (l x loS/fig DNA). The
amount of hybridization in each spot was determined by scanning the resulting
autoradiogram. (A) RNA blot; (B) linear relationship between the amount of RNA
in each spot and the amount of hybridization. (a) 4.00 ,.g control RNA; (b-j)
induced RNA 0.125, 0.25, 0.5, 0.75, 1.00, 1.50, 2.00, 3.00, and 4.00 fig.
(Chen et al. 1988 with permission)as cellular myc (c-myc) genes (Watson et al. 1983a,b; Stanton et al.
1984).
The fact that c-myc genes are conserved among many species oforganisms studied suggests that they may play an important rolein cellular growth and/or differentiation (Bishop 1983). Therefore,
comparison of myc gene sequences from widely divergent species
is an important step towards our understanding of the evolution and12Acknowledgments

three c-myc genes have inserted sequences in the second exon uniqueto themselves. There are two such unique regions in the humanc-myc sequence (HUI and HU2), whereas chicken and trout eachcontain a single unique region (CUI and TUI, respectively) (Van
Beneden et al. 1986). In conclusion, the human and chicken c-myc
sequences show more homology to each other than to fish. Thisobservation is consistent with a phylogenetic model in which birds
and mammals share a common lineage that diverged from the line
leading to modern fishes.

_

The authors would like to thank D. M. Davies and A. Oaks forcritically reading this manuscript. This study was supported by
grants to the senior author from the Natural Science and Engineering Research Council of Canada, the Department of Fisheries andOceans, and the U.S. Geological Survey.

Citations

Restriction polymorphism in c-myc genefrom fish tumors_

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1983. Cellular oncogenes and retroviruses. Ann. Rev. Biochem. 52:301-354.
Chen, T.T.
1983. Identification and characterization of estrogen-responsive gene productsin the liver of rainbow trout. Can. J. Biochem. Cell BioI. 61:802-810.
Chen, T.T., P.C. Reid, R.J. Van BenOOen, and R.A. Sonstegard.
1986. Effect of Aroclor 1254 and Mirex on estradiol induced vitellogenin production in juvenile rainbow trout (Sa/rna gairdner). Can. J. Fish. Aquat. Sci.
43: 169-173.
Chen, T.T., D.A. Howard, L.B. Agellon, C.M. Liu, and S.L. Davies.
1988. Estrogen controlled gene expression: Induction of two estrogen-responsive
genes in the liver of rainbow trout (S. gairdneri). Physiol. Zool. (in press).
Erikson, J., A. ar-Rushdi, H.L. Drwinga, P.C. Nowell, and C.M. Croce.
1983. Transcriptional activation of the translocated c-myc oncogene in Burkitt
lymphoma. Proc. Nat!. Acad. Sci. (USA) 80:820-824.
Eroschenko, V.P., and W.O. Wilson.
1975. Cellular changes in the gonads, livers and adrenal glands of Japanese quailas affected by the insecticide kepone. Toxicol. Appl. Pharmacol. 31:491-504.
Gelman, E.P., M.C. Psallidopoulos, T.S. Papas, and R. Dalla-Favera.
1983. Identification of reciprocal translocation sites within the c-myc oncogene
and immunoglobulin u locus in a Burkitt lymphoma. Nature 306:799-803.
Goldfarb, M., K. Shimizu, M. Perucho, and M. Wigler.
1982. Isolation and preliminary characterization of a human transforming genefrom T24 bladder carcinoma cells. Nature 296:404-409.
Harnlyn, P.H., and T.H. Rabbitts.
1983. Translocation joins c-myc and immunoglobulin r I genes in a Burkitt lymphoma revealing a third exon in the c-myc oncogene. Nature 304: 135-139.
Hickey, J.J., and D.W. Anderson.
1968. Chlorinated hydrocarbons and eggshell changes in raptorial and fish-eating
birds. Science (Wash. D.C.) 162:271-273.
Lautenberger, J.A., R.A. Schulz, C.F. Garon, P.N. Tsichlis, D.O. Spyropoulos,
T.W. Pry, K.E. Rushlow, and T.S. Papas.
1981. The transforming sequences of avian myelocytomatosis virus (MC29).
J. Supramol. Stroct. Cell. Biochem. 16:193-207.
Leatherland, J.F., R.A. Sonstegard, and H.V. Holdrinet.
1979. Effect of dietary Mirex and PCB's on hepatosomatic index, liver lipid,
carcass lipid, and PCB and Mirex bioaccumulation in yearling coho salmon(Oncorhynchus kisutch). Compo Biochem. Physiol. 63:243-246.
Linial, M., and M. Groudine.
1985. Transcription of three c-myc exons is enhanced in chicken bursal lymphoma cell lines. Proc. Nat!. Acad. Sci. (USA) 82:53-57.
Maxam, A.M., and W. Gilbert.
1977. A new method for sequencing DNA. Proc. Natl. Acad. Sci. (USA)74:560-564.
Peakall, D.B.
1975. PCB's and environmental effects. CRC Crit. Rev. Environ. Control, p.
469-511.
Phillips, D.H., and C.J. Marshall.
1984. Activation of a cellular proto-oncogene by in vitro modification with achemical carcinogen. In Proc. RNA Tumor Viruses Meeting, p. 112. Cold
Spring Harbor Lab., Cold Spring Harbor, NY 11724.
Safe, S.
1984. Polychlorinated biphenyls (PCB's) and polybrominated biphenols (PBB's):
biochemistry, toxicology, and mechanism of action. CRC Crit. Rev. Toxicol.
13:319-395.
Santos, E.
1984. Malignant activation of a k-ras oncogene in lung carcinoma but not innormal tissue of the same patient. Science (Wash. D.C.) 223:661-664.

Both human Burkitt's lymphoma and mouse myeloma cells display
specific chromosomal translocations involving immunoglobulin geneloci and the c-myc gene (Erikson et al. 1983; Gelman et al. 1984;
Stanton et al. 1984). As a result of the c-myc gene translocation,
the levels of myc mRNA are greatly elevated in the tumor cells.
Although the biological function of the c-myc gene remains to beelucidated, it is believed that abnormal expression of this gene could
lead to oncogenesis. Lymphosarcoma has also been found in several
fish species, such as northern pike and muskellunge collected fromthe field (Sonstegard 1976). Studies conducted by Whang-Peng etal. (1976) showed karyotypic abnormality associated with lymphosarcoma of northern pike. Therefore, it would be of interest to investigate whether rearrangement or translocation of the c-myc geneis associated with these tumors. High-molecular-weight DNA
isolated from normal and tumorous tissues of northern pike wasused to prepare Southern blots. These blots were hybridized to nicktranslated trout c-myc sequences. A 5.8 Kb fragment generated by
EcoRI digestion of the DNA sample from the normal kidney tissueof pike hybridized to the probe. On the other hand, two fragmentsof 6.2 and 4.4 Kb from feral lymphosarcoma DNA hybridized tothe same probe. The findings suggest that rearrangement and/ortranslocation of the c-myc gene may have occurred in the northern
pike lymphoma cells.
Activated oncogenes are found in association with chemically induced or naturally occurring neoplasm in mammals. For instance,
activated c-ras gene is found in human bladder and lung carcinoma(Goldfarb et al. 1982; Santos 1984), and an activated c-myc geneis found in human Burkitt's lymphoma (Erikson et al. 1983; Gelman
et al. 1983; Stanton et al. 1984). Furthermore, a c-ras gene has
been activated by treatment with chemicals in vitro (Phillips andMarshall 1984). These findings suggest that activation of a specificoncogene can be regarded as an early sign of oncogenesis. In addition, chemicals that can activate any specific cellular oncogene invivo or in vitro should be considered prime suspects as carcinogens.
As mentioned earlier, large populations of fishes have tumors of
one type or the other; hence, there is a need for investigating therelationship between neoplasia in fishes and chemical pollutants present in waters. Besides, there is an urgent need to devise reliable
and sensitive methods for assessing the carcinogenic potentials ofchemical pollutants in contaminated waters. We are actively working
toward developing an in vitro bioassay for this purpose.

13

Schwab, M., K. AIitalo, K. K1empnauer, K.H. Varmus, J.M. Bishop, F. Gilbert,
G. Brodeur, M. Goldstein, and J. Trent.
1983. Amplified DNA with limited homology to myc cellular oncogene is sharedby human neuroblastoma cell lines and a neuroblastoma tumor. Nature 305:245-248.
Shilo, B.Z., and R.A. Weinberg.
1981. DNA sequences homologous to vertebrate oncogenes are conserved in
Drosophila melanocasler. Proc. Nat!. Acad. Sci. (USA) 78:6789-6792.
Sonstegard, R.A.
1976. Studies of the etiology and epizootiology oflymphosarcoma in Esox (Esoxlucius L. and Esox masquinongy) from the United States. Prog. Exp. Tumor
Res. 20: 141-155.
1977. Environmental carcinogenesis studies in fishes of the Great Lakes of North
America. Ann. N.Y. Acad. Sci. 298:261-269.
Stanton, L.W., P.D. Fahrlander, P.M. Tesser, and K.B. Marcu.
1984. Nucleotide sequence comparison of normal and translocated murine c-myc
genes. Nature 310:423-425.
Tata, J.R., and D.P. Smith.
1979. Vitellogenesis: a versatile model for hormonal regulation of gene expression. Rec. Prog. Hormone Res. 35:47-56.
Watson, D.K., E.P. Reddy, P.H. Duesberg, and T.S. Papas.
1983a. Nucleotide sequence analysis of the chicken c-myc gene reveals homologous and unique coding regions by comparison with the transforming gene ofavian myelocytomatosis virus MC29. Agag-myc. Proc. Nat\. Acad. Sci. (USA)80:2146-2150.
Watson, D.K., M.e. Psallidpoulos, K.P. Samuel, R. Dalla-Favera, and T.S.
Papas.
1983b. Nucleotide sequence analysis of human c-myc locus. chicken homologue,
and myelocytomatosis virus MC29 transforming gene reveals a highly conserved
gene product. Proc. Nat!. Acad. Sci. (USA) 80:3642-3645.
Van Beneden, R.J., D.K. Watson, T.T. Chen, J.A. Lautenberger, and T.S.
Papas.
1986. Cellular myc (c-myc) in fish (rainbow trout): Its relationship to othervertebrate myc genes and to the transforming genes of the MC29 family ofviruses. Proc. Nat!. Acad. Sci. (USA) 83:3698-3702.
Wasserman, M., D. Wasserman, S. Cucos, and H.J. Miller.
1979. World PCBs map: Storage and effects in man and his biologic environment in the 1970s. Ann. N.Y. Acad. Sci. 320:69-123.
Whang-Peng, J., R.A. Sonstegard, and C.J. Dawe.
1976. Chromosomal characteristics of malignant lymphoma in northern pike
(Esox lucius) from United States. Cancer Res. 36:3554-3560.

14

Lipids have been regarded only as energy sources in fish diets until
recently. Recent studies, however, have strongly emphasized theimportance of lipids in fish diets as sources of essential fatty acids(EFA) and as carriers of fat-soluble biologically active substances(Watanabe 1982). In contrast to fish and other higher animals, theprawn Penaeus japonicus is unique in that it requires both sterols
and some phospholipids for good growth and survival (Teshima
1978, 1983). The present paper reviews our current investigationon the nutrition of sterols and phospholipids, with particular emphasis on the nutritive values of sterols other than cholesterol andalso the mechanism by which dietary phospholipids exert a growthenhancing effect.

Necessity of dietary sterols
and phospholipids for
growth of the prawn,
Penaeus japonicus Bate
SHIN-ICHI TESHIMAAKIO KANAZAWA
Faculty of Fisheries
Kagoshima University
4-50-20 Shimoarata
Kagoshima. 890 Japan

Sterol nutrition_

The prawn P. japonicus (Kanazawa et al. 1971, Teshima et al.
(983), and the American lobster Homarus americanus (Castell etal. 1975) have been shown to require dietary sources of sterol fornormal growth, resulting from a lack of de novo sterol synthesis
in crustaceans (Teshima (978). Previous studies have also shownthat juvenile prawns utilized some C28 and C29 sterols as substitutes
for cholesterol to some extent, suggesting the possible C-24
dealkylation of these sterols to cholesterol, a C27 sterol, in the body
(Teshima 1983, Teshima et al. 1983). However, little is known of
the effects of sterols other than cholesterol on the feed conversion
efficiency (FCE), protein efficiency ratio (PER), and retention rate(%) of dietary lipids and sterols in the prawn body.
To approach this, recent feeding trials were conducted using
prawn juveniles, weighing about 0.25 g each (Teshima and Kanazawa 1985b). The prawns were maintained on test diets (Table 1)
containing 0.5 % levels of cholesterol, (J-sitosterol, a 9: 1 mixtureof (J-sitosterol and cholesterol, and 24-methylcholesta-5,22-dienol
as a sterol source, or a sterol-free diet in 3D-liter aquaria for 32
days at 24-28°C. The base ration oftest diets was the same as thatreported previously, except for lipid sources and the binder (gluten
M; Riken Vitamin Co., Ltd., Japan) (Kanazawa et al. 1977). Test

ABSTRACT
From the viewpoint of comparative biochemistry and nutrition,
lipid nutrition of the prawn Penaeus japonicus is unique in thatit requires dietary sources not only of sterols but also of some
phospholipids for growth and survival. Our reexamination ofsterol requirements for juvenile prawn has showm that theabsence of sterols in diets results in a marked decrease in weight
gain, feed conversion efficiency (FCE), and protein efficiency
ratio (PER). In terms of weight gain, FCE, and PER, cholesterol
had the highest nutritive value as a sterol source, with
24-methylcholesta-5,22-dienol, (J-sitosterol-eholesterol (9:1), and(J-sitosterol following in descending effectiveness. Cholesterol
was more effectively retained in the prawn's body than
24-methylcholesta-5,22-dienol and (J-sitosterol.
Some phospholipids in the diets were shown to be indispensable for sustaining growth and survival of the prawn; especially the larvae, which suffered 100% mortality prior to attaining
the mysid stage when fed on diets without phospholipids. Feeding
experiments have revealed that the requisite for effective
phospholipids was possession of choline and inositol groups andunsaturated fatty acid moieties in the molecules. Recently, we
have conducted feeding experiments using 14C-Iabeled lipids toclarify the mechanism by which dietary phospholipids enhance
the growth of the prawn. Results suggest that retardation of
growth in the prawn receiving a phospholipid-deficient dietresults from the insufficient transport of dietary lipids, particularly cholesterol, in the body. Understanding nutritional requirements of the prawn win contribute to improvement of techniques
for rearing larval and juvenile prawns using live feeds andartificial diets.

Table 1
Composition of test diets and results of a 32-day feeding trial on juvenile P.
japonicus prawns.
Test
diet I
D-I
D-2
D-3
D-4
D-5

Dietary sterol
0.5% Cholesterol
0.5 % {J-Sitosterol
0.45% {J-Sitosterol +0.05 % Cholesterol
0.5% 24-Methylcholesta5,22-dieno1 4
Sterol-deficient

Survival
rate (%)

Weightgain (%)

FCE 2

PER3

90
90
80

200
118
104

0.70
0.57
0.64

1.55
1.26
1.42

75

136

0.65

1.45

70

55.2

0.37

0.82

1Base ration of test diets was the same as that reported previously (Kanazawa
et al. 1977), except for the binder and lipid sources; 8% pollock liver oil usedin the previous study was replaced with 6 % pollock liver oil and 2 % soybean
lecithin. Twenty prawns were used for each experimental group at start.
2Feed conversion efficiency (FCE) = Avg. weight gain (g)/Avg. food intake(g).

3Prolein efficiency ratio (PER) = Avg. weight gain (g)/Avg. protein intake (g).
424-Methylcholesta-5,22-dienol was a mixture of the 24R- and 24S-isomers(about 1:1, w/w).

15

Table 2

D-l

Body retention rates (%) of dietary proteins, lipids, and total sterols in P.
japonicus prawns fed different sterols.
Body retention rate (%) I
Dietary sterol

150

0-4

...

0-2
0-3

s::

'"

0>

+'

..c

Cholesterol
fl-Sitosterol
fl-Sitosterol-Cholesterol (9: I)
24-Methylcholesta-5,22-dienol
Sterol-deficient (diet D-5)

100

'"

Proteins

LipidsTotal sterols

33.9
20.3
21.8
26.7
9.5

64.5
32.7
39.2
39.4
-2.9

91.7
47.8
52.8
79.3

'Body retention rate (%) = B X IDOlA
A = Intake of dietary nutrient (g)/one prawn
B = Increase in the nutrient of body (g) lone prawn

'"

3

0-5

50

o

10

20

30

Feeding period (days)

Cho 1es tero 1 (CH)"':':":"":"';:':;'::;::';::::::';"'::,:",::::1 CH~-5itosterol (51)

U.illlI5mIIllllIllll

Total sterols

Total sterols

ft-5 i tos tero 1-<:::;:':;:::::':'::';;:":::::::':"'::;:":"::::::;::::"::"::;'::':::;:::;:::;::';:::;::;;;':::::1 CH
Cholesterol (9:1) • 51

Figure 1
Weight gains (%) of P. japonicus prawns reared on diets containing different
sterols. 0-1 to 0-5 indicate test diets (see Table 1).

Total sterols
24-Methylcholesta-.
5,22-dienol(24-MC)

diets contained 6.0% pollock liver oil, 2.0% soybean lecithin, and
0.5 % cholesterol as lipid sources.
Figure I shows the growth of the prawns receiving different
sterols. Weight gain was markedly low on a sterol-deficient diet
(diet D-5). The addition of each sterol examined improved theweight gain. However, cholesterol was most effective in improving the weight gain as previously observed in the larvae (Teshima
et al. 1983), with 24-methylcholesta-5,22-dienol, the 9: 1 mixtureof {3-sitosterol and cholesterol, and {3-sitosterol following. As for
the FeE and PER, cholesterol gave the highest values among thesterols examined (Table I). Interestingly, however, 24-methylcholesta-5,22-dienol resulted in an FCE and PER equally as highas cholesterol.
After the feeding trial, lipid, sterol, and protein concentrationsof prawn bodies were analyzed to determine body retention rates(%) of these dietary nutrients. Prawns receiving dit:ts containing{3-sitosterol or 24-methylchoiesta-5,22-dienol had small amounts of
24-methylenecholesterol (3.3-4.5% of total sterols), one of the possible intermediates in the C-24 dealkylation of {3-sitosterol and24-methylcholesta-5,22-dienol to cholesterol. Body retention rates
of dietary proteins and lipids were notably lower on the steroldeficient diet than on the sterol-supplemented diets (Table 2). Body
retention rates of total dietary sterols varied with the kind of sterols.
Cholesterol gave the highest body retention rate of total dietary
sterols, with 24-methylcholesta-5,22-dienol, {3-sitosterol-cholesterol(9: 1), and {3-sitosterol following with descending values.
Figure 2 shows the body retention rates of individual sterols.
Cholesterol was retained in the whole body more effectively than
{3-sitosterol and 24-methylcholesta-5,22-dienol. In prawns receiving a mixture of {3-sitosterol and cholesterol (9: 1), the retention rateof dietary cholesterol exceeded 100%. This suggests the bioconversion of {3-sitosterol to cholesterol in the body. because the prawn
is incapable of de novo sterol synthesis and all sterols occurring

24-MC
Total sterols- ' 0 - - -.....
5'0----'10'-0---.1J----.-,tooRetenti on (%)

Figure 2
Body retention rates (%) of individual sterols in P. japonicus prawns reared ondiets containing different sterol sources.

in the tissues are of dietary origin and/or their metabolites (Teshima1983).
Kanazawa et al. (1971) have demonstrated that survival rates ofjuvenile prawns receiving ergosterol, stigmasterol, or {3-sitosterol
were high, but growth was inferior when compared with that ofprawns receiving cholesterol as a sterol source. In addition, prawn
larvae have also been shown to grow well with high survival rates
on the microparticulate diets containing 22-dehydrocholesterol,
24-methylenecholesterol, ergosterol, or isofucosterol (Teshima etal. 1983).
Considering these facts and observations, we conclude the following. First, dietary sources of some C 28 and C 29 sterols are likely
to be utH ized by larval and juvenile prawns for growth after beingtransformed to cholesterol via 24-methylenecholesterol anddesmosterol in the body in a sequence similar to that found in insects (Svoboda et aI. 1978). It therefore seems reasonable to believethat a nutritive value of C28 and C29 sterols as substitutes forcholesterol depends on how easily they are converted to cholesterolin the body after being ingested. Since 24-methylcholesta-5,22dienol had a higher nutritive value than {3-sitosterol to both juveniles<Table I) and larvae (Teshima et aI. 1983), the former may be con16

Table 3
Weight gain, survival rate, feed conversion efficiency (FeE), and body retenlion rate (%) of dietary lipids in prawns receiving phospholipid-added (A)and phospholipid-deficient (D) diets.

150

Diet A

RemarkNumber of prawns
Feeding period (day)Initial avg. body weight (g)Final avg. body weight (g)Body weight gain (%)
Survival rate (%)
FCE (gain/diet intake)
Body retention rate (%)2
Total lipids
Cholesterol
Neutral lipids except cholesterol

100

c
.~

'"

en
+-'
..<:

en

Diet D •

OJ

3

-8
o
co

50

./
.~
20

Diet 0'(PL-deficient)

15
30
\.03
2.51
144
100
0.49

15
30
\.72
62.3
80
0.22

4\.8
88.0
29.0

30.3
32.3
2\.6

\.06

'Basa! ration of diets A and 0 was similar to that reported previously (Kanazawa et aI. 1977) except for a binder (gluten M; Riken Vitamin Co., Ltd.). Diet
A contained the following ingredients (g/loo g): casein 50.0; glucose 5.5; sucrose
10.0; a-starch 4.0; glucosamine HCI 0.8; sodium citrate 0.3; sodium succinate,O.3;
DL-methionine \.0; L-tryptophan 0.5; pollock liver oil 6.0; cholesterol 0.5;
vitamin mixture 3.0; mineral mixture 8.6; soybean lecithin 3.0; cellulose 6.5 (equal
to 100%). To this, 8.0% gluten M was added. Diet 0 contained the same composition as diet A, except for the absence of 3.0% soybean lecithin.
2Body retention rate (%) = B X loo/A
A = Intake of dietary nutrient (g)/one prawn
B = Increase in the nutrient of body (g)/one prawn.

O+------.------r--------r
10Diet A'(PL-added)

30

Feeding period (days)
Quite recently, we have examined the effects of dietary phospholipids on the growth, body retention rates of dietary lipids, and bodycomposition of juvenile prawns as part of defining the nutritionalrole of dietary phospholipids (Teshima and Kanazawa 1985a). In
a 3D-day feeding trial using juveniles weighing about 1 g each,weight gain was markedly lower on a phospholipid-deficient dietthan on a 3% soybean lecithin-added diet (Fig. 3, Table 3) . The
FCE, survival rate, and retention rates of dietary lipids and sterols
were also lower on the phospholipid-deficient diet than on thephospholipid-added diet (Table 3). Thus, the prawn was shown to
be incapable of effectively utilizing dietary lipids, especiallycholesterol, for growth when fed the phospholipid-deficient diet.
Since the digestibilities of dietary lipids such as triglycerides andcholesterol in the prawn are high, more than 85% (Teshima andKanazawa 1983), we thought that dietary phospholipids might takesome part in the lipid transport in the body. Our next experiments,
therefore, investigated the effect of dietary phospholipids (3 % soybean lecithin) on the postprandial variation in lipid concentrationsof the hepatopancreas, hemolymph, muscle, and so on, after feedingof diets containing 6% pollock liver oil and 0.5% cholesterol asbasal lipids. Three and six hours after feeding, the lipid concentrations of hepatopancreas and hemolymph were higher in prawns
receiving the phospholipid-added diet than in those receiving thephospholipid-deficient diet (Fig. 4) . The increase in lipid concentrations of the hepatopancreas and hemolymph in prawns receiving the phospholipid-added diet was due mainly to the rise intriglycerides, cholesterol, and phospholipids such as phosphatidylcholine. As for the muscle, however, the two diet groups did notshow a notable difference in the postprandial variation in lipid compositions. These results indicate that dietary phospholipids accelerate
the mobilization of lipids such as dietary triglycerides and cholesterol
from the digestive gland and hepatopancreas to the hemolymph andother extrahepatic tissues. To confirm this, the postprandial variation in radioactive lipid classes was investigated after feeding of

Figure 3
Weight gains (%) of P. japonicus prawns receiving phospholipid-added(A) and phospholipid-deficient (D) diets.

verted to cholesterol in the body more easily than the latter. Second,
dietary cholesterol plays an important role in the effective utilization of dietary lipids and proteins in the prawn, as the lack ofcholesterol in diets results in a marked decrease in protein and lipid
levels in the body. We assume that sterols may function primarily
as components of subcellular membranous structures in the prawn
as well as in insects (Clayton 1964, Svoboda et al. 1978). Sincecholesterol was retained in the body more effectively than
24-methylcholesta-5,22-dienol and (3-sitosterol (Fig. 2), cholesterol
may be required to fill a structurally specific space in the subcellular
membranes of this prawn. The role of cholesterol and other sterols
in crustacean species remains to be more fully elucidated in the
future.

Roles of dietary phospholipids_

Some phospholipids in diets have also been demonstrated to be indispensable for sustaining growth and survival of crustaceans suchas the prawn (Kanazawa 1983, Teshima 1985) and the American
lobster (Conklin et al. 1980, D'Abramo et al. 1981); especially toprawn larvae which suffered 100% mortality prior to attaining the
mysid stage when they were fed a phospholipid-deficient diet. We
have also shown that the requisite for effective phospholipids inprawn larvae was possession of choline or inositol groups in addition to unsaturated fatty acid moieties in the molecules (Kanazawa
et al. 1985, Teshima 1985). However, it was not clear why dietaryphospholipids exerted a growth-enhancing effect in the prawn P.
japonicus.
17

(%)
V>

'"'<lic::"."--./.

0+''""'<li

:r

2.0

0

3

CH

0.2

0.5

0.3

0

6 hr

3

6 hr

0.5

(%)

(%)

(%)

0.3

~
TL

.c::

"'-

E
>,

"0

0.2

E

<li

:r

PC

0.7

<:=:

0.4

u

'""'- 3.0

(%)

(%)

TL

4.0

~
-.-CH •

0.04

0.02

0.1

3

0

6 hr

~
~.
PC

0.3

.

0.1
i

0

3
Holding period

6 hr

0

3
Holding period

6 hr

0

3
Holding period

6 hr

Figure 4
Postprandial variation in total lipid (TL), cholesterol (Cm, and phosphatidylcholine (PC) concentrations (% of fresh matter) in the hepatopancreas andhemolymph of P. japonicus prawns after feeding of phospholipid-added (0) and phosphOlipid-deficient (.) diets.

tripalmitin- 14C and cholesterol- 14C with or without dietary phospholipids.
When the prawns received tripalmitin- 14 C, the inclusion ofphospholipids (3 % soybean lecithin) in the diets resulted in an increase in radioactive phospholipids, especially phosphatidylcholine,
in both the hepatopancreas and hemolymph (Teshima et al. 1986a).
The supplementation of phospholipids to diets also brought on an
increase in radioactive triglycerides in the hepatopancreas but notin the hemolymph. Figure 5 (Teshima et al. 1986b) shows the postprandial variation in free and esterified sterol fractions of thehepatopancreas and hemolymph after feeding of cholesterol- 14 C.
When fed the phospholipid-added diet, the radioactivities of hepatopancreatic free and esterified sterols increased quickly and reachedmaximum levels 3 and 1 hours after feeding, respectively, and thendecreased, while those of free and esterified sterols in the hemolymph increased with the lapse of time and reached maximum levels
9 hours after feeding. Thus, when fed the phospholipid-deficientdiet, dietary cholesterol remained as a free sterol for a long timein the hepatopancreas and entered into the hemolymph slowly, andalso the formation of cholesteryl esters in the hepatopancreas proceeded at a slow rate. These results suggest that dietary phospholipids accelerate the incorporation of dietary cholesterol to thehepatopancreas, the formation of cholesteryl esters in the hepatopancreas, and the movement of hepatopancreatic cholesterol to thehemolymph.
We have shown that lipid transport in the prawn P. japonicus
was basically conducted in the form of lipoproteins containing abundant phospholipids (Teshima and Kanazawa 1980). Considering thepresent observations and information available to date, we believethat dietary phospholipids are necessary for the smooth transportof dietary cholesterol, an essential nutrient, rather than dietary
triglycerides in the body. Although dietary lipids such as cholesterol

and triglycerides could easily enter the hepatopancreas regardlessof the presence of phospholipids in diets (Teshima and Kanazawa
1980), they (especially cholesterol) may not be released effectively from the hepatopancreas to the hemolymph when diets are lacking in phospholipids. We suggest that the prawn P. japonicus
possibly cannot satisfactorily synthesize the phospholipids that are
utilized as constituents of lipoproteins responsible for transport ofcholesterol and other lipids.
There is no information on the mechanism by which dietarycholesterol is transported in crustaceans. In higher animals suchas mammals, most of the dietary cholesterol comes into the blood
system after being esterified by the action of lecithin:cholesterolacyltransferase (LCAT), which catalyzes the transfer of an acylgroup from the (J-position of phosphatidylcholine to the 3{J-hydroxygroup of cholesterol. Mankura et al. (1980) have demonstrated thatthe hemolymph of the crab Portunus trituberculatus has a low but
substantial LCAT activity and also that the LCAT activity of thiscrab is highly increased by the addition of hepatopancreatic lecithinfrom the carp Cyprinus carpio. We assume that the retardation of
growth in prawns receiving the phospholipid-deficient diet ispossibly due to the insufficient transport of lipids, especiallycholesterol, in the body. In the prawn, dietary phospholipids,
specifically phosphatidylcholine containing unsaturated fatty acidmoieties, may be utilized as constituents of lipoproteins responsible for cholesterol transport and also may accelerate LCAT activity
in the hemolymph.

18

600

.:

50

'+-

'0
I.-

~ 400
Vl

M

0

Hepatopancreas

<U
<U
l.LL

X

30

200

E

0.

-0

10
-0

<U
I.<U

>

3

0
U
<U

.:0.--,
24 hr

9

9

3

'r----,
24 hr

I.-

>,
+'

80

>

:;:;
u

.:

'+-

'"
a:
'" +'
0

-0

I.<U
Vl

60

<U

>,
I.-

<U

+'

""7-

____e
10
Hemolymph

6

V)

40
2
~

3

9

~

3

24 hr

9

24 hr

Holding period

Holding period

Figure 5
Postprandial incorporation of radioactivity into free sterol and esterified sterol fractions of the hepatopancreas and hemolymph
in P. japonicus prawns after feeding of cholesterol-4-'4C in (0) phospholipid-added diets and (e) phospholipid-deficient diets.

Conclusions_

Citations

The prawn P. japonicus required dietary sources of both sterols
and some phospholipids for growth and survival. The lack of dietary
sterols resulted in a marked decrease in weight gain, FCE, and bodyretention (%) of dietary proteins, lipids, and sterols in juvenile
prawns. As a sterol source, cholesterol had a higher nutritive value
than (3-sitosterol, (3-sitosterol-cholesterol (9: I), and 24-methycholesta-5,22-dienol. The nutritive values of C 28 and C 29 sterols forthe prawn are likely to depend upon how easily they are converted
to cholesterol in the body.
The lack of dietary phospholipids in diets also reduced the growthof the larval and juvenile prawns. As a phospholipid source,
phosphatidylcholine and phosphatidylinositol containing unsaturated
fatty acid moieties were effective in improving growth and survivalof prawn larvae. To clarify the role of dietary phospholipids in theprawn, the postprandial variation in lipid compositions of thehepatopancreas, hemolymph, muscle, etc., was invesligated after
feeding diets containing tripalrnitin- 14C and cholesterol- 14C with orwithout 3 % soybean lecithin. It seems likely that dietaryphospholipids are required for the smooth transport of dietary lipids,
especially cholesterol, through the hemolymph.

Castell, J.D., E.C. Mason, and J.F. Covey.
1975. Cholesterol requirements of juvenile American lobster (Hamarus
americanus). l. Fish. Res. Board Can. 38: 1431-1435.
Claylon, R.B.
1964. The utilization of sterols by insects. l. Lipid Res. 5:3-19.
Conklin, D.E., L.R. D'Abramo, C.E. Bordner, and N.A. Baum.
1980. A successful purified diet for the culture of juvenile lobsters: The effectof lecithin. Aquaculture 21 :243-249.
D'Abramo, L.R., C.E. Bordner, D.E. Conklin, and N.A. Baum.
1981. Essentiality of dietary phosphatidylcholine for the survival of juvenile
lobsters. J. Nutrition III :425-431.
Kanazawa, A.
1983. Penaeid nutrition. In Pruder, G.D.• C. Langdon, and D.E. Conklin (eds.),
Proceedings of the Second International Conference on Aquaculture Nutrition,
p. 87-105. Spec. Pub\. 2, World Maricult. Soc., La. State Univ., Baton Rouge.
Kanazawa, A., N. Tanaka, S. Teshima, and K. Kashiwada.
1971. Nutritional requirements of prawn. II. Requirement for stero\. Bull. Jpn.
Soc. Sci. Fish. 37:211-215.
Kanazawa, A., S. Teshima, and S. Tokiwa.
1977. Nutritional requirements of prawn. VII. Effect of dietary lipids on growth.
Bul\. Jpn. Soc. Sci. Fish. 43:849-856.
Kanazawa, A., S. Teshima, and M. Sakamoto.
1985. Effects of dietary lipids, fatty acids, and phospholipids on growth andsurvival of the prawn (Penaeus japanicus) larvae. Aquaculture 50:39-49.
Mankura, M., D. Dalimunthe, and M. Kayama.
1980. Comparative biochemical studies on plasma cholesterol. II. Relationshipbetween plasma esterified cholesterol and lecithin: cholesterol acyltransferase
activity. Bull. lpn. Soc. Sci. Fish. 46:583-586.
Svoboda, J.A., M.J. Thompson, W.E. Robbins, and J.N. Kaplanis.
1978. Insect steroid hormone metabolism. Lipids 13:742-753.

19

Teshima, S.
1978. Essential fatty acids and necessity of sterols in crustaceans. In JapaneseSociety of Scientific Fisheries (cd.), Fish culture and dietary lipids. Suisangaku
SeL 22, p. 60-77. Koseisha-koseikaku. Tokyo (in lpn.).
1983. Sterol metabolism. In Pruder, G.D., C. Langdon, and D.E. Conklin (cds.),
Proceedings of the Second International Conference on Aquaculture Nutrition,
p 205-216. Spec. Pub!. 2. World Maricult. Soc., La. State Univ., Baton Rouge.
1985. Lipids. In Yone, Y. (cd.), Fish nutrition and diets. Suisangaku SeL 54,
p. 20-30. Koseisha-koseikaku, Tokyo (in Jpn.).
Teshima, S., and A. Kanazawa.
1980. Lipid transport in crustaceans. Min. Rev. Data File Fish. Res. Kagoshima
Univ. 1:1-25.
1983. Digestibility of dietary lipids and cholesterol in the prawn. Bull. Jpn. Soc.
Sci. Fish. 49:963-966.
1985a. Role of dietary phospholipids in the utilization of lipids and cholesterolby the prawn (Penaeus japonicus). Abstracts of Papers. Biological Assessmentof Nutritional Requirements and Availability in Fish. Satellite Symposium, XIll,
International Congress of Nutrition, held at Brighton, U.K., 17-18 August 1985.
1985b. Sterol nutrition in the prawn, Penaeus japonicus - I. The nutritive valuesof cholesterol, 24-methylcholesta-5,22-dienol, and {J-sitostero!. Abstracts,
autumn meeting, lpn. Soc. Sci. Fish, p. 105.
Teshima, S., A. Kanazawa, and H. Sasada.
1983. Nutritive value of dietary cholesterol and other sterols to the larval prawn,
Penaeus japonicus. Aquaculture 31:159-167.
Teshima, S., A. Kanazawa, and Y. Kakuta.
1986a. Role of dietary phospholipids in the transport of ["C] tripalmitin in theprawn. Bull. Jpn. Soc. Sci. Fish. 52:519-524.
1986b. Role of dietary phospholipids in the transport of ["C] cholesterol in theprawn. Bull. Jpn. Soc. Sci. Fish. 52:719-723.
Watanabe, T.
1982. Lipid nutrition in fish. Compo Biochem. Physio!. 73B:3-15.

20

Applications of endocrinology
to salmon culture: Hormonal
induction of spawning ofadults and hormone patterns
during developmentof juveniles

The many applications of endocrinology in fish culture can be
categorized into two major types. One involves establishing the functions of hormones and measuring their activities during normal
physiological processes. The other involves the application ofhormones (or their synthetic agonists or antagonists) in order to control some aspect of the animal's physiology. Two examples of thesetypes of applications will be discussed. The changes in blood plasma
concentrations of hormones during the parr-to-smolt transformation of juvenile salmon reveal a complex pattern of endocrine
activities that may be used to predict the optimal time for transferring salmon from fresh- to seawater. The use of hormones to
induce spawning of captive broodstock salmon in either fresh wateror seawater is a good example of applications of hormones tofacilitate broodstock husbandry.
The developmental period known as the parr-to-smolt transformation (smoltification) prepares the freshwater resident juvenile
salmon for downstream migration and entry into the estuary andocean. Smoltification is associated with a variety of morphological,
biochemical, and behavioral changes in the fish. This developmental
process appears to be influenced primarily by photoperiod andtemperature, among other environmental factors. Control of smoltification is mediated at least in part through neuroendocrine andendocrine mechanisms. Histological studies have implicated involvement of the thyroid, pituitary, interrenal, pancreas, Corpuscles ofStannius, ultimobranchial, urohypophysis and catecholaminergic(head kidney) tissue. Application of radioimmunoassay to determine blood plasma concentration of hormones has verified a rolefor some of these glands and implicated others as possible mediatorsof smoltification. Increased activity of the thyroid during smoltification is associated with increases in plasma thyroxine (T4) and,depending on the species, triiodothyronine concentration. Plasma
cortisol levels increase subsequent to or coincident with the increasein circulating T4. In some cases, plasma estradiol concentration
has been shown to follow a pattern of increase and decrease similar
to that ofT4. Daytime levels of plasma melatonin appear to be constant throughout smoltification. Plasma levels of insulin increasein parr immediately prior to the beginning of smoltification. A
similar increase in insulin-like growth factor I (IGF I) may also
occur at this time. Plasma levels of norepinephrine and epinephrine
become elevated near the end of smolt development. The large
number of pituitary-dependent and independent hormones thatchange during smoltification suggests that there is a hypothalamicor general endocrine activation during juvenile development ofsalmon (Dickhoff and Sullivan 1987). The appropriate order orsequence of activation of the various endocrine glands during normal development remains to be established. Furthermore, the interactions of various hormones that change during smoltification need
to be examined.
The natural process of spawning in teleost fish is accomplishedthrough neuroendocrine and endocrine pathways. Environmental
stimuli (photoperiod, temperature) are perceived by the brain which
regulates the release of neuropeptides and catecholamines which
in turn control the release of gonadotropic hormone (GtH) by thepituitary gland. Pituitary GtH released into the blood acts on the
gonads to promote the release of gametes (spermiation and ovulation). Release of GtH by the pituitary is stimulated by the brain
peptide, gonadotropin-releasing hormone (GnRH). In teleost fishes
spawning can be induced by the injection ofGtH or GnRH. A synthetic analogue of GnRH, desGlylO, D-Ala 6GnRH, (GnRHa), isparticularly effective in inducing spawning of salmonid fish.

WALTON W. DICKHOFF
National Marine Fisheries Service, NOAANorthwest and Alaska Fisheries Center
2725 Montlake Blvd. E.
Seattle, Washington 98112
andSchool of Fisheries WH-JO
University of Washington
Seattle, Washington 98195

21

Acknowledgments

Salmonids normally spawn in fresh water, although some species
may spawn in brackish water. Attempts to spawn salmonids maintained in seawater have yielded mixed results. Frequently maturing adults retained in seawater will die before spawning. If spawning does occur in seawater, the gametes are often not viable. There
are several biological mechanisms that may be operating to inhibit
spawning in seawater or be responsible for a reduction in gamete
fertilizability or viability. Residence in seawater may block the activation of neuroendocrine and endocrine pathways that normallycontrol spawning. Alternatively, prolonged seawater residence may
cause ionic or osmotic imbalances in the blood or tissues and havedirect adverse effects on the gonads. Experiments were carried out
to test these possibilities (Dickhoff unpubl.). Four-year-old Atlantic salmon (Sa/rna safar) of the Gaspe stock were maintained infloating seawater netpens in Puget Sound near Manchester, Washington. Near the time of sexual maturation, two groups of fish wereretained in seawater (29°/00), two groups were transferred to fresh
water, and a third set of two groups was transferred to floating
netpens in which a layer of dilute seawater (17 to 19°/00) wasestablished. One group in each of the three environments was injected with GnRHa (10 f1g/kg) on days 1 and 3. The control groups
were injected with 0.9% saline. In comparison with controls. spawning was advanced in all groups injected with GnRHa. Prespawning mortality was high in the group maintained in full-strengthseawater and injected with saline. The cumulative percent fertilization and survival to the eyed stage for gametes and embryos was
similar in hormone- and saline-injected groups in fresh water andin dilute seawater for the hormone-injected group that was spawned within one week after injection.
Fertilization and survival of embryos were reduced in the groups
in dilute seawater at day-1O after injection and in the group in fullstrength seawater. These results indicate that spawning of salmonids
in seawater can be induced by injection of GnRHa. Egg fertilizability
and gamete viability may be reduced when adults are maintainedin full-strength seawater or for a prolonged period in dilute seawater.
The results support the hypothesis that in maturing salmonids inseawater the neuroendocrine pathway controlling spawning is inhibited by the central nervous system.

_

This work was supported in part by Washington Sea Grant (RIA42) and the National Science Foundation (Grant DCB 8416224).
The author wishes to thank the following for their assistance: F.W.
Waknitz, J.L. Mighell, C.V.W. Mahnken, E. Plisetskaya, P. Swanson, C.V. Sullivan, and A. Gorbman.

Citation_

DickhofT, W.W., and C.V. Sullivan.
1987. Thyroid involvement in salmon smoltification with special reference tometabolic and developmental processes. In Dadswell, MJ., et aI. (cds.), Common strategies ofanadromous and catadromous fishes, p. 197-210. Symp. ser.
I, Am. Fish. Soc., Bethesda, MD 20014.

22

Several species of seaweeds belonging to the genus Porphyra areused for food ("Nori"). The complete life history of these species
was not known until Drew (1949), an English algologist, discovereda small shell-boring algae (Conchocelis) thought to belong in a different genus from Porphyra but which is, in fact, the summer formof Porphyra. Later research has demonstrated that the chromosome
number of the Conchocelis is diploid; that of the thallus, which isactually used for food, is haploid; and the chromosome number
used for culture is n = 3.
In current Nori culture, the thallus stage is cultured, controlled,
and produced at sea from autumn to spring, and the Conchocelis
stage is cultured and controlled as seeds in land equipment fromspring to autumn. Through the whole process, two applications are
considered in the use of protoplasts as seeds. One application is
the productive technical system which, concretely, is able toeliminate the Conchocelis stage from the process of Nori culture
by direct use of a budding body of protoplasts as seeds. We canpreserve the thallus of Porphyra from several months to several
years by semidehydrofreezing at -20°C. This is done from early
spring, when the Nori culture ends, to early autumn when the sporesare placed in culture nets. When spores are needed, the thallus isthawed to provide protoplasts that are set directly in culture nets.
Another application attempts to improve the breed through partial use of a breeding technique called cell fusion, consisting oftreating the protoplasts to bring about artificial variation in genes.
As mentioned above, Porphyra is a haploid-chromosome-number
stage; thus, improving it by crossbreeding is much more difficult
than in the case of land plants. To date, only selection has been
introduced as a breeding technique. With cell fusion, however, newcombinations of genes can be produced without sexual reproduction. For this reason, there is a strong hope that cell fusion using
the haploid-chromosome-number stage can be used as a plant
breeding technique.
Among the more popular and larger algae, protoplasts of thefollowing species have been isolated: Bryopsis plumosa, Enteromorpha intestinalis, Enteromorpha sp., Porphyra yezoensis, andLnminaria japonica.
Algal cell wall structures, as well as their chemical components,
are very different from those of land plants. Many land plants arecomposed of cellulose and pectin, and the enzymes sold on themarket to dissolve them are largely ineffective on the cell wallsof marine algae. Also, there are remarkable differences between
species of algae. That is why we need to discover enzymes fordissolving cell walls that are suitable to each algae.

Isolation and developmentof protoplast in Porphyra
HITOSHI KITO
Seikai Regional Fisheries Research Lnboratory
Fisheries Agency
49 Kokubu-machi
Nagasaki, 850 Japan

Components of the Porphyracell wall and the search fordissolving enzymes_

There is a thin membrane of protein in the outermost structure of
the Porphyra cell wall, and beta-l-4 mannan, a polysaccharide, isinside this membrane. Further in, there is beta-I-3 xylan in a rnicrofiber structure. Moreover, porphyran is the substance that makes
up the cell wall. Thus, polysaccharides with different structuresfrom land plants are in the cell wall. The digestive enzymes of small
animals that eat marine algae and enzymes produced by marine
bacteria effectively dissolve these polysaccharides. However, wehave so far been unable to develop refined enzymes that effect a
specific polysaccharide.
23

Table 1
Former results of protoplast isolation in Porphyra.
Results of protoplast
Species

Enzyme or organisms fromwhich enzyme was reduced-------_._------Yielded quantity

P. yezoensis

Pseudomonas sp.
(isolated from Porphyra sp.)P. yezoensis

Digestive enzyme of sea urchin

P. perforata

AAP (Abalone Aceton Powder)>100 cells/field*

Agarase<2-5 cells/field

Macerase
Macerase + AAP>100 cells/field

Pectinase
Pectinase + AAP<2-5 cells/field>200 cells/fieldSulfatase
Sulfatase + AAP>100 cellslfield

Development

Number not counted, but
positively recognized

References
Fujita et al. 1982

Saga and Sakai 1984

Regenerated to thalli

Pol ne-Fuller et al. 1984

<2-5 cells/field<2-5 cells/field

P. perforata

AAP'" 10% of cells in thematerial

Rcgenerated, concluded life cycle

Polne-Fuller and Gibor1984

P. yezoensis

Papain + enzyme bacilli fromsea bottom + Aeromonas sp.
+ bacteria from seaweeds

Number not counted, butmany recognized

Cell division

Hatate et al. 1986

P. yezoensis

Pseudomonas sp.
(isolated from Porphyra sp.)

Number not counted, but
positively recugnized

Regenerated tothalli, monospore

Fujita and Migita 1985

P. yezoensis

Papain and AAP

10% of cells in thematerial

Regenerated tothalli, monospore
conchocelis

Kito 1985*Microscopic field used x 10 object lens.

In past experiments, the enzymes used in most cases to dissolve
cell walls of marine algae were crude ones extracted by the researcher. The only enzyme on the market is one extracted by acetonfrom abalone (Abalone Aceton Powder, Sigma Co.). The enzymes
used and subjects of extraction in attempts to dissolve the Porphyracell wall are shown in Table 1.

Extraction method of the enzyme
and its characteristics

in the vicinity of 5.5 to 6.0. The dissolving power of this powder
is a little stronger than the enzyme produced by marine bacteria.
Because of its commercial availability, AAP is considered to be
the most useful at present.

Isolating protoplasts_

The technique for isolating protoplasts from the Porphyra plantlet
is shown in Figure 1. In this process, the germ-free technique isomitted because many marine algae have viscous substances on theirbody surfaces, making it difficult to produce germ-free plantlets.
To experiment with completely germ-free plantlets, the plantlet will
have to be reared from a budding body in germ-free culture.

_

For the purpose of dissolving the Porphyra cell wall, we isolated
the bacteria that could dissolve zylan and mannan from cultured
Porphyra plantlets. Two strains with especially strong dissolving
power were selected from these bacteria. They were culturtd inlarge quantities and the enzyme produced in the culture solution
collected. The dissolving power of these enzymes against Porphyra
crude fiber was examined.
Both strains were identified as Vibrio fisheri. It was found that
adding about 2 % crude fiber substance educed from Porphyra tostandard culture medium, Zobe1l22l6E, substantially increases the
enzyme's dissolving power. These enzymes showed the highest activation at pH 8.
We do not know the process of preparation of Abalone Aceton
Powder (AAP). What we do know is that this powder does not react
to the thin membrane of protein that forms the outermost layer ofPorphyra plantlets, but dissolves the layer of mannan, xylan, andporphyran inside this thin membrane. Optimum pH of reaction is

Culturing a budding bodyfrom protoplasts
The number of cells isolated as protoplasts varies considerably withthe age (days after beginning the culture) of plantlets used. A plantletof "'40 days with a length of "'3-5 cm is most suitable. In this case,approximately 10% of the cells are isolated as protoplasts (see Figure2a). Protoplasts obtained by reaction of the enzyme solution are
washed thoroughly in seawater, then collected together by slowly
centrifuging (at 600 rpm for 5 min), and cultured. SWM-3 is a good
culture solution. The first stage of culture consists of setting theprotoplast5 in a 5-cm-diameter petri dish. These are stationary24

cultures at a temperature of 20°C and an illumination of "-'SOOO
lux with fluorescent lamps.
Five days after beginning the culture, protoplasts begin cell division and gradually develop into germlings of plural cells. When
germlings consist of about 10 cells each (see Figure 2b), they areseparated and placed in culture vessels with many separate chambers
and maintained at ISoC and "-'SOOO lux. Further, when germlings
attain "-' 1.0 mm in length, we transfer them into culture bottles (30mL each) and place them into shaking culture. After that, depending on their growth, we move them into Porphyra culture vessels
with an air-pipe at the bottom of a round-bottomed flask containing "-'SOO mL culture medium.

I
2% AAP
0.3 mol manitol
0.5% Potassium Dextran sulfatebase seawater, pH 6.0

Sample:Porphyra sp.
thallus cut into
3x3 cm sizeII
rinse 3 times
with sterilized
seawaterreaction of enzyme;
shake slowly at 20°C
for 1 hour

2% Papain
0.5% Potassium Dextran sulfatebase seawater, pH 6.0

filter with
30-1'flI plastic gauzeII

centrifuge with
0.1 mol manitol
seawater at 600 rpm
for 5 min at 5°C

reaction of enzyme;
shake slowly at 20°C
for 15 minutes

Early budding of protoplasts
and growth to plantlet

By culturing protoplasts of Porphyra yezoensis in a small petri dish,the volume of each germling begins to increase gradually anddevelop into an egg shape. After about one week, cell division begins(see Figure 2b).
In the first cell division, at the budding time of general conchospores and monospores, two cells of the same size are formed. But
in the case of protoplasts, a boundary cell wall is formed and thesizes of the divided cells are uneven. In many cases the boundarycell wall is indistinct. Such unevenness of cell sizes disappears during further division and development to a plantlet. In the early stages,
the cells form many layers and develop to a massive germling withmany folds.
When this massive germling attains "-'SO 11m in diameter, one
to several large transparent cells develop in the margin of the germling, and one or more rhizoids extend from it (see Figure 2c). By
setting it into a shaking culture, this germling grows into a unilayered
juvenile plantlet with many folds (see Figure 2d). The juvenile
plantlet of about S-mm body length consists of many layers of cells,
and releases monospores. These monospores develop into a plantlet
with cell divisions the same as that of a normal juvenile plantlet.

I
centrifuge withseawater at 600 rpm
for 4 minrinse 2-3 times
with sterilized
seawaterI
Protoplast_ _ _1_ _- ,culture with SWM-3 solution
at 20°C under 5000 lux
fluorescence for 2 weeks

cell fusion
experiments

individual culture,with slow shaking culture

Figure 1
Protoplast isolation technique.

~c;~

(,
I~

_

I

\

,-,:,

.

G

,\

b

,<

f'

'1I\ _cadFigure 2(a) Protoplast; (b) Germlings of protoplast, 13-d-old after liberated; (c) Germlings of protoplast, 30-d-old after liberated;(d) Juvenile thallus, 65-d-old after liberated. Scale: a-c, 50 I'm; d, I mm.

2S

Citations

The plantlets from protoplasts grow much slower ("-15 cm during 120 days of culture) than the budding body from conchosporesand monospores, but there are no differences in growth mechanisms.
At 90 days of age, recognizable reproductive cells are formed.
Conchocelis are obtained by removing part of a carpospore intoa small test tube for culture.

Discussion_

Drew, K.M.
1949. Conchocelis phase in the life-history of Porphyra umbilicalis (L.) Kutz.
Nature 164:748-749.
Fujita, Y, T. Thompson, and S. Migita.
1982. Isolation and culture of protoplast from Porphyra and Enteromorphaprepared by bacterial enzyme. Abs(r. Annu. Meet. Jpn. Soc. Sci. Fish., 211
p. (in Jpn.).
Fujita, Y., and S. Migita.
1985. Isolation and culture of protoplast from some seaweeds. Bull. Fac. Fish.
Nagasaki Univ. (57):39-45 (in Jpn.).
Hatate. H., Y. Aoki, T. Araki, and M. Kitamikado.
1986. Preparation of bacterial enzymes capable of degrading the cell wall of
red algae Porphyra yezoensis. Bull. Jpn. Soc. Sci. Fish. 52(3):545-548 (in Jpn.).
Kito, H.
1985. Isolation and development of protoplast in Porphyra. Res. J. Food Agric.
8(9):20-24 (in Jpn.).
Polne-Fuller, M., M. Biniaminov, and A. GiOOr.
1984. Vegetative propagation of Porphyra perforala. Hydrobiologia 116/117:
308-313.
Polne-Fuller, M., and A. GiOOr.
1984. Developmental studies in Porphyra. I. Blade differentiation in Porphyra
perforala as expressed by morphology, enzymatic digestion, and protoplast
regeneration. J. Phycol. 20:609-616.
Saga, N., and Y. Sakai.
1984. Isolation of protoplast from wminaria and Porphyra. Bull. Jpn. Soc. Sci.
Fish. 50(6): 1085.

_

In attempts to isolate protoplasts with unicellular algae, some of
the many researchers report that fused cells begin to form cell walls
and at times even propagate. However, in multicellular algae production for food in Japan, observation of the beginning of protoplast
cell division is rare.
As mentioned above, the thallus is haploid in chromosome number. Therefore, if the diploid Conchocelis stage could be obtained
from the clonal plantlet from this protoplast by accelerating selffertilization of each plantlet, it would be easy to produce a homozygote and improve the breed. Because the thallus is haploid, itshould also be possible to produce a homozygote by self-fertilizationof a normal thallus. However, according to recent reports, meiosis
of Porphyra is observed at the first cell division after budding of
conchospores. In this case, four kinds of cells whose genes are different from one other will be present in the Porphyra plantlet.
Therefore, we need to first make protoplasts and then form plantlets
from them and make homogenes by self-fertilizing the plantlets.
It is thought that selection can increase the effectiveness of thistechnique.
Up to now, the Conchocelis has been cultured by boring into
oyster shells which have functioned well to release conchospores
effectively and to attach them evenly in culture nets. However, thesummer stage of the Conchocelis production could be omitted,
although it would have a great impact on the entire system of Nori
culture as it is practiced in Japan at present. Therefore, it is necessary
to look at the problem from many viewpoints.
In the cell fusion experiment, we obtained protoplasts from redand green-pigment mutant species, used these mutant species asmarkers, and repeated the experiment with the polyethylene glycol(PEG) method for land plants. At present, we can obtain the fusedcell. After this, it is important to culture the fused cells to adult thalli.
Finally, we think extreme caution is necessary in producing
organisms through biotechnology that do not presently exist in thenatural world and discharging them into the sea. Even if this industry disposes of whole products in the form of aquatic culture,
it is absolutely impossible to completely and reliably isolate partof a water mass; therefore, genetic mixtures with wild species may
occur. It is necessary to investigate the use of newly produced seeds
used in sea farming for release in the sea. It is important to preservegene resources, and a plan for gene preservation for useful aquatic
species is being developed. It was begun in 1985 by a group ofnational fisheries laboratories with the financial help of the Japanese
Agriculture, Forestry, and Fisheries Council.

26

Genetic engineering andbiotechnology of economicallyimportant seaweeds

Increased commercial utilization of seaweeds in the United Statesand other developed countries will most likely depend upon the successful cultivation of newly developed, genetically improved strains.
Genetic engineering techniques have the potential to produce valuable new strains of seaweeds not otherwise possible by classicalbreeding methods. Genetic engineering involves the in vitro transfer
of genes from one organism to another in the absence of sexual
reproduction. Methods by which gene transfer can be accomplishedinclude protoplast fusion-somatic hybridization, microinjection,
direct gene transfer, and the use of vectors such as the Ti plasmidof Agrobacterium. Although such techniques are being applied toa great many land plants today, they are just beginning to be
developed for seaweeds. Indeed, the current status of genetic
engineering in seaweeds is much like it was for land plants in the
1960s; that is, the basic knowledge and techniques for protoplast,cell, and tissue culture are still in an early stage of development.
Nevertheless, rapid progress has been made in these essential supportive techniques in the past few years and the future for suchresearch looks bright. The purpose of this report is to briefly review
the current status of work in this area, and to describe recent researchin my laboratory on protoplast production and tissue culture ofphycocolloid-producing red seaweeds.
Protoplast and tissue culture research in seaweeds is a relatively
new field, starting less than 10 years ago. To date, there has beena total of 16 species (9 genera) of seaweeds from which protoplasts
have been isolated (see review by Cheney 1986) Examples include
Enteromorpha, Monostroma, Ulva, Laminaria, Macrocystis, andProphyra. Our laboratory has recently reported on methods for producing large numbers of viable protoplasts from the agarophytes
Gracilaria tikvahiae and G. lemaneiformis (Cheney et al. 1986,
Cheney 1984). Protoplast-produced cell masses up to the 16-32 cellstage have been grown in culture; however, efforts to regenerate awhole plant have been unsuccessful to date. We and several others
have reported (presumed) protoplast fusion, although unequivocal
evidence of heterokaryon production is thus far lacking.
The ability to grow a plant as undifferentiated callus or as single
cells rather than organized into a thallus is a recent development
in seaweeds. To date, a total of 21 species of seaweeds have reportedly been regenerated from cell or tissue culture (Cheney 1986).
Tissue culture has been reported for Enteromorpha, Ulva, Laminaria, Undaria, Macrocystis, Porphyra, and Chondrus. We have developed tissue culture methods for the carrogeenophyte Agardhiella
subulata. This is the first time a true tissue culture system (capableof repeated subculture and controlled plant regeneration) has beendeveloped for an anatomically complex, higher red alga.
Alt,hough clearly it will be some time before genetic engineering
techniques can be applied to economically important seaweeds ona practical basis, protoplast and tissue culture technology can andwill undoubtedly play an important role in the future of seaweed
genetic improvement and mariculture.

DONALD P. CHENEY
Biology Department
Northeastern University
Boston, MA 02115

Additional note_

Since this report was first prepared, considerable progress has been
made in the protoplast and tissue culture of red seaweeds in ourlaboratory. In particular, we've developed methods for the successful regeneration of Gracilaria protoplasts to whole plants, as well
as a true tissue culture system for the carrageenophyte Agardhiella.

27

Protoplasts have now been successfully regenerated to wholeplants for several strains of Graci/aria tikvahiae. Several factors
proved important in obtaining these successful results, including
the careful selection of starting material, the gradual dilution ofculture media after protoplast production, and the control of bacterial
growth with a specially formulated antibiotic mixture. Interestingly, protoplast regeneration did not occur in axenic cultures, as is
usually the case in higher plants. Protoplast regeneration only occurred in those cultures in which bacteria growth was not eliminatedbut rather controlled with antibiotics. The antibiotic mixture weused contained a total of eight different antibiotics. These were
specifically selected from a wide variety of antibiotics by using a
new method we've termed the "one-step, antibiotic disk method"(Bradley et al. In press). This method provides a simple and rapid
technique for determining which antibiotics are effective against
a plant's bacterial flora. Further details of our protoplast culture
methods will be described elsewhere.
Upon the successful development of protoplast regeneration
methods, we initiated protoplast fusion experiments with Gracilaria.
Protoplasts from a wild-type, red pigmented Gracilaria tikvahiae
strain were fused with protoplasts from a green mutant strain using
the chemical fusant polyethylene glycol (PEG). The methods weused were basically similar to those commonly used with higher
plants, incorporating a high pH-high Ca ++ dilution step after
protoplast agglutination (e.g., see Kao and Michayluk 1974).
Protoplast agglutination, but not complete fusion, was observed
microscopically. After subsequent culture, several red and green
bicolor gerrnlings developed. These eventually developed into red
and green chimeric whole plants. Such chimeras were produced
at too great a frequency to have been caused by either mutations
or reversions. Since bicolor heterokaryons were not observed microscopically, the development of these chimeras may simply have beencaused by the adherence of red and green protoplasts to each otherwithout fusing. Regardless, the production of such red and green
bicolor chimeras is unusual. To our knowledge, this is the first reportof chimera production from protoplasts in a seaweed. Protoplast
fusion in Gracilaria is currently under further investigation in ourlaboratory .
A second area in which considerable progress has been made isin the tissue culture of Agardhiella. Since Agardhiella shares anumber of similarities with the commercially farmed seaweed
Eucheuma, we have been using Agardhiella subulata as a model
for Eucheuma. Our tissue culture of Agardhiella is unusual in thattwo distinct types of tissue growth have been produced in culture:
true callus and masses of elongated, uniseriate filaments. Callus
consisting of masses of unorganized, pigmented, spherical cells havebeen produced from medullary cores grown in enriched seawater.
Filaments (up to 5 rom long) develop from outer cortical cells ofcross-sectional disks cultured in ASP-12-NTA media. Such filaments
can be easily subcultured to form filamentous masses. Both callus
and filamentous masses have been subcultured for over a year andregenerated into whole plants. A preliminary report on the filamentous tissue culture system has been presented elsewhere (Cheney
et al. In press).
One of the most interesting features of the filamentous tissue
culture system is its relatively fast growth rate. Under optimal
growth conditions, the ftlamentous masses grew at rates quite comparable to that of whole plants; that is, around 1-3 % per day based
upon wet weight. This is a fast growth rate for a tissue culture system
and allowed us to conduct chemical analyses of the filamentous
tissue. In particular, we were interested in determining how the
carrageenan composition of the tissue culture compared with thatof whole plants. Our results have been described in detail elsewhere(Cheney et al. In press). However, basically we found that theftlamentous tissue culture produces a type of iota carrageenan similar
to that found in whole plants but perhaps slightly higher in its content of precursor molecules. Additional biochemical studies areunderway with the Agardhiella filamentous tissue culture system.
Furthermore, this system is also being used to produce mutants invitro.

Acknowledgments_

This work was supported in part by a grant from the National
Science Foundation, PCM-83l2607, and was conducted with theable help of Peter Bradley and Clif Duke.

Citations_Bradley, P., D. Cheney, and N. Saga.
In press One step antibiotic disk method for obtaining axenic cultures ofmulticellular marine algae. Plant Cell Tissue Organ Cult. 12.
Cheney, D.
1984. Genetic modification in seaweeds: application to commercial utilizationand cultivation. In Colwell, R., E. Pariser, and A. Sinskey (eds.), Biotechnology
ill the marine sciences, p. 161-175. Wiley-Interscience, NY.
1986. Genetic engineering in seaweeds: applications and current status. Nova
Hedwegia 83:22-29.
Cheney, D., E. Mar, N. Saga, and J. van der Meer.
1986. Protoplast isolation and cell division in the agar-producing seaweed
Grucilaria (Rhadophyta) J. Phycol. 22:238-243.
Cheney, D., A. Luistro, and P. Bradley.
In press. Carrageenan analysis of tissue cultures and whole plants of Agardhiellasltbu/ala. Hydrobiologia 151/152.
Kao, K., and M. Michayluk.
1974. A method for high-frequency intergeneric fusion of plant protoplasts.
Planta 115:355-367.

28

Of the 10 species of Haliotis that inhabit Japan, four species, H.
discus hannai, H. discus, H. sieboldii, and H. gigantea, are economically significant. The geographical distribution of these species
is shown in Figure I. Haliotis discus hannai ranges along the northern areas such as Hokkaido and the Pacific littoral, near which acold current flows, and the other species along the littoral near which
a warm current flows.
The landings and yields of abalone for 10 years between 1973
and 1983 are shown in Figure 2. Annual landings fluctuated between
4,607 and 5,839 tons, averaging 5,626 tons. Annual yields varied
between $41.0 million and $83.8 million, averaging $72.6 million.
Thus, abalones are important species for coastal fisheries and a staple
product in Japanese Aquaculture Centers established for seed production. Numbers of seed abalones produced and released in 1983
are shown in Table 1.
The seed producing systems in Japan vary from prefecture toprefecture according to their localities. In principle, however, they
are based on the method introduced by Kikuchi and Dki (1974a,b),
and Seki (1980). Such technical systems have the following problems
to be solved.
Annual production of the northern abalone species by each farmis 1.217 million compared with 496,000 for the southern species,
which means that production of the former is 2.5 times as great
as the latter. This difference is thought to be caused by the fact
that, although research on gonadal maturation of the southern species
has been conducted, adult conditioning is not as efficient in southern
as in northern areas.
Northern growers begin intermediate rearing of seed abalones,
distributed to them by seed abalone farms, in the spring and release
them before winter because seed abalones stop growing in winterand their rearing becomes difficult to control. Thus, the sizes ofMass culture of Ulvella lens
as a feed for abalone,
Haliotis discus hannai
KANJI TAKAHASHI
Kesennuma Miyagi Prefectural Fisheries
Experimental Station
Hajikami-Uchinuma 119
Kesennuma, 98802 Japan

AKIMITSU KOGANEZAWA
Tohoku Regional Fisheries Research Laboratory
Shiogama, Japan••/l!.thtlcutl hwmai

o l!.di~C\lH"'1i.~• .!!.~

CHISHIMA COI.O
CURRENTISEA OF JAPANTSUSH I MA WARM CURRENT

/

•

.o.t~

/"
PACIFIC OCEAN
KURaSH 10 WARM CURRENT/
Figure 1
Distribution of industrially significant abalones in Japan.

29

90

IIf

6000

o

•

70
5000

60

E

llf

{f.

c
50 :

>-

en
LU
a:

40

0

a.

en

4000

0
0

T

I

1973

1980

1975

N

J

LL

0

a:

1983

4

10

LU

lD

:;;
::>

z

Figure 2
Abalone fisheries in Japan.
Solid line = landings; dotted line = yields.

310/{,'--...;:,

Table 1
Abalone seed (n) produced and released in 1983.
Northern species

Southern species
2

H. discus hanna;

H. discus

H. seiboldii

H. gigantea

Total

10

•
2

\

~

3

4

5

6
7

TIME(DAYS)

Production

13,394,000

10,911,000

518,000

024,823,000

Release11,888,000

6,018,000

423,000

5,000

18,334,000

II

23

4

No. of prefectures34

released juveniles depend on the size of the juveniles distributedby the seed fanns. When seed is collected during the natural spawning season from August to September, the average size of distributed
juveniles one and a half years later in the spring will be 15 mm.
Though the desired size of released juveniles is more than 30 mm,
juveniles of intennediate rearing are released when smaller than
30 mm under certain circumstances.
Therefore, to increase the juvenile recapture rate, it is necessary
to increase the size of distributed seed abalones. As a means to that
end, early seed collection in June and July, which hastens the growthof early juveniles by taking advantage of summer water temperatures, is thought to be effective. However, the conventional method
to produce food for early juveniles by controlling algal communities
is difficult because of low water temperatures during the algae
culture period.
Therefore, the authors developed a method to guarantee a stable
and systematic diet for early juveniles through the mass culture of
Ulvella lens which can be obtained as the second-phase algae community, except in summer, At present, this method is incorporated
into the seed abalone production system in the northern areas andhas resulted in stabilization of the production process, as well as
the production of larger seed abalones and increased landings. This
paper reports on the ecological aspects of Ulvella lens and themethod of its mass culture.

Figure 3
Relationship between water temperature and the number ofzoospores released by sporophytes of the green alga, Vlvellalens. Sporophy1es were immersed in l-L beakers kept at constant temperatures of 8°C (L'.), lZOC (0), ISoC (0), 20°C(.), and 2SoC (*), by means of water baths on 9 March
1983. Vinyl chloride plates were set at setting substrate forzoospores. Density counts on the plates were begun on thesecond day following temperature stimulation.

Ecology of Ulvella lens_Relationship between spawning of zoospores
and water temperature
The relationship between spawning and water temperatures wasstudied when the culture water temperatures of adult sporophytes
were 7.4°e (Experiment I) and 9.2°e (Experiment 2), and theresults are shown in Figures 3 and 4. In Experiment I, the daily
settlement of zoospores at the immersion water temperature of 8°e
was less than 1,000 and no daily variations were recognized. The
settlement increased daily at 12, 15,20, and 25°e, with maximum
settlement at 20 and 25°e on the fourth day and at 12 and 15°e
on the fifth day. The total settlement for 5 days was highest at 20 o e.
In Experiment 2, no settlement occurred at the immersion watertemperature of 30 o e. At water temperatures of 12, 15, 20, and25 °e, maximum settlement occurred on the third day. The total
settlement for 5 days was highest at 15°e.
30

1rf

6

10

5

10

5

10

rn
w

'"c..

en

0

w

rn

a:

0
0

0
n.

N

en

u.

0

0
0

ffi Ill'

N

'"::>

::;

4

15 10

z

a:

w

III

::E
::>

z

3

10

llr
2

10

o

*

.--.--.--.--.
2

3

4

5

6

7

T1ME(DAYSJTIME(OAYS)

Figure 4

Figure 5

Relationship between water temperature and the number ofzoospores released by sporophytes of the green alga, Ulvellalens. Sporophytes were immersed in l-L beakers kept at constant temperatures of 10°C (*J, 12°C (0), 15°C (0), 20°C(e), 25°C (*), and 30°C (.), by means of water baths on
18 April 1983. Vinyl chloride plates were set at setting substrate for zoospores. Density counts on the plates were begun
on the second day following temperature stimulation.

Relationship between water temperature and the number ofzoospores released on mature spawning by sporophytes of thegreen alga, Vlvella lens. Sporophytes previously spawned atconstant temperatures of 15°C (e) and 20°C (0) on 20-23
April 1983 were thereafter kept under natural water·temperature condition (10°C) and respawned on 2-6 May to assess
fertility of the sporophytes. Comparisons with Figures 1 and2 show comparable fertility under multiple spawning.

Respawning of sporophytes

The settlement at 10°C in Experiment 2 rapidly increased to 56
times greater than that at goC obtained in Experiment 1. It can beinferred from this result that very few zoospores are spawned atwater temperatures lower than 10°C. Therefore, securing adultsporophytes to cultivate the diet for seed collection must be completed before water temperature falls below 10°C.
The settlement at 15°C was 2.3 times greater than at l2°C in
Experiment I, and 4 times greater in Experiment 2. Thus, the lowest
water temperature suitable for obtaining spores is somewhere around
15°C. Maximum water temperature appears to be about 20°C,
because settlement at 20°C was higher than that at 25°C, and no
settlement was seen at 30°C in Experiment 2. Judging from this,the suitable water temperatures for zoospore release seem to be
between 15 and 20°C, a range that can be maintained by atmospherictemperature from late April in standing immersion water.

Adult sporophytes that had spawned zoospores at 15 and 20°C in
Experiment 2 were kept in running water at an average 10.1 °C for
10 days, and their respawning of zoospores at the immersion watertemperature of 20°C was investigated. The result of the zoospore
settlement is shown in Figure 5. Maximum settlement occurred on
the fourth day, one day later in Experiment 2, and the number produced was greater. Apparently sporophytes of a different developmental stage were growing because of the long time since adultsporophyte culture was begun. It is obvious, therefore, that adultsporophytes, which have spawned zoospores can be used again if
they are kept in natural seawater at about 10°C.

31

/
/
'/
5

10

/

(9.n~.

'/

'/

/
/

"::;:
u

w
0..

4

10

t/l
W
Cl::

/
4/

'/
/

~

'/

Vl

/

~

o

"-

3

10

o

/

10

/

....
Z

N

/

o

/
/.

0:

/:

/
/
/

"-

/

:;:

~

,

~

'/

2

Vl
Z

OJ

~ I~ ~ t

t/l

~

~

/

0:

/

0..

10

/

'/

o

j ~,

'/

/

Cl::

(J

/

/

....
«:

z

/~
2

0

/j
/

i

/~
/:
/
/

/
/

/

/

o '/

10

O~'

/

/

L.....

6

10 /

o

12

18

24

6

r2

18

24

6

o

12

T lME (HOURS. DAYS)

."

B

12

18

24

."

B

12

18

24

6

12

Figure 7

TIME (HOURS. DAYS)

Relationship between natural photoperiod and the number of zoospores releasedby sporophytes of green alga, Ulvella lens, spawned at constant temperatures of
21°C on 1-4 May 1983. Under natural photoperiod, it was observed most spores
were released I hour following sunrise. Similar results were observed under artificial photoperiod (see Figure 6). (-. -) number of zoospores, (... o· .. )illumination intensity, ( ~) light, ( ~) darkness.

Figure 6
Relationship between artificial photoperiod and the nnrober of zoospores reitasedby sporophytes of the green alga, Ulvella lens, spawned at constant temperaturesof 20 0 e on 24 April 1983. Under a photoperiod of 12 hours of light ( ~)and 12 hours of darkness ( ~ ), it was observed most spores were released
I hour following light stimulation.

Daily periodicity for zoospore release~atural photoperiod-The settlement of zoospores and the varia-

tion of light intensity in the natural photoperiod are shown in Figure
7. Daily settlements were 240 on the second day, 21,380 on thethird day, and 901,960 on the fourth day. The daily settlement increased with an increasing number of immersion days and on the
fourth day in particular rose to 42 times greater than the previous
day, while daily variations of settlement showed an identicaltendency irrespective of number of immersion days. The settlement
reached its maximum one hour after daybreak when the light intensity rose over 0 lux and gradually decreased as the light inten~ity increased, reaching zero by 4 hours after daybreak. The total
settlement occupancy rates measured one and two hours after
daybreak were 93.5% on the third day and 97.8% on the fourthday. Thus, the spawning of zoospores in the natural photoperiod
commences at daybreak, lasts two hours, and ends in three hours.
Therefore, it is inferred that removal of adult sporophytes cantake place during the period of zoospore settlement without concern for spawning of zoospores and that one settlement examination per day is sufficient.

Artificial photoperiod-Photoperiod was set for a 12-hour lightperiod from 5 a.m. to 5 p.m. and a 12-hour dark period from 5
p.m. to 5 a.m. The change of settlement with the passage of timeis shown in Figure 6. The maximum settlement for each day occurred at 6 a.m., one hour after the light period began. The settlement rate (settlement at the hour/daily total settlement x 1(0) at
this hour was 85.5% for the second day, 99.7% for the third day,and 99.8 % for the fourth day. Zoospores were spawned within one
hour after the dark period shifted into the light period. An extremely
small number of zoospores settled at 5 p.m. in the dark period on
the fourth day, but never on other days. These results indicate thatthe spawning of zoospores can be induced by light stimulation andcontrolled by adjusting the photoperiod.

32

Figure 9
Shaded culture UlvelJa lens on the 14th day (x 1000). Pyrenoid (P) is clearly foundand interceUular space (IS) is not enlarged.

Figure 8
Eight-day-old discoid germling of Ulvella lens (x 1000). Initial zoospore ceU (ZsC)
is formed in the center.

Light intensity and cell morphology
Eight-day-old discoid germlings of 29.3 Ii diameter with initial
zoospore cells formed on the central parts (Fig. 8) were cultured
without fertilizer in running water in an outdoor tank covered with
90 % shading cloth obtained from the market. The results of theshaded and nonshaded cultures on the 14th day are shown in Figures
9 and 10, respectively.
The shaded and nonshaded culture groups differed in cell morphology. In the shaded culture group, pyrenoid is clearly present
and has a small intercellular space. On the other hand, no pyrenoid
is found in the nonshaded group, although cytoplasmic granules
are present. There is less chloroplast than in the shaded culture group
and a large intercellular space. The reason pyrenoid does not occur in the nonshaded culture group seems to be because starch grains
are not formed around a pyrenoid matrix. The formation of granules
and the large intercellular space appear to be caused by the dissociation of chloroplast and cell walls, respectIvely.
As far as cell morphology is concerned, the shaded culture group
grows more normally than the nonshaded group. From this, it isconcluded that after formation of the initial zoospore cell, shaded
culture is superior because abnormal growth is caused by too muchsunlight in the nonshaded culture.
The disc diameter of the shaded culture group was 101.5 Ii andthat of the nonshaded group was 172.6 Ii, but their daily growth
was 5.6 Ii and 11.1 Ii, respectively. Thus the nonshaded culturegroup grew better than did the shaded culture.
When Ulvella lens is cultured in the stagnant, nutriment-added
culture solution, Plasinocladia rrmrina propagates itself so muchas to color not only the Ulvella-growing plates but also the entire
culture tank green. This type of algae community is thought to beunsuitable as feed for early abalone juveniles because it is unstableand often falls off the substrate or covers early juveniles and causes
them to fall off. The shaded culture of Ulvella lens prevents
Plasinocladia rrmrina and diatoms from growing, thus stabilizing
the culture. Thus, it is advantageous to grow Ulvella lens in shaded
rather than in nonshaded culture, despite its slower growth.

Figure 10
Nonshaded culture Ulvella lens on the 14th day (x 1000). No pyrenoid is foundand cytoplasmic granules (CpG) are present. Intercellular space (IS) is enlarged.

Development of zoospores
The zoospores of Ulvella lens are pear-shaped with a red eyespot.
Their four flagella of equal length protrude from the anterior endof the head. They lose their flagella and become 5-lilength spores
4-5 minutes after settlement (Fig. ll-l). The spores germinate in
24 hours and become a 2-3 cell gerrnJing on the third day (Fig.
11-2) and a 4-8 cell disc on the fourth day. By the 8th day, thedisc grows to 21-32 Ii diameter, averaging 26.6 Ii, and initial
zoospore cells can be recognized on discs of 30 Ii diameter(Fig.H-3). Then individual variations in size occur as they grow,and on the 19th day when they have grown to 60.0-107.5 Ii, withan average diameter of 76.6 Ii, zoospore spawning is observed (Fig.
11-4).

33

Seed collection and thegrowth of abalone juveniles_

The question of whether or not cultured Ulvella lens can be used
as a feed for early abalone juveniles was investigated using a seedcollection tank (2.4 x 1.5 xO.6 m, containing 300 plates) used for
abalone production. The results are shown in Table 2.

Larval inducement by
mucus secreted from the pedal sole
In Experiment I (Table 2), there were marked differences in setting time, depending on the presence or absence of mucus. Thus,
the sole mucus essential to cultured Ulvella lens must be securedby transferring juveniles onto Ulvella-growing plates and used forseed collection in order to make all swimming larvae settle in aplanned setting density.

Spawning of zoospores and the capture rate
After transferring setting larvae into a seed collection tank, zoospore
spawning was controlled by making the photoperiod dark until thefollowing day in Experiment 4(1) (Table 2). In Experiment 5(1),there was no zoospore spawning because Ulvella lens was at its
spore stage. From the results of Experiment 4(1) and 5(1), it isinferred that the capture rate of larvae can be increased by controlling zoospore spawning.

Growth of early juveniles
The results of Experiment 2 (Table 2) show that early abalone
juveniles grew better in the light and dark photoperiod [2(1)] thanin the continuous light photoperiod [2(2)] for 7 days until they weretransferred to an outdoor tank following seed collection.
The results of Experiment 3(2),4(1), and 5(2), in whichjuveniJes
were reared in the light and dark photoperiod, suggest that they

Figure 11
Development of Vlvella lens under sunlight. (I) Spore with red eyespot (x 1000);(2) Two-cell stage germJing with a pyrenoid (x 1000); (3) Nine-day-old discoid
germling with initial zoospore cell. After this stage, gennlings were cultured under
90% shading conditions (x 1000); (4) 19-day-old adult sporophyte forming
unilocular sponangia. Spores are detected (x 400); (E) Eyespot; (P) Pyrenoid; (S)Spore; (US) Unilocular sporangium; (ZsC) Initial zoospore cell.

Table 2
Seed collection and juvenile growth of abalone, Haliotis discus hannai. on Vlvella lens cultured artifically.
Environmental conditionsof larval collection

Collector
for selll ing larvae

Captured rate

Mucus ofabaloneCreeping period
of abalone(days)

Time ofstagnant water(hours)Released
spores.

Daily photoperiod
for juvenile rearing

34
34

+

4

5
5

+++
-t-++

t=7=1

45
45

+
+

6
6

8
8

+++
+++

44
44

+
+

7
7

7
7

4

37
37

+
+

9
9

8
8

5

I
24

+
+

8
8

Exp.
no.

2

Culture periodof Vlvella lens(days)

Shell lengthof juvenile-----juveni.le
Larva
(%)
(%)

Avg.
(1')

Range
(1')

67.1
94.2

63.9
90.3

459
459

440-470445-470t T\;;d

96.1
96.0

92.4
92.8

512
485

465-530460·505++++++

tbw:.~:.;;j

92.4
92.2

91.5
90.0

512
522

490·530
500-540++++~t;:.:d

92.7
99.7

90.4
97.3

522
502

500-545470-525

100.0
93.9

96.0
90.4

481
479

470-510460·520

+-++

I

I
3

I

I

I

I

f:·:·::::·:·:1

rnsr~..
.:.:.:....:
f:·:·::::·:·:1

Note: After larval settlement was observed by the naked eye, water supply was started. Larval loss is measured on the following day after water supply.
Photoperiod: c::==:::J Continuous light: !:.. . . . :;.:......:.:~ Continuous darkness: I f!..!..!·j 8 n light and 16 darkness.
Collected juveniles are transferred into outside rearing tanks on the 8th day after larvae are placed in collecting tanks.

34

grew differently depending on the developmental stages of Ulvellalens. However, further detailed studies are necessary because,
although the rearing-water temperature was the same in all the experiments, they were performed on different days.
The average shell length of abalone juveniles was 512-522 I.l at
the time they were transferred into outdoor tanks in Experiment2(1),3(2), and 4(1). Cultured Ulvella lens is thought to be a suitable
feed for early juveniles, as compared with their shell length of about
480 I.l in the past.

Mass culture of Ulvella lens_Securing adult sporophytes
When the abalone juveniles are transferred onto plastic plates
covered with Navicula or Nitzchia to continue rearing them, adult
Ulvella lens can be secured after wintering providing the juvenile
density is appropriate. To secure the plastic plates covered withadult Ulvella lens, juveniles must be transferred onto the plates whenwater temperatures exceed 15°C, because water temperature
decreases as the juveniles feed on diatoms and the desired watertemperatures for zoospore spawning are above \2 0 e.

Figure 12
Adult sporophyte plates are put into holder containing spore settlement plates
in alternate position. (AsP) Adult sporophyte growing plates; (SP) Spore settlement plates.

Securing zoospores
Releasing of zoospores-After removing the abalone juveniles
setting on adult sporophyte plates by using a narcotic (Ethyl Aminobenzoate), these plates are put into holders which contain spore
settlement plates in alternate positions (Fig. 12). The holders are
immersed without aeration in stagnant water of outdoor culture tanks(Fig. 13). When the immersion water is stagnant, suitable watertemperatures for zoospore spawning can be maintained by atmospheric temperatures without any other heat sources.
Removal of adult sporophytes-When the culture water temperature of the adult sporophyte rises above 10°C, the maximum settlement occurs by the fourth day after immersion. The adult sporophyte
plates are removed no later than the fourth day to prevent Spirobis
foraminousus from settling on them. The mass release of zoospores
can be observed by the naked eye unless it is rainy or windy becausea thin yellow film is formed on the surface of the water by the aggregation of spores (Fig. 14).
The developmental stage for removal of adult sporophytes ranges
from the spore stage to the 5-cell gerrnling, and the dominant stages
are the spore stage and the 2-cell gerrnling.
When adult sporophytes are reused, they must be stored in running water, avoiding sunlight to prevent diatom propagation.

Figure 13
Holders containing adult sporophytes and spore settlement plates are immersed
without aeration in stagnant water of outdoor tank.

Table 3Composition of culture medium forUlvelta.

Figure 14

Ingredient

AmountNaNO}
NaH 2 PO.
Clewat1,000 gm
200 gm
500 gmKl
Tap water

12,000 cc

60 gm

Culture medium is added in the 10-t
culture tank.

Thin yellow film formed by aggregation of zoospore. Film was removed at thecenter.

35

Cultivation of spores
Aeration is started after removal of adult sporophytes and thenutrients indicated in Table 3 are added to the culture water (Fig.
15). Spores are then cultivated in stagnant culture water without
shading for 7-8 days.

Cultivation of young sporophytes
When the disc diameters reach 30-40 1.1 and initial zoospore cells
are formed, the plates are moved alternately to fill the vacant partsof holders. The holders fllled with the plates are moved into clean
tanks for storage. To prevent Plasinocladia marina propagation,
culture tanks are shaded with 90% shading cloths (Fig. 16), andcultivation of young sporophytes is then continued with aeration
by running water. The culture period is determined by a required
disc diameter greater than 200 1.1 and greater than 60% zoosporangium regions. The culture period averages about 40 days.

Figure 15
Nutriment is added to culture water by a watering pot after removal of sporophyte
plates.

Transferring juveniles
The abalone juveniles are transferred onto Ulvella growing plates
7 days before seed collection to secure mucus secreted from thesole (Fig. 17). The shading culture causes no problems in preparing seed collection plates because juveniles react to the light intensity of 90% shading.
The juvenile density necessary to obtain abalone mucus without
the consumption of Ulvella lens was not investigated in detail. When
10 juveniles of an average 15 rom shell length were transferred onto
each plate (40 x 30 cm) and their plates changed every seven days
to supply them for seed collection, the juveniles' intake of Ulvellalens was detected microscopically, but not by the naked eye. Thisis thought to be because the juveniles selectively fed on diatoms
growing on the surface of Ulvella lens.

Summary_

The diet needed for earlier collection of abalone in June and Julycannot be obtained by the prevalent culture method because oflow
water temperatures. Therefore, we have developed the mass cultureof Ulvella lens for feeding the early abalone juveniles.
This new method contributed to the development of the seedabalone production system, wherein collected juveniles are detachedfrom the collectors during Fall and made to grow at high density
on artificial food before the spawning season. By this technicalsystem the productive expansion and increase of seed abalones wereachieved. At present it is possible to produce 2 million abalonesof 2 cm length each year in Miyagi Aquaculture Center, Japan.

Figure 16
Culture tanks are shaded with 90% shading cloths after initial zoospore cells wereformed.

Citations_

Kikichi, S., and N. Uki.
1974a. Technical study on artificial spawning of abalone, genus Haliolis. I. Relation between water temperature and advancing sexual maturity of Hations discus
hannai. Bull. Tohoku Reg. Fish. Res. Lab. 33:69-78 (with Engl. summ.).
1974b. Technical study on artificial spawning of abalone, genus Haliolis. 2.
Effect of irradiated sea water with ultraviolet rays on inducing to spawn. Bull.
Tohoku Reg. Fish. Res. Lab. 33:79-86 (with Engl. summ.).

Seki, T.
1980? An advanced biological engineering system for abalone seed production.
Tn 1m. Symp. Coastal Pacific Marine Life, Bellingham, WA, October 15-16,
1979. p. 45-54. Cosponsored by W. Wash. State Univ. and Office of Sea Grant,
NOAA. [Avail. Library, W. Wash. State Univ., Bellingham, WA 98225.]

Figure 17
Abalone juveniles detached by narcotic are transferred onto adult Vlvella growing plate to secure the sole mucus.

36

The planktonic larvae of many marine invertebrates preferentially
set in habitats that support growth and reproduction of adults ofthe species. Chemical cues induce set in many invertebrates
including the oysters, Crassostrea virginica and C. gigas.
Oyster set can be divided into two clearly definable phases: settlement and metamorphosis. The settlement phase can be subdivided
into three stages, i.e., two behavioral and one physiological components. The first stage of settlement, during which a competentlarva swims with its foot extended forward, is termed "swim/
search. " During the second stage settlement, "crawl/search," thelarva begins traversing the surface in characteristic crawling patterns, with the target area becoming increasingly focused. In thelast settlement stage, "cementation," the larva secretes a cement
which affixes it permanently to the surface. The second phase ofoyster set, metamorphosis, is characterized by the morphogenetictransition from larva to juvenile or spat. Prior to cementation, set
is reversible and is contingent upon presence of the appropriate cue.
If an oyster fails to encounter a suitable external cue, it does not
set, and dies within 15 days.
Three species of bacteria, a new marine species (LST), Vibrio
cholera HTX, and Hyphomonasjannashiana VP 1, synthesize a cue
for swim/search and crawl/search behavior. Synthesis of this cuecorrelates with melanin production, and the specific cue is believed
to be L-DOPA, or a DOPA mimetic molecule, since L-DOPA isa precursor of melanin and purified L-DOPA triggers searching
behavior.
LST is the only species tested that produced a polysaccharide
adhesive viscous exopolymer (PAVE), serving as a marine biofIlm,
to induce or entice oyster larvae to attach and metamorphose. Thus,a neurotransmitter precursor may cue behavioral aspects of oyster
set, and PAVE may cue physiological aspects, notably cementing
to the surface.
Requirement of a chemical cue (or cues) in set of marine invertebrates is well documented. Only few such cues, however, haveactually been identified. Fewer still are in systems that haveaquaculture potential. Of these, the oyster (discussed here) andabalone (Morse 1984) have immediate potential to increase aquaculture productivity, especially where inefficient and low set affects
the aquaculture potential.
Surfaces coated with LST enhanced set several thousand percent,compared with glass surfaces, and several hundred percent better
than non-acclimated cultch. It is hypothesized that LST providesa) an appropriate searching cue, b) an appropriate cementing cue,c) nutritive value, and d) antimicrobial activity serving to control
harmful organisms. Other organisms that synthesize DOPA, e.g.,
Aeromonas, Streptomyces, Azotobacter, and Proteus, are unsuitable
for aquaculture application because they are pathogenic for theoyster, halointolerant, and/or their growth is suppressed in oyster
habitats.
It is now possible to prepare several liters of LST for hatcheryuse, but we cannot yet scale up to the 50,OOO-gallon quantities thatare required for commercial use. A significant problem is that
secondary metabolism, including exopolymer synthesis, is repressed
when LST is transferred to seawater from the growth medium, orfrom lyophils, even when the receiving seawater is copiotrophic,i.e., contains abundant oyster metabolic products. If controls
regulating synthesis of cues could be bypassed and production
amplified by cloning, then scale-up of LST production for hatcheryuse would be feasible. Also, if, indeed, a specific determinant present in PAVE, e.g., mannuronic acid, acts as the cue for larvae

Bacterial products andpolysaccharide films
as cues and enhancers
of oyster set
RONALD M. WEINERLEVENT DAGASANMICHAEL P. LABARERITA R. COLWELL
Department of Microbiology
University of MarylandCollege Park, Maryland 20742

DALE B. BONARSTEVEN L. COON
Department of Zoology
University of MarylandCollege Park, Maryland 20742

37

Acknowledgments

metamorphosis, then artificial cultch can be manufactured. Investigations of these possibilities are in progress.

_

This material is based on work supported by the National Science
Foundation under Grant No. PCM-8316178 and research sponsoredin part by NOAA Office of Sea Grant, Department of Commerce,
under Grant NO. NA 84-AA-D-00014.

Citation_

Morse, D.E.
1984. Biochemical and genetic engineering for improved production of abalones
and other valuable molluscs. Aquaculture 39:263-282.

38

ABSTRACT

Juvenile hormone
in Crustacea

Juvenile hormone (JH) is known to play important roles in insect development and reproduction. We and others have shownthat JH analogues, such as methoprene, also have potent effects on these same processes in crustaceans, suggesting that
there may be JH-like compounds in this arthropod class. One
possible source of such a crustacean JH is the mandibular organ(MO), a tissue shown by others to affect reproduction in the
spider crab, Libinia emarginata.
To test this hypothesis, we incubated MOs from several
crustaceans, including Libinia, Callinectes sapidus, and Homarus
americanus, in Patin's or lobster saline for 2 hours supplemented
with methyl-labeled methionine. Secreted products were extracted from the culture medium and analyzed by HPLC. MOsfrom all three species secreted radio-labeled methyl farnesoate(MF). The chemical identification was confirmed by GCIMS.
The synthetic rate for MOs from Libinia ranged from 3 to 38
ng/gland' hour. Radio-labeled JHIII was also secreted by Libinia
MOs, at a rate that was less than 0.1 % of that observed for MF.
Stereoisomer analysis of this material revealed the presence ofboth enantiomers, suggesting that it may have been formed by
the nonspecific chemical oxidation of MF. MF was also detectedin hemolymph samples from Libinia, using a GCIMS procedure,with circulating levels of 10-50 ng/mL. These smples also contained small amounts of JHIII, 3-30 pg/mL, but these were probably produced by the oxidation of MF during analysis.
Our data demonstrate that MF is a tissue-specific and major
secretory product of the crustacean MO. Since MF has JH activity in insects, and its concentration appears to correlate withthe reproductive state of the organism, this compound may be
a crustacean JH. Alternately, MF may be a prohormone, similar
to the ecdysone, that is converted to some other compound, such
as JHIII, by peripheral tissues.
The control of metamorphosis and reproduction seems, atleast in part, to be regulated by the MO and its secretory
product(s). Analysis of the crustacean MO, its secretions, andhow they affect reproduction should lead to better strategies
for crustacean aquaculture. (Supported in part by Sea Grant
NA82AA-D-OOOI8.)l

HANS LAUFER
Department of Molecular and Cell Biology
University of Connecticut
Storrs, Connecticut 06268

andMarine Biological Laboratory
Woods Hole, Massachusetts 02543

DAVID BORST2
Department of Molecular and Cell Biology
University of Connecticut
Storrs, Connecticut 06268

FRED C. BAKER
DAVID A. SCHOOLEY
Zoecon Research Institute
Palo Alto, California 94304'The information in this abstract is included in the following article: Laufer, H.,
D. Borst, F.C. Baker, C. Carrasco, M. Sinkus, C.C. Reuter, L.W. Tsai, and D.A.
Schooley. 1987. Identification of a juvenile hormone-like compound in a crustacean.
Science (Wash., D.C.) 235:202-205.
'Present address: Department of Biological Sciences, Illinois State University,
Normal, IL 61761.

39

Recent advances in nursery
culture of bivalve mollusks
in North America

The successful culture of bivalve mollusks is dependent upon theavailability of large quantities of set or seed. In areas where dense
sets reliably occur, the problem is reduced to the labor and logisticsof harvesting the set. In North America, the culture of bivalves has,for the most part, relied upon more controlled methods of providing
seed for the industry. Large quantities of hatchery-reared set canbe supplied throughout the year but costs of production, and subsequent value of the set, place an economic imperative on high survival and fast growth of set to field planting size (Brown et al. 1983).
The success and popularity of mollusk hatcheries in North America
and Western Europe has thus led to the development of nurserysystems capable of accepting and capitalizing upon the hatcheryproduced set.
The large majority of field growout techniques used in commercial hard clam aquaculture require a minimum initial clam seed sizeof 7-10 mm SL (Castagna and Kraeuter 1977,1981; Claus 1981;
Manzi 1985) while scallops and surf clams must be 10-20 mm(Rhodes et al. 1981) and oysters should be 1-5 g whole wet weight(Bayes 1981). It is generally not economically feasible to raise seed
to this size in hatcheries. Nurseries are the link connecting hatcheries
with field growout operations, providing the intermediate growth tobring hatchery-produced set to the size seed required for field growout activities. The semi-controlled conditions provided by nurserysystems are necessary in culture operations that depend on high survival and fast growth for economic viability. In this regard, nurseries
are pivotal to the success of commercial bivalve mariculture. This
paper reviews the recent advances in bivalve nurseries in North
America and provides some comparative data and performance
characteristics from experimental and commercial-scale systems.

l

JOHN J. MANZINANCY H. HADLEYSouth Carolina Wildlife and Marine Resources Department
P. O. Box 12559
Charleston, South Carolina 29412

ABSTRACT
Nursery systems in common use in North America for the cultureof bivalve mollusks are surveyed. Special attention is given to
the use of upflow culture in recirculating systems for young postset and in open flowthrough systems for seed production. The
first year's operation of a commercial nursery system using
upflow culture is discussed. Results showed that seed clam,
Mercenaria mercenaria, growth in upflow culture was directly
related to water flow rates. Biomass increases as high as 1400%
per month were achieved in upflow systems at stocking densities of 0.3 to 0.5 g/m 2 , and flow rates of 80 to 120 Llmin' kg.
Results from commercial and experimental upflow systems indicate that they are attractive alternatives to raceways. The
potential benefits include maximizing space utilization, low construction cost, ease of maintenance, and operational longevity.

Post-set maintenance_

Perhaps the most critical period in the production of bivalve seedoccurs between the time the larvae metamorphose (set) and the time
the set attain a size of one millimeter. Most hatcheries retain themetamorphosing larvae and recent set in their larval rearing containers. Culture water is changed daily, and the cultures are fed
relatively large amounts of cultured algae (lOS-106 celis/mL·day).
Alternatively, post-set cultures are provided with a steady low flowof prefiltered (10-25 /-lm) raw seawater if ambient phytoplankton
concentrations are suitable. Eyed oyster larvae are often placed inshallow trays with shell particles covering the bottom. This resultsin "cultchless" spat that can be handled similarly to other bivalves.
After a short time interval, the post-set are transferred to a numberof different post-set rearing systems, depending on the hatchery's
production system and location. Most large production facilities
continue static water culture of post-set in flat-bottom tanks. Others
use water tables that introduce a flow of coarsely filtered ambient
seawater across a broad, shallow area. These water tables or raceways can promote excellent growth and high survival when ambient conditions are suitable.
Recently, recirculating upflow culture units have been introducedin post-set culture. These units utilize an upward vertical flow ofwater through a cylinder suspended in a reservoir tank (Fig. 1).
Fine nylon mesh (100-150 /-lm) is stretched over the bottom of thecylinder and an airlift penetrates a side wall. The mesh supports
the young post-set (200-300 /-lm SL) in the path of an upflow current created by the airlift pump. The water in the reservoir is thus
recirculated up through the cylinder and back out into the reser-'Contribution No. 203 from the South Carolina Marine Resources Center.

41

cial bivalve culture, primarily for oyster and scallop growout.
Although the techniques and designs vary, they all attempt to holdseed up in the water column near higher concentrations of phytoplankton and away from benthic predators. Rafts are probably theoldest field nursery systems in North America. They are generally
simple in design, consisting of a culture unit or units, flotation
devices, and an anchoring system. Rafts are normally deployed inchains or pods to decrease expense and provide some mutual protection. Despite their traditional use, suspended culture systems ingeneral, and rafts in particular, have become less used in commercial mariculture operations. The general consensus is that, although
they work, suspended culture systems are expensive to constructand maintain and are extremely susceptible to storm damage.
Recently, ponds and impoundments have been reexamined as field
nursery systems. Shrimp and fish culture has been extremely successful in ponds, and there is growing interest in biculturingmollusks with shrimp or compatible fish. In the southeastern UnitedStates, where pond culture of shrimp is beginning in earnest,
bivalves offer a possible offseason crop allowing use of ponds ona year-around basis. A recent study (Stevens et al. 1985) evaluatedthe use of shrimp culture ponds in South Carolina for overwintering bivalve seed from the northeastern states. Results indicated thatseveral bivalves, including seed hard clam, M. mercenaria, exhibit
high growth rates and low mortality in ponds used as overwintering nurseries. In general, 5-6 months of culture in trays deployedin vacant shrimp ponds in South Carolina yielded seed size increasesof 3-10 mm and survivals ~80%.

airliftreservoir:.'. ::.
'.:':'.:':'.: .. ':,:':"

Figure 1
Recirculating upflow culture unit for post-set culture. Schematic shows crosssection of a typical unit in a reservoir tank. Arrows indicate direction of water flow.

voir via the airlift pump. At the Waddell Mariculture Center in South
Carolina, preliminary experiments with recirculating upflow culture
for juvenile Mercenaria spp. indicated that such units are effective
for post-set as small as 300 flm SL. At least one commercial clam
mariculture operation has incorporated recirculating upflow culture
for post-set introducing the clams to the system as soon as they set(190-230 flm in size).
Recirculating upflow culture requires appreciable energy expenditures. The Waddell Mariculture Center uses seawater heating andchilling units to maintain optimum reservoir temperatures. Reservoir water must be changed daily and algae must be available insufficient quantities to maintain efficient feeding concentrations(0.5-1.0X las cells/mL·day). In addition, post-set populations require frequent inspection and thinning to maintain uninterrupted
growth. Despite the demand on resources, recirculating upflow
culture of post-set bivalves appears to offer controlled rapid growthand high survival. Space and water requirements are not large, andtotal time required to bring post-set to sizes large enough (~1 mm)
for flow-through nurseries is minimized. These systems are thus
reasonable and possibly preferable methods of post-set culture forboth research and commercial facilities.

Field nursery systems

Onshore nursery systems_

Hatcheries in North America have long used onshore nurserysystems to grow young seed to commercially salable sizes. Onshore
nursery systems provide a more controlled environment for rearing young bivalve seed. They require adequate siting and sufficienttechnical input, however, to insure that uninterrupted and appropriate quantities of high-quality estuarine waters are available.
Onshore nurseries have traditionally used raceways or tiers ofshallow trays, continuously supplied with raw, ambient seawater.
Raceways are generally long shallow tanks or troughs, constructedof epoxy-coated wood, fiber glass, or concrete. Seawater, pumped
from an adjacent source, enters the raceway at one end and isdirected horizontally along the tank to drains at the opposite end.
In shallow raceways, a single layer of seed is spread over the bottom and the water level is adjusted by standpipes so that the horizontal flow of water is just deep enough to cover the seed. This insures proper water mixing and efficient ratios of water use per unit
biomass of seed. In deeper raceways, racks or tiers of plastic mesh
are used to layer seed and thus take advantage of the increased tankcapacity. Deeper raceways also commonly use baffles to mix thewater as it flows along the structure.
Tiers of shallow trays have been used in bivalve nursery culture
on a commercial scale. Rectangular fiber glass trays, 7-10 cm deep,
with substantial sidewall lips are supported in a rack-like structure.
Each tray is supplied with substrate (usually sand) or small meshplastic netting stretched across a wooden frame, to support a layerof bivalve seed. Ambient seawater is delivered to each trap which
in turn overflows through a standpipe or sidedrain into a commontier drain. Although excellent for small seed, water flows are
generally not sufficient for the biomass loads of larger seed.
Maintenance is also a problem with tiers of trays, making them
impractical in commercial nursery systems.

_

Field nurseries utilize protected natural areas for the intermediate
growth of bivalve seed. Shallow subtidal or low intertidal areas are
suitable for a number of types of bottom nursery culture. Castagna
and Kraeuter (1981) describe low intertidal bottom nurseries using
aggregate cover and plastic mesh tenting to provide protection frompredators. Tray and rack nursery culture systems are also employedin low intertidal or shallow subtidal areas. Trays, constructed ofwood or plastic frames with mesh of various materials and sizes,
are placed directly on the bottom or stacked in tiers, two to several
trays high. These systems provide good protection but require appreciable labor to maintain adequate water flows through the trays,
particularly in areas where silt or fouling are problems.
Suspended nursery culture has a long history in bivalve culture
in North America. Rafts, suspended trays, lantern nets, and pearl
nets have been used and to some degree are still used, in commer42

Upflow nursery systems for bivalve seed have been in use since
the mid-1960's (Bayes 1981) but commercial interest in thesesystems has been revived in North America only in the past five
years (Manzi and Whetstone 1981, Manzi et aI. 1984, Manzi etal. 1986). Upflow systems utilize a vertical water flow directed up
through a seed mass, rather than a horizontal flow across the seed
as in raceway systems. Two upflow culture systems are in common use in flowthrough bivalve nurseries: active flow that forceswater under pressure up through a layer of seed, and passive flowthat "pulls" water up through a seed mass. Figure 2 compares
schematic illustrations of typical passive and active upflow culture
systems. Active systems are constructed of closed-bottom cylinders
plumbed to accept water, under pressure, at or near the bottom of
the cylinder. The seed are suspended above the water intake ona plastic screen of appropriate mesh size to retain the seed. The
water is forced up through the screen, partially fluidizing the seed
mass, and exits through drains positioned near the top of thecylinder. Active (or force) flow cylinders are normally employedin banks and used primarily for very small seed. Passive upflow
systems use open-ended containers (usually cylinders of PVC pipe)
suspended in a reservoir. A screen with an appropriate mesh size
forms the bottom of the container and supports the seed mass, which
covers the screen, often to a depth of several centimeters. Water
entering the reservoir can exit only through drains located in theupper section of each container, although some reservoirs also use
a single standpipe to maintain a desired water column depth. Incoming water enters the reservoir and is drawn up into each upflow
container, accelerating rapidly as it passes through the seed massand decelerating upon reaching the overlying pool between the seedmass surface and the unit drain. If flow rates are appropriate, wastes
and silt are swept through the seed mass and settle as a loose layer
at the seed mass surface as the flow decelerates in the upper section of each cylinder.

Inflow

Culturecylindert
Raw Seawater

Reservoir
ScreenPassive Flow

Cu Iturecy Ii nder

ScreenI nflo w -a:::=::J

Forced Flow

Nursery culturecomparisons_

Figure 2
Diagrammatic representation of forced and passive upnow culture systems (after
Manzi el al. 1984).

In general, field nursery systems are less successful than onshore
facilities. Although they require simple low-energy input, they haveseveral distinct disadvantages, including high maintenance requirements, incomplete predator protection, limited access, minimal comtrol of stocked populations, and susceptibility to environmental
perturbations, vandalism, and theft. In contrast, onshore systems
provide maximum protection, access and control over stocked
populations, but require appreciable energy input. Survival andgrowth of bivalve seed in land-based systems are high, thus providing economic justification for the greater operational costs. Onshore systems are also generally more longevous than field systems,
allowing the amortization of capital construction costs over several
years. Raceway and upflow culture are the principal land-based
nursery systems in use in North America; therefore, comparisonof some of their attributes is appropriate.
The use of raceways is a longstanding tradition in bivalve nursery
operations in North America, and recent research attests to theirefficacy in seed growth of hard clams, M. mercenaria (Hadley andManzi 1984), surf slams, Spisula solidissima, and bay scallops,
Argopecten irradians irradians (Rhodes et aI. 1981). In comparisonsof growth rates of seed M. mercenaria in subtidal field units andin raceways in South Carolina, Hadley and Manzi (1984) reported
similar growth at high stocking densities (20,OOO/m 2) but significantly greater growth in raceways at low densities (740/m 2).

These differences between growth in field unils and raceways adjacent to one another and deriving water from the same well-mixed
estuary are difficult to resolve. One factor that obviously differs
is the water-flow characteristics in subtidal field units and landbased raceways. Raceway flow rates are constant and unidirectional.
Hadley and Manzi (1984) reported good seed mean growth atflow rates as low as 3L H20 per L seed/min, but water flowrates
two to three times higher were necessary for maximum growth.
Rhodes et al. (1981) reported flow requirements of 5 and 6.5 L
H2 0 per L seed/min for surf clams and scallops, respectively.
Water flow requirements for nursery culture depend, to a large
extent, on food supply per unit volume of water. Rhodes et aI. (1981)
reported that growth of surf clams in raceways was controlled byfood supply and recommended that flowrates be adjusted so thatat least 1 jlg/L of chlorophyll should remain in the nursery effluent
to assure adequate food supply. Kirby-Smith (1972) suggested thatat least 60% of the incoming chlorophyll should remain in the effluent for bay scallops to grow well in raceways. Hadley and Manzi(1984) demonstrated a strong correlation between chlorophyllremoval and growth of hard clam seed in raceways.
The relationships between water flowrates, food availability, andstocking density emphasize a primary problem with raceway culture.
43

Table 1

Table 2

Mean total increase in length (nun) of seed clams (initial mean size =, 3.9nun) grown in raceways at four densities, February-August 1981 (data from
Hadley and Manzi 1984).

Relationship between water flowrate (L/min' kg) and clam seed biomass increase (g/I00 g) in an experimental-scale upflow culture system (data from
Manzi et aI. 1986).

Density (clams/m2)

Initial density (kg/m2)

Approx.
mean distance (em)from inflow

740

2200

6600

19980

Y

13
42
71
100

16.44
13.68
11.39
10.61

15.09
13.87
11.83
10.91

13.23
10.24
9.72
9.20

10.29
9.03
8.49
7.34

13.76
11.70
10.36
9.52

Y

13.03

12.92

10.60

8.79

2.5
Days

5.0

10.0

20.0

Flow Growth

Flow Growth Flow Growth Flow Growth

120
33
19
14

266.8
75.1
26.4
15.5
837.5

58
16
10
8

256.5
67.1
14.9
7.7
636.9

29
9
6
6

213.1
47.7
6.2
3.7
409.4

15
6
5
5

125.6
32.7
5.4
1.0
218.5

40

209.4t

20

159.2

11

102.4

7

54.6

---_.

30 (22.1)'
60 06.8)90 (\5.7)
120 (\0.4)
Cumulative

Y

--_.

The effective flowrate, or the rate at which new water is available
to the seed, decreases as the water travels down the raceway. An
example of this effect was reported by Hadley and Manzi (1984)
who correlated growth of hard clam seed in raceways with distancefrom inflow (Table 1). As distance from the inflow increased, therate of growth of seed clams at all densities decreased. Maximum
growth occurred at the lowest stocking densities nearest the inflow
and minimum growth in the highest densities nearest the outflow.
Growth rates of seed clams at the same density were notic,~ablydifferent when separated by as little as 29 cm from the inflow.
Significant decreases in growth rate occurred over the first meterof raceway length. Thus, flow rates per unit biomass in rac~way
culture must be adjusted in relation to distribution of the seed. This
is often difficult to put into practice, as increased flow rates and/or
maintenance requirements may be difficult to justify economicallyin many extant raceway culture systems.
The performance characteristics of upflow culture systems forbivalve seed have received recent attention (Rodhouse et al. 1981,
Rodhouse and O'Kelly 1981, Manzi 1985, Manzi et al. 1984, 1986).
These systems apparently create conditions for rapid growth andhigh survival at relatively dense concentrations of bivalve seed.
Bayes (1981) indicated that as much as 1000 kg of cultchless oyster
seed could be maintained in an upflow container with a volume of
only 1.5 m3 • In a study of upflow culture at experimental flowrates and stocking densities, Manzi et al. (1986) showed a positive
correlation between flow rate and growth of hard clam seed (Table2) . At four initial stocking densities, equivalent to 2.5, 5.0, 10.0,
and 20.0 kg/m2 , seed clams exhibited high growth rates at flowrates equal to or greater than 29 Llmin' kg when ambient watertemperatures were relatively high (y = 22.1°e). At lower temperatures ('V15-17°e) flowrates of at least 10 Llmin· kg of biomass
were necessary for acceptable seed growth. These data, however,are confounded by ambient chlorophyll levels, or food availability. Table 3 presents an experimental relationship between growth
and ambient chlorophyll removal for the data presented in Table
2. In general, growth in upflow culture units was directly related
to ambient chlorophyll-a available to the seed and effe'~tive
chlorophyll-a removal. Since this makes energetic sense, in thatan individual clam expends less energy for more food, a comparisonof chlorophyll-a removal would be valid for density effects within
specific time intervals. At optimum ambient water temperatures,moderate growth in clam seed occurred at flowrates ~9 Llmin' kg
and chlorophyll-a availability between 100 and 500 j.lg/min· kg. Exceptional growth occurred at flowrates ~ 16 Llmin' kg and ambient
chlorophyll-a availability of 500 j.lg/min· kg or greater. Whenchlorophyll-a concentrations were highest (first 30 days), greatest
growth occurred when chlorophyll-a removal exceeded 150 j.lg/min'Mean water temperature ('C) for each 30-day period.
tCumu1ative percent increase divided by the number of months (4).

Table 3
Relationship between ambient chlorophyU-a and clam seed biomass increasein an experimental-scale upflow culture system (data from Manzi et aI. 1986).
Days

Density(kg/m2)

Ambient Chi-a(Jlg/min'kg)

Chi-a removal(Jlg/min'kg)

Growth(g/IOO g)

30

2.5
5.0
10.0
20.0

2064
999
500
249

168
161
159
118

267
257
213
126

60

2.5
5.0
10.0
20.0

472
234
134
93

66
51
48
46

75
67
48
33

90

2.5
5.0
10.0
20.0

128
67
43
34

31
37
24
23

26
15
6
5

120

2.5
5.0
10.0
20.0

141
81
56
44

13
22
19
17

16
8
4
I

(n)

for each kg of biomass maintained in the system (Table 3) . At theselevels, the lowest stocking density (2.5 kg/m2) removed only 8.2%of the available chlorophyll-a, while the highest density (20 kg/m2)
removed nearly 48 %. Kirby-Smith (1972) reported that scallop
growth was reduced when more than 40 % of the available
chlorophyll-a was utilized. Data presented here from Manzi et al.
(1986) suggest that growth of seed clams in upflow culture may
be reduced when chlorophyll-a reduction exceeds 25-30%.
Data directly comparing raceway and upflow culture are not yet
available, but previous data from raceways (Hadley and Manzi 1984)
and from experimental upflow units (Manzi et al. 1986) indicate
similar performance of the two systems for clam seed nursery
culture, at least at the higher tlowrates. In experimental scale uptlow
units, mean monthly rates of biomass increase (MBI) varied from73 to 126% at temperatures of 16-18°C and flowrates of 7-118
Llmin' kg. In raceways, seed grew at mean rates of 125 to 132 %(MBI) at water temperatures of 13-16°C and flowrates of 56-1483
Llmin' kg. A direct comparison of growth data and water flowrates
from both studies is presented in Figure 3. The raceway study design
44

Table 4
10

Ideal (I) and actual (A) stocking schedules for a commercial hard clam nurseryin South Carolina, March-December 1983 (after Manzi et al. 1984).

• RACEWAYo UPFLOWu

Residual(kg)

Net production
(kg)

Input
(kg)

E
c

Space
utilization*

----

"-

Month

E
:I:

March
April
May
June
July
Aug.
Sept.
Oct.
Nov.
Dec.

I-

:;:
0

cr

<!l

0
0

I

10

100

HOH (Ll/CLAMS (Ll/MINUTE

Figure 3

Total

Mean monthly biomass increases (%) of seed clams raised at various waterflow
rates (Llmin . kg) in raceways and upflow columns. (Data from Hadley and Manzi
1984, Manzi et at. 1986.)

4
4
4
4

16

158.7
1.5
2.2
8.3

170.7

A

A

3.6
14.0
87.5
196.0
312.4
352.0
283.5
175.0
55.0

-11.3
-10.3
10.6
207.0
262.7
209.3
113.3
-11.7
43.1

7.6
25.6
54.6
104.6
142.0
144.0
115.0
65.0
20.0

158.7
59.6
51.5
37.5
89.3
227.0
247.0
267.0
255.5
215.4

1,479.0

812.8

20.0

215.4

4
25
59
94
100
98
66
31
8

68
20
33
27
75
100
100
100
97
92

'Percent of total space available.

passive-upflow cylinders. These were stocked initially at 500 to
850 g/unit (0.2-0.35 g/cm2) or approximately 50,000 to 85,000
seed at 3 mm SL, and provided with a flow of 25-35 Llmin.
The nursery had a theoretical instantaneous capacity of 4 to 16
million seed clams, depending on seed size. An idealized stocking
schedule for the nursery is compared with the actual stocking in
Table 4. Ideally, 16 million I-mm seed would have been imported
over a 4-month period from April to July. Assuming a 75 % survival to planting size (7-8 mm), net production would be 1480 kg
or a 9250% biomass increase in 9 months. Instead, the nurserystocking period was preceeded by the introduction of 158 kg ofresidual seed from previous nursery operations, and the majorityof new seed (> 12 million) were imported and stocked in the nurseryin July. From April to December 1983, 3.5 million seed weretransferred from the nursery to field culture units as they attainedplanting size. At the end of December, almost 4 million seed (215kg) still remained in the nursery. Net production for the year was
813 kg, and survival was approximately 38 %. The low production
and poor survival were primarily the results of an inappropriate
stocking schedule. Hatcheries were unable to meet scheduled commitments, resulting in only 3.8 million seed arriving during the ideal
stocking period (Table 4) . The bulk of imported seed, over 12
million, arrived during a 2-week period in July when ambient watertemperatures were 29-30°c, which is above the range suitable for
growth of M. mercenaria. The nursery was thus understocked during
the spring and early summer when best growth occurs and nursery
carrying-capacity is greatest, and stocked at full capacity duringlate summer, fall, and winter when poorer conditions exist.
Growth of seed clams in the nursery system was directly related
to stocking densities and water temperatures. The smaller seed «3mm) stocked in active flow units exhibited mean monthly biomass
increase rates (% MBI) that were relatively high throughout thesummer. The MBI over the first 60 days of each stocking ranged
from a low of 277% toahighofl147% at21 and 30°C, respectively. Apparently the high flowrates and fluidizing effect attainedin these cylinders fostered growth even under high-temperature conditions. Growth rates in the larger passive cylinders were lower.
Seed larger than 3 mm grew from 115 to 336 % (MBI) over the
first 60 days of culture at temperatures of 24 to 29°C and flowrates
ranging from 17 to 117 Llmin' kg.

employed very low stocking densities and subsequent high effective flowrates, while the upflow unit study used relatively high stocking densities and resultant low effective flowrates. Despite these
discrepancies, an analysis of the individual and combined data setsshow similar responses of seed clam growth to water flow (Fig.
3). Upflow units, however, can support much greater biomass perunit area than raceways. In the raceway study, an area of 0.1 m2
was used to support a maximum biomass of 31 g, while an upflow
column of similar area could support 150 times that biomass. The
two systems performed similarly in the one aspect most important
to commercial interests: the length of time required to raise seed
to field planting size. Both systems produced planting size seed (7-8mm) in a minimum of 60 days of culture.
In conclusion, a comparison of raceways and upflow units forthe nursery culture of hard clam seed indicated that both systems
provide conditions for good seed growth aad survival. Upflow
systems, however, provide several distinct advantages over raceways, including ease of maintenance, effective space utilization,
longevity of service, and ease and economy of construction. Upflow
units are reasonable and economic alternatives to the more traditional raceway culture of bivalve seed.

Upflow culture:
Commercial application

A

A

_

A commercial-scale application of upflow culture in a hard clamnursery was recently reported (Manzi et al. 1984). The nursery consisted of 60 forced-flow cylinders, arranged in banks of 20, and
160 passive-flow cylinders, arranged as 8 unit "batteries" in 20
reservoir tanks. A 25-hp centrifugal pump supplied approximately
360 m3/hr (1600 gpm) of high-salinity estuarine water to thenursery.
Active upflow cylinders (20-cm diameter) were used for thesmallest seed clams «3 mm) and were usually stocked with recently
purchased I-mm seed at densities of 100,000 to 300,000 per unit
(0.3-1.0 g/cm 2). Each unit received water at a flow rate between
II and 15 Llmin, a flow just sufficient to partially fluidize the seedmass. As the seed grew, fluidization became less effective and seedclams were transferred to the larger diameter (46 and 56-cm)
45

tected natural areas, are simple and inexpensive but are difficult
to maintain and are subject to predation and environmental damage.
Onshore nurseries, while more expensive to construct and operate,
provide reliable rapid growth and high survival. Onshore systems
have traditionally employed raceways, but upflow nurseries havebecome popular in the last few years because they are easier to maintain than raceways and make optimum use of space.

Table 5
Recommended stocking densities and now rates for various size seed damsin active and passive upnow culture cylinders in a commercial nursery sfstem
in South Carolina (data from Manzi et al. 1984).

Unit type

Seed
size
(mm)

Initialdensity(g/cm')

Initialflow(Llmin' kg)

Final
density(g/cm')

Final
fl0W(Llmin'kg)

Active flow
(20 cm dia.)

<2
2-3

0.31
0.46

120
80

1.08
1.23

34
30

Passive flow
(56 cm dia.)

<2
2-3
3
4
5
6

NR
0.16
0.20
0.35
0.47
0.61

80
70
41
30
23

0.62
0.65
0.69
0.82
1.24

23
22
21
18
12

Acknowledgments_

This work was sponsored by NOAA, National Sea Grant College
Program Office, U.S. Department of Commerce, and the South
Carolina Sea Grant Consortium.

Citations

NR = Not recommended for this size seed.

_

Bayes, J.C.
1981. Forced upwelling nurseries for oysters and clams using impounded water
systems. In Claus. C .• N. DePauw, and E. Jaspers (eds.), Nursery culturingof bivalve molluscs, p. 73-83. Spec. Publ. 7, Eur. Maricult. Soc., Bredene,
Belgium.
Brown, J.W., J.J. Manzi, H.Q.M. Clawson, and F.S. Stevens.
1983. Moving out the learning curve: an analysis of hard clam, Mereenaria
mereenaria, nursery operations in South Carolina. Mar. Fish. Rev. 45(4-6):
10-15.
Castagna, M., and J.N. Kraeuter.
1977. Mereenaria culture using stone aggregate for predator protection. Proc.
Natl. Shellfish. Assoc. 67:1-6.
1981. Manual for growing the hard clam Mereenaria. Spec. Rep. 249, Va. Inst.
Mar. Sci., Gloucester Point, VA 23062.
Claus, C.
1981. Trends in nursery rearing of bivalve molluscs. In Claus, C., N. DePauw,
and E. Jaspers (eds.), Nursery culturing of bivalve molluscs, p. 1-33. Spec.
Publ. 7, Eur. Maricult. Soc., Bredene, Belgium.
Hadley, N.H., and J.J. Manzi.
1984. Growth of seed clams, Mereenaria mereenaria, at various densities in
a commercial scale nursery system. Aquaculture 36:369-378.
Kirby-Smith, W.W.
1972. Growth of the bay scallop: the influence of experimental water currents.
J. Exp. Mar. BioI. Ecol. 8:7-18.
Manzi, J.J.
1985. Clam aquaculture. In Huner, J.V., and E.E. Brown (eds.), Crustacean
and mollusk aquaculture in the United States, p. 275-310. AVI Publ. Co.,
Westport, CT.
Manzi, J.J., and J.M. Whetstone.
1981. Intensive hard clam mariculture: A primer for South Carolina watermen.
South Carolina Sea Grant Consort. Mar. Adv. Publ. 81-01, Charleston, SC
29401.
Manzi, J.J., N.H, Hadley, C. Battey, R. Haggerty, R. Hamilton, and M. Carter.
1984. Culture of the northern hard clam, Mereenaria mereenaria (Linne), in
a commercial-scale, upflow nursery system. J. Shellfish. Res. 4(2): 119-124.
Manzi, J.J., N.H. Hadley, and M.B. Maddox.
1986. Seed clam, Mereenaria mereenaria, culture in an experimental-scale
upflow nursery system. Aquaculture 54:301-311.
Rhodes, E.W., R. Goldberg, and J.C. Widman.
1981. The role of raceways in mariculture systems for the bay scallop,
Argopeeten irradians irradians, and the surf clam, Spisula solidissima. In
Claus, C., N. DePauw, and E. Jaspers (eds.), Nursery culturing of bivalve
molluscs, p. 227-251. Spec. Publ. 7, Eur. Maricult. Soc., Bredene, Belgium.
Rodhouse, P.G., and M. O'Kelly.
1981. Flow requirements of the oysters Os/rea edulis and Crassostrea gigas
Thunb. in an upwelling column system of culture. Aquaculture 22 (1,2): 1-10.
Rodhouse, P.G., B. Ottway, and G.M. Burnell.
1981. Bivalve production and food chain efficiency in an experimental nurserysystem. 1. Mar. BioI. Assoc. U.K. 61 :243-256.
Stevens, F.S., J.J. Manzi, R.L. Walker, and R.A. Kraus.
1985. Growth of overwintering northern shellfish in mariculture ponds andestuarine systems in the Southeast. Abstracts, 77th Annual Meeting, Natl.
Shellfish. Assoc., Norfolk, VA, June 17-20, p. 15.

Growth rates observed at the various stocking densities, flowrates,
and water temperatures experienced during the first year of clamnursery culture were used by Manzi et al. (1984) to determine appropriate stocking densities for upflow culture in South Carolina(Table 5). While these recommended densities are appropriate forthis particular system, the carrying capacities of any nursery depend
upon site-specific characteristics. In this nursery. active flow
cylinders could be stocked at 0.31-0.46 g/cm 2 (depending on seedsize) at flowrates approximating 80-120 Llmin' kg. Seed stockedin these units had to be redistributed frequently, at about 2-weekintervals, so that densities did not exceed 1.1-1.2 g/cm 2 and flowrates were not reduced below 30 Llmin' kg. Seed clams smaller
than 2 mm did not grow well in the larger diameter passive-upflow
cylinders. Small seed produce byssal threads that tended to precipitate the formation of seed clumps. The clumping resulted in unevenseed distribution and subsequent "fountaining" of water throughthe seed mass. Larger seed grew well in passive-upflow culture
if the seed were redistributed frequently to avoid densities greaterthan 0.6-0.7 g/cm2 and flowrates lower than 23-30 Llmin'kg(Table 5). In general, seed clams could be left in the upflow cylinders
until the stocked volume, or biomass, doubled and then redistributed
to reduce the biomass to an appropriate initial density.
Although the first year of operation of a commercial upflow
nursery system yielded results that were below expectations (Manziet al. 1984), the system demonstrated that seed clams can growrapidly at high densities in upflow culture. Biomass increases wereas high as 1400% (MBI) in active-flow cylinders and 800% (MBI)in passive-flow cylinders, and compared favorably with growth rates
reported previously for raceways and experimental upflow systems.
Poor survival over the first season can be attributed, at least in part,to factors that can be eliminated with better planning.

Summary
Bivalve nurseries are used to bridge the gap between hatcheries andfield operations, providing protected growth conditions for postset and small seed at a reasonable cost. In North America, post-set
are generally maintained within the hatchery in shallow flowthroughraceways, static flat-bottomed tanks, or recirculating upflow
cylinders. When post-set reach a size of ""' 1 mm SL, they are transferred to field or onshore nurseries. Field nurseries, located in pro46

Seaweeds are important marine resources and have many uses. During the last decade, they have been regarded as a promising biomass
for fine chemicals and alternative energy production. Recently,
cultivation of economically valuable seaweeds has become a big
industry in Japan. 'Nori' (Porphyra), 'Wakame' (Undaria) , and'Kombu' (Laminaria) are three major marine food-crops, and theirannual yields are about 300,000, 120,000, and 30,000 tons (dryweight), respectively. The seaweed-cultivation industry is estimatedto be worth over $1 billion per year.
Tissue culture and genetic engineering hold extraordinary promise
for aquaculture of seaweeds. We expect much help from these innovative techniques in breeding and seedling production. In orderto develop improved seaweed strains, we are performing the following several projects: tissue culture (including callus formation andregeneration, and suspension cell culture); cell engineering (protoplast formation and regeneration, somatic hybridization using
protoplasts, microinjection of foreign nucleus and cytoplasmic
organella); and genetic engineering (mutant production and selection, gene cloning and sequence analysis, improvement of vector
and recipient cell systems, transformation using Ti-plasmid). We
are very optimistic of obtaining good results by applying thesetechniques newly introduced into the marine sciences.

Tissue culture and genetic
engineering for seaweed
aquaculture
NAOTSUNE SAGAYOSHIAKI SANBONSUGA
Hokkaido Regional Fisheries Research Laboratory
Fisheries Agency of Japan
116 Katsura-koi
Kushiro City, Hokkaido, 085 Japan

ABSTRACTIn this paper, the following several subjects on economicallyvaluable seaweeds are briefly reviewed: Tissue culture (callus
formation and regeneration, suspension cell culture); cellengineering (protoplast formation and regeneration, somatic
hybridization using protoplasts); and genetic engineering(mutant production, improvement vector, and recipient cellsystem). To date, tissue culture has been achieved in 10 generaand 16 species of seaweeds, and protoplast isolation in seaweeds
has been achieved in 9 genera and 16 species. The studies ofsomatic hybridization using protoplasts and transformation
using recombinant DNA techniques in seaweeds have just begun.

Tissue culture ofseaweed_Acquisition and isolation of axenic strains
The easiest way to obtain algal strains is to depend on culture collection centers. There are more than 50 algal culture collection
centers in the world (Haines et al. 1982). In these centers moststrains are microalgae, and marine macrophytes (so-called seaweeds)
are a minor group. For example, UTEX is a famous culture collection center, and there are only 120 seaweed strains out of 2,200
algal strains.
If we cannot find needed strains in a culture collection center,
we must isolate our own strains. Several methods to obtain axenic
strains of algae were summarized by Chapman (1973). The pipettewashing method, a basic one, is usually used for isolation of axenic
strains, but it is time-consuming, and skilled technique is required.
On the other hand, methods employing chemical sterilants for obtaining an axenic culture were published: antibiotics (Provasoli 1958,
Boalch 1961, Iwasaki 1961, Tatewaki and Provasoli 1964); iodine(Fries 1963); potassium tellurite (Ducker and Willoughby 1964);
sulfa drugs (Kanazawa 1968); and antibiotics and sodium hypochlorite in combination (Druehl and Hsiao 1969). These methods,
although convenient, were inadequate in terms of efficiency ofsterilization and toxicity of chemicals.
A convenient and secure method for obtaining axenic culture,
the one-step selection method, has been established by Saga andSakai (1982a). Following are the procedures of this method using
the brown alga, Dictyosiphon foeniculaceus (Fig. I):
I) Fertile plants are wiped with a clean gauze, rinsed several
times in autoclaved seawater, and cut into lengths of "'I cm;
2) Ten pieces of the organisms are transferred into a glass vessel
containing 100 mL ABM* and stocked in a refrigerator at 5°C for
2 days;• ABM = antibiotic mixture.

47

f\
b3--

ABM sol.
Piece of olant

Slack In freezer for 2 doys

wi th glass st tel<ST3 agar olate

Put zoosPOres on OgClrolate wah micro olooet

Figure 1
Procedure for one-step selection method of isolating axenic strains of seaweeds.

3) One piece is rinsed several times in sterile ASP* l2-NTA
medium, and numerous zoospores are released within 10 minutes;4) Two or three drops of the medium containing many zoosporesare put on an agar plate with a pipette and spread over an agar surface with a glass rod. The agar plate is cultured for I month;5) After I month the germlings will develop into 0.5-1.0 mm
masses of profusely branched fIlamentous thalli, and bacteria whichsurvived step 2 above grow to 3-5 rnm diameter colonies. Clean
algal masses growing on an agar plate should be selected and inoculated into 10 mL ASP 12-NTA medium in test tubes. This treatment serves both as an isolation of axenic algal strains and a sterility
test for bacteria.
6) These cultures are maintained in an incubator for I month,and "-'3 rnm masses of axenic microthalli can be obtained.
Recently, Bradley et al. (1986) have developed an improved onestep selection method using antibiotic disks.

Table 1
Media for seaweeds (enriched seawater type, 100 mL).
Erd-schreiber'
Distilled water
Seawater
NaNOJ
Na-glyceroPO,
Na2HPO,·12Hp
Fe (as EDTA; 1: I mol)Fe-sequestren
PH metals'
Vitamin BJ2Thiamine
BiotinVitamin mix ESS 5
Soil eXlract
Tris
HEPES
KI
pH

Media for axenic cultures
There are two major kinds of media for seaweeds: one is an enrichedseawater type (Table I), and the other is an artificial seawater type(Table 2). Following are the representative media of the enrichedseawater type: Erd-Schreiber (F~yn 1934), PES (Provasoli 1968),
PESI (Tatewaki 1966), and ESS (Saga and Gibor 1986). ErdSchreiber and ESS are usually used for general seaweeds, PES issuitable for culture of red and green algae, and PESI for cultureof brown algae.

100 mL
10 mg

PES2

PESI 2

ESS J

100 mL

IOOmL

60 mL

350 mg
50 mg

350 mg
50 mg

600 mg
80 mg

2.5 mg

2.5 mg

25 mL
10 fIg
0.5 mg
5 fIg

25 mL

2 mg
40 mg
40 mL

1 mL
5 mL
500 mg

500 mg

7.8!OO fIg
7.8

I g

7.8

'Use directly.
2For PES or PESI medium add 2 mL PES or PESI stock solution to 100 mL
autoclaved seawater, respectively.
JFor ESS medium add I mL ESS stock solution to !OO mL autoclaved seawater.
'I mL PH metals contains: Na2-EDTA I mg. Fe (as CI-) 0.01 mg, B (as
HJBOJ) 0.2 mg, Mn (as CI-) 0.04 mg, Zn (as Cn 0.005 mg, Co (as Cn 0.001
mg.
51 mL vitamin mix ESS contains: Vitamin B'2 10 fIg, Biotin 10 fIg, Thiamine· HCI I mg, Nicotinic acid I mg, Ca-pantothenate I mg, P-aminobenzoic
acid 100 mg, Inositol 10 mg, Thymine I mg.

*ASP = artificial seawater of Provasoli.

48

Table 2
Media for seaweeds <artificial seawater type, 100 mL).

NaCI
NaSO.
MgSO.. 7H,o
MgCI 2 '6H,oKCl
Ca <as 0-)
NaN03<NH.h SO•
K2HPO.
K3PO.
NaH 2PO.· H2O
Na2Si0 3 ' 9H,o
NaC03 'H 2O
NaHC03Na2 -glyceroPO.
Fe <as Cn
PH metals I
SII metals 2
P8 metals 3
Metal mix ASS'
Vitamin BI2
BiotinThiamine' HCI
Vitamin mix S3 5
Vitamin mix 8A 6
Vitamin mix ASS 7
KDS solution 8
KDTM solution9
Tris
GIyci1-g1ycine
HEPES
pH

ASP1

ASP2

ASP6

ASP7

ASP12

KDX

ASSI

2.4 g

1.8 g

2.4 g

2.5 g

2.8 g

1.9 g
0.32 g

2.5 g

0.6 g
0.45 g0.06 g
40 mg
10 mg

0.5 g

0.8 g

0.9 g

0.06 g
10 mg
5 mg

0.7 g
0.4 g
0.07 g
40 mg
10 mg

2 mg

0.5 mg0.07 g
15 mg
30 mg

0.07 g
30 mg
5 mg

1.0 g
0.87 g
50 mg
8.5 mg0.66 mg

0.07 g
30 mg
10 mg

I mg
0.7 mg
2.5 mg

I mL

7 mg

7 mg

15 mg

10 mg

2 mg

I mg

3 mL

I mL
1 mL

O.I!,g0.02!,g
0.1 !'g
10 !'g

15 mg
3 mg

50!'g
3 mL

8.8 mg0.14 mg

10 mg
2 mg

I mL
I mL
0.Q2 !'g

0.2!,g0.05!,g

1 mL

1 mL
0.1 mL0.05 mL

0.1 mL
1 mL
I mL
0.1 g

0.1 g

O.J g

0.1 g

0.1 g
75 mg

7.6

7.8

7.8-8.0

7.6

7.8-8.0

8.3-8.4

100 mg
8.0

I J mL PH metals contains: Na 2-EDTA 1 mg, Fe <as CI-) 0.01 mg, B <as H3 B03 ) 0.2 mg, Mn <as 0-) 0.04 mg, Zn(as 0-) 0.005 mg, Co <as 0-) 0.001 mg.
2 1 mg SH metals contains: Br (as Na+) I mg, Sr <as CI-) 0.2 mg, Rb (as 0-) 0.02 mg, Li <as 0-) 0.02 mg, Mo<as Na+) 0.05 mg, 1 <as k+) 0.001 mg.
3 1 mg P8 metals contains: Na 3H-EDTA 3 mg, Fe (as 0-) 0.2 mg, B <as H3B03) 0.2 mg, Mn <as 0-) 0.1 mg, Zn(as 0-) 0.05 mg, Co (as 0-) 0.001 mg, Mo (as Na+) 0.05 mg, Cu <as 0-) 0.002 mg.
'1 mL metal mix ASS contains: Fe (as Fe-sequestren) 100 !,g, B (as H3B03) 100 !'g, Mn (as 0-) 100 !'g, Zn <as CI-)
10 !'g, Co <as 0-) 1 !,g, Mo <as Na2 MoO.) 10 !'g, Cu (as 0-) I !,g, Br <as K+) 1 mg, Sr <as CI-) 100 !,g, Rb (as 0-)
10 !'g, Li (as 0-) 10 !,g, I <as K+) I!,g.
51 mL vitamin mix S3 contains: Thiamine' HCI 50 !,g, Nicotinic acid 10 !'g, Ca-pantothenate 10 !,g, p-aminobenzoic
acid I !'g, Biotin 0.1 !,g, Inositol 500 !'g, Folic acid 0.2 !'g, Thiamine 300 !,g.
6 1 mL vitamin 8A contains: Thiamine' HCI 200 !,g, Nicotinic acid 100 !'g, Putrescine'2HCI40 !,g, Ca-pantothenate 100!'g, Riboflavin 5 !,g, Pyridoxine' 2HCI 20 !'g, p-aminobenzoic acid 10 !,g, Biotin 0.5 !'g, Choline-H 2 -citrate 500 !'g, Inositol
1 mg, Thimine 800 !'g, Orotic acid 260 !'g, Cyanocobalamine 0.05 !,g, Folic acid 2.5 !'g, Folinic acid 0.2 !,g.
7 1 mL vitamin mix ASS contains: Thiamine·HCI 100 !'g, Nicotinic acid 100 !,g, Putrescine'2HCI 10 !,g, Ca-pantothenate
100 !'g, Riboflavin 10 !,g, Pyridoxine'2HO 10 !'g, Pyridoxamine'2HO 10 !'g, p-aminobenzonic acid 10 !,g, Biotin 1 !'g,
Inositol 1 mg, Choline-H2 -citrate 100 !,g, Thymine 100 !'g, Orotic acid 100 !'g, Cyanocoba1amine I !'g, Folic acid 1 !'g,
Folinic acid 0.1 !,g.
8 1 mL KDS solution contains: KBr 7.84 mg, KCI 54.2 mg, Sr0 2 '6H2 0 1.95 mg, Cyanocobalamine 0.01 !,g, Biotin
0.05 !,g, Thiamine' HC! 10 !,g.
9 1 mL KDTM solution contains: H·EDTA 668.4 !,g, H 3 BO. 1.14 mg, FeSO.. 7H,o 199.0 !'g, CuSO.. 5H 2 0 3.9 !'g,
Na2 MoO.·2H 20 12.61', MnCI 2 '4H,o 36 !,g, ZnSO.. 7H,o 44 !'g, CoCI 2 '6H,o 4.0 !'g, NH.V0 3 2.3 !'g, KI 3.9 !,g.

Tissue culture

Following are the representative media of the artificial seawater
type: ASP1, ASP2, ASP6, ASP7, ASP12 (Provasoli et al. 1957),
KDX (Bonneau 1977), and ASS1 (Saga and Gibor 1986). ASPI
and ASP7 are suitable for culture of green algae, ASP2 and ASP6
for culture of red algae, and ASPI2 for brown algae (Tatewaki1971). ASS1 is suitable for culture of general seaweeds and also
good for a basal medium of algal tissue culture.

The first report of seaweed tissue culture, presented by Chen andTaylor (1978), involved tissues of the red alga, Chondrus. In theirstudy, nonpigmented medullary cubes regenerated into complete
thalli. In the same year, single cells were obtained from callus-liketissue of the brown alga, Laminaria, and they regenerated into whole
Laminaria plants (Saga et al. 1978). This was the first establishedclone plant production using tissue culture methods in seaweeds.
Tissue culture of seaweeds to date has been achieved in 10 genera;
16 species so far as we know (Table 3).

49

Phylum

Table 3

Table 4Present status of tissue culture study in seaweeds.

Composition of ASP C-I medium! (100 mL).

Species

CultureRhodophyta

Chondrus crispus
Eucheuma uninatum
Porphyra perforata
P. yezoensis

TissueTissueCell
Tissue and cell

Phaeophyta

Dictyosiphon
foeniculaceus
Laminaria
angus/ata
L. angustata
L. angustala
L. digitata
L. hyperborea
L. japonica

Tissue and cell

Chen and Taylor 1978
Polne-Fuller et aJ. 1986
Polne et aI. 1984
Zhao and Zhang 1981,
1984
Saga et aI. 1982

Sporophyte cell

Saga et aJ. 1978

Diploid tissue
Haploid tissue
TissueTissueTissueSaga and Sakai 1983
Saga and Sakai 1982b
Fries 1980
Fries 1980
Fang et aI. 1983,
Chen 1984
Lee 1985
Chen 1984,
Saga (in prep. a)
Polne-Fuller et aJ. 1986
Mooney and van
Staden 1985
Fries 1984
Zhang 1982, Fang et
aI. 1983, Chen 1984
Saga (in prep. a)

Chlorophyta

L. saccharina
Macrocystis
pyrifera
Sargassum muticum
S. heterophyllum

TissueTissueFucus spiralis
Undaria pinnatifida

TissueTissueEnteromorphaintestinalis

TissueTissueTissueNaCI
KCl
MgS04 '7HP
MgCI'6H zO
Ca (as Cl-)NaNO)
K 3 P04
Naz ' glyceroP04
Na 3Si0 3 ' 9Hp
PH metalsz

References

SH metals)
Vitamin B,zBiotinThiamine' HCINTAMannitol
Yeast extract
Tris(Agar)4pH

2.8 g
0.07 g
0.7 g
0.4 g
40 mg
10 mg
1 mg
1 mg
15 mg
1 mL

1 mL
0.02 Jlg
0.1 Jlg
10 f'g
10 mg
3 g
0.1 g
0.1 g
1.5 g)
7.8-8.0'Saga et aI. 1982.
in Table 2.
4Agar should be omitted in a liquid medium,Z,3 See footnotes

I

I

g

To obtain axenic primary cultures, there are two possibilities:
one is a punching method that is applicable to relatively large
seaweeds, e.g., members of Laminariales or Fucales; the other isa method using cell lines previously sterilized, possibly apprcpriate
for relatively small seaweeds, e.g., members of Ectocarpales,
Chordariales, or Dictyosiphonales.
Following is the protocol for the punching method using
Laminaria plants (Saga and Sakai 1983):
1) Stipes of the thalli are cut into lengths of 5 cm, wiped witha clean gauze, and rinsed several times in autoclaved seawater;
2) Both ends of the cleaned stipes are dipped in absolute ethanol,
burned with an alcohol lamp, and cut off with a sterile knife;3) The ends are punched out axially by a sterile cork borer (4.0mm diameter), and these columnar medullary tissues are cut into
2-mm thick sections;4) Tissues obtained should be inoculated on 50-mL ASP 12-NTA
medium solidified with 1.0% agar and maintained in an incubatorat 14°C and illuminated with cool white fluorescent lamps at 2,000
lux with a 14-hr and lO-hr dark cycle;
5) A sterility test of the discoid tissues obtained by the aforementioned method is tried using ST-3 medium;6) After 1 month's culture, callus masses 2-3 mm in diametercan be derived from the inocula.

AXENIC PLANT CULTURES

j

~

W
g
j

PROTOPLAST ISOLATION

/

7

0

~

PROTOPLAST FUSION

L- U -U-VJ

f
HYBRID SELECTION

Following is the protocol for the method using axenic strains ofDictyosiphon (Saga et al. 1982):
1) Axenic tissues that develop into a mass 1.0 mm are inoCllated
into an Erlenmeyer flask containing the ASP C-l medium (Table 4);2) After culture for I month on this medium, the inoculum
develops into callus masses "-'5 mm in diameter;
3) Since these calli are comprised of round and fragile cells, small
cellular aggregates and single cells can be obtained 'by suspension
culture or by hand-crushing;

HYBRID REG::NERATIONNEW STRAINS

Figure 2
Procedure for genetic improvement of seaweeds applying somatic hybridization.

50

Table 5
Present status of protoplast isolation in seaweeds.
Phylum
Rhodophyta

Gracilaria tikvahiae
G. iemaneiformis
Porphyra perforata
P. suborbicuiata
P. yezoensis

Phaeophyta

Laminaria japonica
Macrocystis pyrifera
Sargassum muticum
Undaria pinnatifida
Enteromorpha inteslinalis

Chlorophyta
AXENIC PLANT CULTURES

E. linza
Monos/roma angicQvQ

M. nitidum
M. zostericoia
Uiva linza
U. perlusa

PROTOPLAST ISOLATIONI
PROTOPLAST

O ~··

~

Cheney 1984, Cheney el al. 1986
Cheney el al. 1986
Polne-Fuller and Gibor 1984
Tang 1982
Saga and Sakai 1984, Fujita andMigita 1985, Kilo 1985, Hatate
el al. 1986
Saga and Sakai 1984
Saga el al. 1986
Saga el al. 1986
Fujita and Migita 1985
Millner el al. 1979, Saga el al.
1986
Saga 1984, Fujita and Migita
1985
Zhang 1983, Saga (in prep. b)
Fujita and Migita 1985
Saga 1984
Zhang 1983
Saga 1984, Fujita and Migila
1985

Cell and genetic engineeringof seaweeds
VECTOR_

Protoplast isolation
Genetic manipulation of protoplasts will offer new means of breeding seaweeds. Somatic hybridization using protoplasts provides amethod of combining the genomes of different plants that are sexually incompatible (Fig. 2). Also, protoplasts are essential materials
for transformation involving direct gene transfer of foreign DNA
using plasmids as vectors (Fig. 3), The first report of protoplast
isolation in seaweeds was by Millner et al. (1979) for the green
alga, Enteromorpha intestinalis. Protoplast isolation of seaweeds
has been achieved in 9 genera and 16 species to date (Table 5).
Protoplast isolation of green algae is relatively easy, because thecomponents of their cell walls are the same as higher plants, andcellulase and pectinase can be used for protoplast isolation of somegreen algae. Following is the protocol for the protoplast isolationof green algae (Saga 1984) (Fig. 4):
1) Thalli are cut into small pieces, and 1 g is inoculated in 10
mL enzyme solution containing 2 % Cellulase Onozuka R-lO (Kinki
Yakult Mfg. Co. Ltd.), 1.2 M sorbitol at pH 6.0;
2) Incubation should be carried out at room temperature (f\J20°C)
and continued for several hours with shaking (90 excursions/min);3) Isolated protoplasts should be separated from debris by filtration through nylon mesh (60 /-1m pore size), collected by centrifugation at 500 xg for 5 min, and washed several times with 0.1 M Tris
buffer containing 1.2 M sorbitol and 3.5 mM CaCI 2 at pH 8.0;
4) Resulting pellet should be resuspended in Tris-sorbitol buffer and stored at 5°C. Figure 5 shows protoplasts isolated from
Monostroma angicava using the above procedure.
Protoplast isolation of red or brown algae is relatively difficult,because their cell wall components are quite different from thoseof higher plants, and cellulase or pectinase cannot be used for protoplast isolation of red or brown algae. For successful isolation of
their protoplasts, we must look for sources of enzymes that will
digest their cell walls. The most promising enzyme sources are gut
juice of some marine herbivorous invertebrates or culture filtrate

I

G

References

Species

TRANSFORMATIONREGENERATIONNEW STRAINS

Figure 3
Procedure for genetic improvement of seaweeds applying transformation.

4) These cellular aggregates or single cells regenerate into complete thalli.
Micropropagation using tissue culture methods promises toprovide an improved system for the mass-production of seaweed
seedlings in the future.

51

CLEAN TISSUEIlJ
HYPERTONICENZYMEBUFFER

TREATMENTSOL.UTION

FIL TRATETHROUGH
NYLON MESH

Figure 4
Procedure for protoplast isolation from seaweeds.

Figure 5
Freshly isolated protoplasts from gametophytes ofMonoslroma angicava. Bar = 100 I'm. (Saga unpub!.)

in 500 mL ice-cooled acetone and homogenizing with a blender,
then filtering through filter paper, rinsing with 100 mL ice-cooled
ether. maintaining in a vacuum desiccator for several days, andstoring in a freezer (-20°C).

of some marine epiphytic bacteria which can digest the thalli of
red or brown algae. Following is the protocol for the protoplast
isolation from red or brown algae (Saga et al. 1986):
I) Thalli are cut into small pieces and I g is inoculated in lO-mL
solution of an abalone enzyme mixture;2) Incubations are carried out at room temperature for 10 hours
with rotary shaker (25 rpm), and subsequent procedures are similar
to those described above for the green algae;
3) Prescription of the abalone enzyme mixture is a) 20 g abalone
acetone powder in 100 mL 0.1 M Tris buffer at pH 7.0, homogenized for 10 min, stirred with magnetic stirrer overnight, andcentrifuged at 12,000 xg for 20 min; b) 2 % Cellulase Onozuka R-lO,
1 % Macerozyme R-lO, and 1 % pectinase (Sigma Chern. Co.)
dissolved in the supernatant, and this enzyme mixture dialyzed witha cellulose visking tube against deionized water for 12 hours, withdialysis tubes changed every 3 hours; c) after dialysis, the solution
is made to contain 1.0 M sorbitoL 50 mM MgCl z ' 6H zO, 50 mM
KCI and 10 mM CaCl z, and finally adjusted to pH 6.0;
4) Abalone acetone powder is prepared by collecting abalone
locally, immersing 100 g of the digestive tract and hepatopancreas

Protoplast regeneration
There have been several reports on successful regeneration of protoplasts into complete thalli in green algae (Zhang 1983, Saga 1984,
Fujita and M;gita 1985, Saga in prep. b). There have been no reports
on ~uccessflll regeneration of pratoplasts into complete thalli in redor brown algae except Porphyra (Tang 1982, Paine-Fuller and Gibor
1984, Fujita and Migita 1985, Saga et al. 1986). In comparison
with Porphyra, it is more difficult to culture protoplasts ofanatomically complex red or brown algae. Cheney et al. (1986)
have recently succeeded in cell division (16-32 stage) in Graci/aria
prutoplasts, and Saga (unpubl.) also has observed cell division in
Laminaria protoplasts. However, Cheney et al. (1986) and Saga
(unpubl.) have not attained protoplast regeneration into whole plants.

52

Fang, T., Z, Yan. and Z. Wang.
1983. Some preliminary observation on tissue culture in Laminaria japonica
and Undaria pinnatifida. Kexue Tongbao 28:247-249.
Fllyn, B.
1934. Lebenszyklus, Cytolgy und Sexualitiit der Chlorophycee Cladophorasuhriana Kiitzing. Arch. Protistenkd. 83: I-56.
Fries, L.
1963. On the cultivation of axenic red algae. Physiol. Plant. 16:695-708.
1980. Axenic tissue cultures from the sporophyte of Laminaria digitata andLaminaria hyperborea (Phaeophyta). J. Phycol. 16:475-477.
1984. Induction of plantlets in axenically cultivated rhizoids of Fucus spiralis.
Can. J. Bot. 62: 1616-1620.
Fujita, y" and S. Migita.
1985. Isolation and culture of protoplasts from some seaweeds. Bull. Fac. Fish.
Nagasaki Univ. 57:39-45 (in Jpn.).
Haines, K.C., K.D. Hoagland, and G.A. Fryxell.
1982. A preliminary list of algal culture collections of the world. In Rosowski,
J.R., and B.C. Parker (eds.), Selected papers in phycology II, p. 820-826.
Phycol. Soc. Am., Inc., Lawrence, KS.
Hatate, H., T. Aoki, T. Araki, and M. Kitamikado.
1986. Preparation of bacterial enzymes capable of degrading the cell wall of
red alga Porphyra yezoensis. Bull. Jpn. Soc. Sci. Fish. 52:545-548 (in Jpn.).
Iwasaki, H.
1961. The life-cycle of Porphyra tenera in vitro. BioI. Bull. (Woods Hole)
121:173-187.
Kanazawa, A.
1968. Axenic culture of multicellular algae-I. Treatment of algae with sulfa
drugs. Bull. Jpn. Soc. Sci. Fish. 34:570-575.
Kito, H.
1985. Isolation and development of protoplast in Porphyra. Kenkyu J. 8(9)20-24(in Jpn.).
Lee, T.
1985. Aposporous gametophyte formation in stipe explant from Laminaria saccharina (Phaeophyta). Bot. Mar. 28: 179-185.
Millner, P., M. Callow, and L. Evans.
1979. Prepararion of protoplasts from the green alga Enreromorpha intestinalis.
Planta 14:174-177.
Mooney, P.A. , and j. Van Staden.
1985. In vitro plantlet formation and multiple shoot induction in Sargassumheterophyllum. S. Afr. J. Bot. 51:41-44.
Polne, M., M. Biniaminov, and A. Gibor.
1984. Vegetative propagation of Porphyra perforata. Proc. Int. Seaweed Symp.
II :308-313.
Polne-Fuller, M., and A. Gibor.
1984. Developmental studies in Porphyra. I. Differentiation and protoplast
regeneration in Porphyra perforata (Rhodophyta). J. Phycol. 20:609-616.
Polne-Fuller, M., N. Saga, and A. Gibor.
1986. Algal cell, callus, and lissue cultures and selection of algal strains. Beih.
Nova Hedwigia 83:30-36.
Provasoli, L.
1958. Effect of plant hormones on Ulva. BioI. Bull. (Woods Hole) 114:375-384.
1968. Media and prospects for the cultivation of marine algae. In Hattori, A.
(ed.), Proc. U.S.-Jpn. Conf., Cultures and Collection of Algae, p. 63-75. Jpn.
Soc. Plant Physiol., Kyoto.
Provasoli, L., j.j.A. McLaughlin, and M.R. Droop.
1957. The development of artificial media for marine algae. Arch. Mikrobioi.
25:392-428.
Saga, N.
1984. Isolation of I'rotoplasts from edible seaweeds. Bot. Mag. Tokyo
97:423-427.
(In prep. a). Tissue culture in marine algae.
(In prep. b). Isolation and culture of protoplasts from the edible marine alga

Cell fusion and transformation
There are very few reports of cell fusion in seaweeds. Reports concerning cell fusion utilizing protoplasts in seaweed include thoseof Saga et al. (1986) for Enteromorpha intestinalis using an electrofusion method, and Zhang (1983) for intergeneric fusion of Viva
and Monostroma. Spontaneous protoplast fusion also has been
observed in Gracilaria (Cheney et al. 1986). Transformation studies
using protoplasts and recombinant DNA techniques have just begun
in several microalgae, and there is no report on the transformation
study in seaweeds (Saga and Gibor 1986). We are now searchingfor a suitable vector for algae and are attempting to isolate anutritionally-deficient and drug-resistant mutant which may be essential to the establishment of a host-vector system in algae.

Conclusion_

Cheney (1984) listed the following four future research needs fordevelopment of algal biotechnology: 1) Improvement in the basic
methodology of seaweed cell and tissue culture; 2) Improvement
in methods of seaweed protoplast isolation and regeneration; 3)
Development of discriminating selection system for the recovery
of hybrid cells; 4) Development of basic molecular biology, andrecombinant DNA techniques for seaweed.
As a developing marine biotechnology, we can expect the following benefits: Effective mass-production of seaweed seedlings using
a micropropagation system and the effective breeding of seaweedstrains using somatic hybridization and recombinant DNA techniques, e.g., disease-resistant, stress-tolerant, high-growth, andspecialty chemical-rich strains. Marine biotechnology will contributenew and exciting advances to the applied marine sciences.

Citations
Boalch, G.T.
1961. Studies on Ectocarpus in culture. I. Introduction and methods of obtaining uni-algal and bacteria-free cultures. J. Mar. BioI. Assoc. U.K. 41:279-286.
Bonneau, E.R.
1977. Polymorphic behavior of Ulva lactuca (Chlorophyta) in axenic culture.
I. Occurrence of Enteromorpha-Iike plants in haploid clones. J. Phycol.
13:133-140.
Bradley, P.M., D.P. Cheney, and N. Saga.
1986. A new method for obtaining axenic cultures of marine algae. Appl. Phycol.
Forum 3(2):4-5.
Chapman, A.R.O.
1973. Methods for macroscopic algae. In Stein, J .R. (ed.), Handbook ofphycological methods, p. 87-104. Cambridge Univ. Press, London.
Chen, j.
1984. Tissue culture from the sporophyte of Laminariajaponica, U"dariu pinnatifida and Macrocystis pyrifera. Mar. Fish. Res. 6:27-33.
Chen, L.C.M., and A, Taylor.
1978. Medullary tissue culture of the red alga Chondrus crispus. Can. J. Bot.
56:883-886.
Cheney, D.
1984, Genetic modification in seaweeds: applications to commercial utilizationand cultivation. In Colwell, R., E. Pariser, and A. Sinskey (eds.), Biotechnology
in the marine sciences. p. 161-175. Wiley-Interscience, NY.
Cheney, D.P., E, Mar, N. Saga, and j, Van Der Meer.
1986. Protoplast isolation and cell division in the agar-producing seaweeds
Graci/aria (Rhodophyta). J. Phycol. 22:238-243.
Druehl, L.D., and S.LC. Hsiao.
1969. Axenic culture of Laminariales in delined media. Phycologia 8:47-49.
Ducker, S.C., and L.G. Willoughby.
1964. Potassium tellurite as a bacteriostatic agent in isolating algae. Nature
202:210.

Monoslroma lJtJgicava.

Saga, N., and A. Gibor.
1986. Pure culture of algae. In Yamada, Y., and Y. Okada (eds.), Plan'biotechnology, p. 55-71. Tokyo Kagaku DOJin (in Jpn.).
Saga, N., T. Motomura, and Y. Sakai.
1982. Induction of callus from the mari"e brown alga Dietyosi/JllOn
foeniculaceus. PI"nt Celi Physiol. 23:727-730.
Saga. N., M. !'olne-Fuller, and A. Gibor.
1986. Protoplasts from seaweeds: Production and fusion. Beih. Nova Hedwigia
83:37-43.
Saga, N., and Y. Sakai.
1982a. A new method for pure culture of macroscopic algae, the one step selection method. Jpn. J. Phycol. 30:40-43.

53

1982b. Tissue culture study in seaweeds. Proc. 31st Annual Meet., Bot. Soc.
lpn., Hokkaido Branch: 4-5 (in lpn.).
1983. Axenic tissue culture and callus formation of the marine brown alga
Laminaria angustata. Bull. lpn. Soc. Sci. Fish. 49: 1561-1563.
1984. Isolation of protoplasts from Laminaria and Porphyra. Bull. lpn. Soc.
Sci. Fish. 50: 1085.
Saga, N., T. Uchida, and Y. Sakai.
1978. Clone Laminaria from single isolated cell. Bull. lpn. Soc. Sci. Fish. 44:87.
Tang, Y.
1982. Isolation and cultivation of the vegetative cells and protoplasts of Porphyra suborbiculara Kjellm. 1. Shandong Coli. Oceanol. 12:37-50.
Tatewaki, M.
1966. Formation of crustaceous sporophyte with unilocular sporangia in
Scytosiphon lomenraria. Phycologia 6:62-66.
1971. Cultures of seaweeds. Kaiyo Kagaku 3(11):40-45 (in lpn.).
Tatewaki, M., and L. Provasoli.
1964. Vitamin requirements of three species of Antithamnion. Bot. Mar.
6: 193-203.
Zhang, D.
1983. Study on the protoplast preparation, culture and fusion of somatic cells
from two species of green algae - Ulva linza and Monos/ramo angicava Kjellm.
l. Shandong Coil. Oceanol. 13(1):57-65
Zhang, J.
1982. Some experiments and observations on the tissue and cell culture of UnOOria pinnatifida. l. Shangdong Coil. Oceanol. 12(3):29-38.
Zhao, H., and X. Zhang.
1981. Isolation and cultivation of the vegetative cells of Porphyra yezoensis.
l. Shandong ColI. Oceanol. 11(1):61-66.
1984. On the cultivation of the isolated vegetative cells of Porphyra yezoensis
Veda. l. Shangdong Coli. Oceanol. 8(3): 197-202.

54

Preliminary investigations on
cryopreservation of marine
bivalve gametes and larvae
LEHMAN L. ELLISCollege of William and Mary
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062

ABSTRACTTechniques for the cryopreservation of gametes and/or embryosof several vertebrate species have been developed, but similar
work on invertebrates has been attempted for only a few species
and reproducible techniques for long-term storage are still being
developed. In this study, embryos and early larvae of oysters,
Crassostrea virginica, and clams, Mercenaria mercenaria, werecooled in several cryoprotectant solutions at rates between -0.2
and -SOC/min to temperatures between 0 and -100°C/min and
immediately thawed or immersed in liquid nitrogen. No survival occurred in embryos or trochophore larvae after immersion in liquid nitrogen. Embryos or trochophore larvae that wereimmediately thawed showed survival after cooling to temperatures down to -60°C, and reduced or no survival after coolingto temperatures below this level. The highest survivorship wasseen in cryoprotectant solutions with Me2SO or ethylene glycol,
and no survivorship was seen in cryoprotectant solutions containing glycerol. Survival was also affected by the freezing ratewith rates between -2 and -SoC/min yielding the most survivors in all cryoprotectant solutions. Vitrification has also been
evaluated as a means of cryopreserving embryos and larvae.
Survival in these experiments has only been about 1% regardlessof whether embryos or larvae were immersed in liquid nitrogen.
The principal factor in low survivorship in these experiments
appears to be damage from osmotic changes, since the embryos
and larvae appear unchanged until the vitrification solution isremoved. In similar work on gametes, sperm from oysters or
clams are motile after thawing in several cryoprotectant solutions; however, the level of fertilization of fresh ova by thawed
sperm is very poor. Ova frozen at controlled rates in cryoprotectant solutions are shrunken and brownish after thawing and do
not bind fresh sperm. Vitrified ova after recovery from liquid
nitrogen retain their original color and size and bind sperm,
but pronuclear fusion does not occur. (Supported in part by
Sea Grant Project R/A-16.)55

Microparticulate diets for larval fish require the following:(I) Small particles of 5-300 lim diameter that are stable in water
until consumed by the larvae.
(2) Nutritionally well-balanced ingredients that are digestible in
the alimentary canal, since digestive systems of some fish are notfully developed in the prelarval stage.
(3) Particle size and specific gravity of diets should be adjusted
to the developmental stages of larvae.
Various types of microparticulate diets have been prepared byseveral workers (Adron et al. 1974, Gatesoupe and Luquet 1977,
Gatesoupe et al. 1977, Metailler et al. 1979, Teshima et al. 1982,
Kanazawa et al. 1982, Kanazawa and Teshima 1983, Dabrowski
et al. 1983, Bromley and Howell 1983, Kanazawa 1985a,b,
Kanazawa et al. 1985). The microparticulate diets reported areroughly categorized into three groups:

Microparticulate dietsfor fish larvae
AKIO KANAZAWA
SHIN-ICHI TESHIMA
Faculty of Fisheries
Kagoshima University
4-50-20 Shimoarata
Kagoshima, 890 Japan

ABSTRACTMicro-encapsulated Micro-bound diet
(MBD)
diets (MED)
Nylon-protein
Carrageenan
Gelatin-gum acacia Agar
Egg albumin
Zein
Glyco peptide
Alginic acid
ChitosanGelatinIn the seed production of aquatic animals for aquaculture, live
foods such as the rotifer, Brachionus plicatilis, and Artemio salina
have been widely used throughout the world. However, it requires much in terms of facilities, maintenance expense, andlabor to safely produce a constant amount of live foods.
Therefore, it is necessary to develop microparticulate diets asa substitute for live foods to further increase the productivityof seed for fish culture. We have attempted to rear larval fishwith microparticulate diets for several years. As a result, seedproduction of fish such as the Ayu, Plecoglossus a/tivelis, starry
flounder, Paralichthys olivaceus, and red sea bream, Chrysophrys
major, was successfully achieved by partial or even complete
substitution of microparticulate diets for live food.

Micro-coated diet
(MCD)
Zein
Cholesterollecithin
Nylon-protein

Materials and preparation of
microparticulate diets_

The nutritional components of microparticulate diets for fish larvae should be determined on the basis of the requirements of thelarval fish for proteins, amino acids, lipids, carbohydrates, vitamins,
and minerals. However, because the requirements oflarval fish arestill undefined, protein resources having a high nutritional value
are used, such as krill meal, squid meal, scallop meal, short-neckedclam extract, chicken egg, skim milk, casein, gelatin, egg albumin,
yeast, and fish meal (Teshima et al. 1982).
Details of the procedures for preparation of microparticulate diets
are described elsewhere (Teshima et al. 1982, Kanazawa andTeshima 1983, Kanazawa 1985c).

Effects of microparticulate dietson growth and survival oflarval starry flounder_Attempts to rear larvae of the starry flounder, Paralichthysoiivaceus, with several microparticulate diets have been carried out.
Three types of microparticulate diets were prepared: the cholesterollecithin microcoated diet (MCD), zein microbound diet (MBD),
and x-carrageenan microbound diet (MBD). The composition of
the three diets were as shown in Tables I and 2. The starry flounderlarvae were grouped into 1000 fish/loo-L tank for feeding trials.
The larvae were reared with microparticulate diets only for 40 days
after hatching at 18-22°C. The feeding amount and size of microparticulate diets were as shown in Table 3. The rotifers, Brachionus
piicatilis, used as a live food were cultured with a marine type ofChiorella. Dietary value was evaluated in terms of growth and survival rate.

57

Table 1
Composition of microparticulate diet for starry flounder.
g/lOO g dry diet

Ingredient

Mineral mixture3
Bonito egg lecithin
Squid liver oil
Soybean oil

24.0
8.0
5.0
10.0
15.0
10.0
5.0
8.0
6.0
3.0
4.0
2.0

Total

100.0

Chicken egg yolk
Short-necked clam extract powder
Bonito extract powder
Milk casein
Egg albumin
Soluble yeast powder
Amino acid mixture I

Vitamin mixture2

o

10

20

30

o

40

Feeding period (days)'Amino acid mixture; see Table 2.
2Halver.
3USP XlI ·and trace elements.

10

20

30

40

Feeding period (days)

Figure 1
Growth of larval starry flounder on microbound (MHO) and microcoated (MCD) microparticulate
diets. 0 = control (live food), 0 = cholesterol-lecithin MCD, C. = Zein MBD, V = x-Carrageenan
MBD, • = no food.

Table 2Composition of amino acid mixture.
Amino acid lgAmino acidg

Arginine
Histidine
Isoleucine
LeucineLysineMethionine
Phenylalanine

0.8
0.2
0.3
0.5

Threonine
Tryptophan
ValineCystine
AlanineNa aspartate
Glycine

0.3
0.1
0.5
0.2
0.3
0.2
0.1

0.7

0.2
0.6

20
0>

E

+-'

£

0>
Q)

~10
>,

'Total amount 5.0 g.

-0

o

ro

Table 3
Diets and feeding amount.
Size of fish(mm)

Size of diet
Feeding amount

t!'m)

10
Microparticulate diet
3.0 - 6.0
0.2 g x 10 limes/day
6.0 - 8.0
0.3
8.0 - 10.0
0.5
1.0
10.0 - 12.0
1.5
12.0 - 14.0
2.0
14.0 - 16.0
2.5
16.0 - 18.0

-125
125-250250-350250-350250-350250-350250-350

30

Figure 2
Effect of soybean lecithin on growth of red
sea bream larvae. 0 = control (rotifer), C.
= microparticulate diet containing 3%
pollock liver oil + 5% soybean lecithin, 0 =microparticulate diet containing 3% or 6%
pollock liver oil.

Control (live food)
Rotifer
2 individuals x mLiday
3.0 - 4.03
4.0 - 5.0
5.0 - 18.05Artemia
5.0 - 8.0
8.0 - 15.0
15.0 - 18.0

20

Days after hatching

Figure I shows the results of the feeding trials. The cholesterollecithin MCD and zein MBD gave good growth and high survivalof the starry flounder larvae. However, growth and survival on thex:-carrageenan MBD were inferior to that of the groups receivingcholesterol-lecithin MCD and zein MBD. These results show thatthe microparticulate diets alone were sufficient to sustain growthand survival of the newly hatched larvae of the starry flounder.

30 individuals x fish/day
60
90

58

Table 4

Table 5

Effect of lecithin on the incidence of malformation of Ayu
larvae.

Digestibility (%) of microencapsulated [14C] ovalbumin in the Ayu
larvae.

Incidence ofmalformation
PugheadProtrusion ofthrax
Twist of jaw
Scoliosis
Twist of caudal
peduncle
Incidence (%)

Rotifer +commercial
diet

Rotifer +microparticulate
diet including
3% lecithinGroup(Days after
hatching)

o
o

3
10

8
10
35

o
o

63

5

3

Necessity of dietary phospholipids
for growth of fish larvae

20

II
32

70

65

'Mean values from three experiments using the Ayu larvae.

After hatching, the Ayu larvae were fed on rotifers until use.
Four stages of postlarvae were used in the feeding experiments(Table 5) . At each larval stage, 100-150 larvae were put in two
beakers, each containing 300 mL of seawater and 3000 microcapsules with [14C] ovalbumin. About I hr after administration of the
MED, the larvae were taken out from one of the beakers, washedwith seawater, and subjected to radioactive measurements to determine the quantity of ovalbumin intake by the larvae (A: expressed
as cpm). Another beaker was covered with a light-intercepting coverabout I hr after administration of the MED and maintained for subsequent 12 hrs. Then, the larvae were removed and the radioactivityof the whole body (B: expressed as cpm) measured. Sampling timesof the larvae were estimated on the basis of microscopic observation, which showed the presence or absence of microcapsules inthe digestive tracts. The apparent digestibility (%) of [14C]
methylated ovalbumin was calculated as follows: Digestibility (%)= B/A x 100.
As shown in Table 5, the digestibility of [14C] ovalbumin determined on the lO-day larvae of the Ayu was only II % of the [14C]
ovalbumin intake. Also, the attempt to determine digestibility on
the 3-day larvae was unsuccessful because more than half of thelarvae died during the treatments, probably due to a physiologically weak vitality at this stage. the so-called "critical period" (Tanaka
1975). However, the digestibilities of [14C] ovalbumin on the
20-day and 70-day larvae were 32 % and 65 %, respectively. These
results suggest that with maturation the postlarvae of the Ayu islikely to break down the membrane of MED, probably by intestinal
peristalsis, and assimilate dietary proteins such as ovalbumin to someextent.

_Kanazawa et al. (1983a) examined the effects of supplemental
phospholipids on the growth of larval red sea bream, Chrysophrys
major, using purified microparticulate diets. Ten-day-old larvae
reared for 20 days showed better growth and survival on a diet containing 3 % pollock liver oil (PLO) plus 5 % soybean lecithin (SBL)
than when given diets containing 3% or 6% PLO alone (Fig. 2).
The SBL-supplemented diet had a higher nutritive value, corresponding to the live food (rotifers) in terms of growth and survival
rate. These results indicate that the attempt to substitute the beneficial
effects of SBL with w3-highly unsaturated fatty acid was unsuccessful, and that lecithin is an essential dietary component for larval fish.
The necessity of dietary phospholipids for growth of larval fish
was investigated on the knife jaw, Oplegnathus fasciatus (Kanazawa
et aI. 1983a), and Ayu, Plecoglossus altivelis (Kanazawa et aI. 1981,
1983b, 1985).
Also, the effects of dietary phospholipids on the incidence ofmalformation in the larval Ayu were investigated (Kanazawa et al.
1981). As shown in Table 4, it was suggested that the incidenceof malformation in larval Ayu, especially scoliosis and twist of jaw,
was reduced by the supplement of lecithin.

Digestibility of protein in ayu larvae
fed a microparticulate diet

Digestibility (%)'_
Microparticulate diets adjusted
to amino acid patterns ofstarry flounder

We have succeeded in rearing the larvae of a prawn, Penaeus
japonicus, to postlarvae on the microencapsulated diet (MED) witha nylon-cross linked protein membrane (Jones et al. 1979). Accordingly, the MED may be expected to be a promising microparticulate
diet for the larvae and juveniles of some fish, although it is questionable whether larval fish assimilate the MED as effectively as
observed in crustacean larvae. As a part of resolving the above question, we planned to clarify the digestibility of protein in the larvaeof Ayu fed the MED containing radioactive protein.
To the homogenate (2.5 g) of whole chicken egg, [14C]
methylated ovalbumin (1.0 /lCi) dissolved in 0.01 M sodium
phosphate buffer (pH 7.2) was added, and the mixture was microencapsulated by the interfacial polymerization procedure of Changet at. (1966). The MED so prepared had a radioactivity of 1.30
dpm/capsule.

_

Generally, proteins having an essential amino acid (EAA) patternsimilar to that of whole body or egg proteins are likely to have ahigh nutritive value for fish. Therefore, we analyzed the amino acidcomposition of whole body proteins of the starry flounder, and thenmade four test diets using various protein sources to simulate theamino acid patterns of the body protein of larval starry flounder.
Four microparticulate diets were formulated with a white fish meal,
brown fish meal, bonito powder, yeast powder, crab meal, gluten
meal, and/or krill meal (Tables 6 and 7). Their nutritive values werecompared with a live food control by a feeding trial during somelarval stages until the bottom-dwelling period.
59Seedling mass production withmicroparticulate diets

Table 8 shows the results of a 38-day feeding trial. The test dietgroups had 36.1-44.8% survival and 19.5-19.7 mm total length,
whereas the live food control receiving the rotifer and Artemia had
51.7% survival and 21.4 mm total length. The incidence (%) ofabnormal pigmentation (whitening of the skin) was lower on thetest diet groups (8.4-14.2 %) than the control group (88.4%). Thus,
the microparticulate diets were found to reduce the incidence ofabnormal pigmentation.

Ayu
Ayu larvae, numbering 100,000 and 400,00, were placed in 50and lOO-ton tanks, respectively. Feeding trials were carried out for
83 days at 11.1-27.0°C. Larvae received the MBD 10 times daily.
Dietary values of diets were evaluated in terms of total length, bodyweight gain, and survival rate. Furthermore, the incidence of malformation was checked 83 days after hatching. Two groups of larval
Ayu were fed the diets as shown in Figures 3 and 4. One group
was fed mostly live food such as rotifer and Artemia, and anothergroup was fed mostly MBD containing one-half rotifer and onefifth Artemia.
The results of growth and survival of larval Ayu reared for 83
days are shown in Table 9 and Figure 5. The survival of the MBD
and live food groups at the end of the feeding trial was 44% and
54 %. respectively. Total lengths of the MBD and live food groups
were 50 and 47 mm, respectively. These results indicate that a
microparticulate diet has a nutritive value corresponding to live foodin terms of growth and survival rates. Also, such abnormalities as
scoliosis were rarely found when the Ayu larvae were reared with
MBD containing lecithin.

Table 6Composition of microparticulate diets for starry flounder.
Diet
4

2
3
(gil 00 g dry diet)

IngredientWhite fish meal
Bonito powder
Squid gristle
Yeast powder
Brown fish meal
Crab meal
Soybean meal
Gluten meal
Krill meal
Egg albumin
Milk casein
Cellulose
Squid liver oil
Soybean lecithin
Mineral mixture
Vitamin mixture

30.0
11.9
9.0
25.0

2.1
8.0
3.0
5.0
6.0

5.2
8.0
3.0
5.0
6.0

5.0
8.0
3.0
5.0
6.0

20.0
10.0
2.5
6.5
8.0
3.0
5.0
6.0

Total

100.0

100.0

100.0

100.0

13.2
8.5

11.9

14.0
45.9
15.0
16.8
22.5
12.0

_

25.0

Red sea bream
We have previously shown the probable utilization of microparticulate diets as a substitute for rotifer and Artemia in the practicalseed production of the Ayu. In the present study, a similar feeding
experiment was conducted on the larval red sea bream with commercial microparticulate diets (Kyowa Hakko Kogyo Co., Ltd.)
using a 50-ton tank. The hatched red sea bream larvae (900,000
fish) were reared on Type A microparticulate diet (particle sizes
250 lim) along with rotifers for 30 days, followed by Type B
microparticulate diet (particle sizes 250-400 lim; a substitute for
Artemia) for 3-6 days, and then by a larger Type C microparticulate
diet (particle sizes 700 lim; a substitute for minced fish flesh) until
56 days after hatching (Figs. 6, 7).
Results of the feeding experiments are shown in Table 10. Whenthe newly hatched red sea bream received a combination of the
microparticulate diet and rotifer (about I: I, ww), 112,000 fish with
10 mm total length were produced with 12.4% survival rates. The
30-day larvae reared successively on the microparticulate diet alone
grew to the 14-mm total length equally as well as the control group
receiving Artemia and krill alone. Larval fish of 14 mm total length
were transferred from the tank on land to a cage in the sea andreared on the microparticulate diet alone until reaching 26.3 mm
total length, the size of shipment. As a result, total replacementof minced fish flesh by the microparticulate diet was found to be
successful in terms of both growth and survival rate.

Table 8
Effect of microparticulate diets on growth and survivalof starry flounder.
--------------~---

Final

InitialTable 9--_._----_.-

Diet no.

Total length

Survival rate

(mrn)

(%)

Growth and survival of larval Ayu fed on microbound diet (MBD).

Total length(mrn)

No. fish--_ _..

1
2
3
4
Control(Live food)

4.9±0.5
4.9±0.5
4.9±0.5
4.9±054.9±0.5

39.2
36.1
44.8
439
51.7

19.6± 1.6
19.5± 1.3
19.6± 1.519.7± 1.5
21.4±2.0

60

Total length
Avg. body weight
(mm)
(mg)
- - - - - - - (H-83) - - - - - - -DietInitialFinal(H-83)

Control

394,000

213,000

54.3

47.1

433

MBD

105,000

46,500

44.4

49.8

410

Survival(%)

ExperimentControl

M8D

Diet

HatFhing

1p, 1010,(H-14)

Rotifer(individual/fish)
Artemia(individual/fish)
Commercial diet(mg/fish)Egg yolk(mg/fish)
Artificial diet(mg/fi sh)
Rotifer(individual/fish)
Artemia(individual/fish)(H-13)

~O

30
,

2488

, 2866, 1124

I

150

,

, 62

, 50

, 117

, 193

,

,

(H-14)
(H-6)
,

!

~O

400

, 565

,563

237

, 145

,142 (H-81)(H-75),

0.75,

1.04 ,

1.61 ,

4.35, 0.05,

,

0.08

,

0.08 ,

0.09 ,

0.42 0.63 (H-83)

2.86

,

5.78

,

6.00 , 7,80

,

63

2.28

,

1520

0.04

,

2.78

,

1581

!

,9.46

I

0.25 , 0.53

0.67,

869

~O(days)

62

52

49

(H-83),

I,I

12,10 (H-81 )I, 1614 (H,-40)(H-40)

,

115

,

53

I

52 (H,-75)

Figure 3
Diets and reeding amount used in each experimental group or larval Ayu.

ExperimentContro1

Diet

Hat,ch i ng

RotiferI

10,(H-1~H-75)30,
240.68 X

I (H-14-H-81)

Artemia--

I

Commerci a1 diet

MBD

20
,

40
,

45350 93860 9Art ifi ci a1 di et

I (H-6-H-8l)

I

Figure 4
Total amount or diets used in each experimental group or larval Ayu.

50.0

500
en

40.0

E

...,
.<::

....,

.<::

0'
C
Q)

en

30.0

~

Q)

~

3=
>,

...,'"

0
I-

-0

20,0

0

co

10.0

400
300
200
100

20

40

60
80
20
Feeding period (days)

40

Figure 5
Growth or larval Ayu red on microparticulate diets.
a = control, b. = microbound diet (MBD).

61

I

I

1(H-4l-H-75) 1.198 X HJlindi.1

Artemi a'

I

I

20640 9

---

E
E

,

I

I(H-l-H-40) 38.8 X lip individual I

Rotifer80(days)

21,274 X 1d3 i ndi vi dua 1(H-14-H-83)

I

70
,

60

I

lip i ndi vi dua1

(H-13-H-83)

Egg yolk

50

60

80

Citations
Experiment

10

H

20

30

40

56(days)50

Adron, J,W., A. Blair, and C.B. Cowley.
1974. Rearing of plaice (Pleuronectes platessa) larvae to metamorphosis using
an anificial diet. Fish. Bull., U.S. 72:352-357.
Bromley, P.J" and B.R. Howell.
1983. Factors influencing the survival and growth of turbot larvae, Scophthalmus
maximus L., during the change from live to compound feeds. Aquaculture
31:31-40.
Chang, T.M.S., F.C. Macintosh, and S.G. Mason.
1966. Semipermeable aqueous microcapsules I. Preparation and propenies. Can.
J. Physiol. Pharmacol. 44: 115- [28.
Dabrowski, K., R. Bardega, and R. Przedwojski.
1983. Dry diet formulation study with common carp (Cyprinus carpio L) larvae.
Z. Tierphysiol. Tierernaehr. FUllermitte[kd. 50:40-52.
Gatesoupe, F.-J., and P. Luquet.
1977. Recherche d'une alimentation anificielle adaptee a I'elevage des stades
larvaires des poissons I. Comparaison de quelques techniques destinees aameliorer la stabilite a I'eau des aliments. 3d Meeting, ICES Working Groupon Mariculture, Brest, France, 4, p. 13-20.
Gatesoupe, F.-J., M. Girin, and P. Luque!.
1977. Recherche d'une alimentation anificielle adaptee a I'elevage des stades
larvaires des poissons II. Application a I 'elevage larvaire du bar et de la sole.
3d Meeting, ICES Working Group on Mariculture, Brest, France, 4, p. 59-66.
Jones, D.A., A. Kanazawa, and S. Abdel-Rabman.
1979, Studies on the presentation of anificial diets for rearing the larvae of
Penaeus japonicus Bate. Aquaculture 17: 33-43.
Kanazawa, A.
1985a. Essential fatty acid and lipid requirement offish. In Cowey, C.B, A.M.
Mackie, and J.G. Bell (eds.), Nutrition and feeding in fish, p. 281-298.
Academic Press, London.
1985b. Nutritional requirements of larval fish. Sea Farming Technol. Res.
14:87-96 (in Jpn.).
1985c. Micropaniculate diets. In Yone, Y. (ed.), Fish nutrition and diets, p.
99-110. Suisangaku ser. 54, Koseisha-Koseikaku, Tokyo.
Kanazawa, A., and S. Teshima.
1983. Development of micropaniculate diets for the larvae of fish, crustaceans,and shellfish. Yoshoku 20:97-[Q[ (in Jpn.).
Kanazawa, A., S. Teshima, S. Inamori, T. Iwashita, and A. Nagao.
1981. Effects of phospholipids on growth, survival rate, and incidence of malformation, in the [arval Ayu. Mem. Fac. Fish. Kagoshima Univ. 30:301-309.
Kanazawa, A., S. Teshima, S. Inamori, S. Sumida, and T. Iwashita.
1982. Rearing of larval red sea bream and Ayu with anificial diets. Mem. Fac.
Fish. Kagoshima Univ. 31:[85-192.
Kanazawa, A., S. Teshima, S. Inamori, and H. Matsubara.
1983a. Effects of dietary phospholipids on growth of the larval red sea bream
and knife jaw. Mem. Fac. Fish. Kagoshima Univ. 32: 109-1 [4.
Kanazawa, A., S. Teshima, I. Kobllyashi, M. Takae, T. Iwashita, and R. Uehara.
1983b, Necessity of dietary phospholipids for growth of the larval Ayu. Mem.
Fac. Fish. Kagoshima Univ. 32: 1[5-120.
Kanazawa, A., S. Teshima, and M. Sakamoto.
1985. Effects of dietary bonito-egg phospholipids and some phospholipids on
growth and survival of the larval Ayu, Plecoglossus altivelis. Z. Angew.
Ichthyol. 4:165-170.
Metailler, R., C. Manant, and C. Depierre.
1979. Micropanicules alimentaires inenes destinees a I'elevage larvaire despoissons marins. Utilization des alginates. In Proc. World Symp. on Finfish
Nutrition and Fishfeed Technology, Hamburg, p.181-190.
Tanaka, M,
1975. Digestive organ of larval fish. In Japan. Soc. Sci. Fish. (ed.), Feeding
and growth of larval fish, p. 7-23. Suisangaku ser. 8, Koseisha-Koseikaku,
Tokyo.
Teshima, S., A. Kanazawa, and M. Sakamoto.
1982. Micropaniculate diets for the larvae of aquatic animals. Min. Rev. Data
File Fish. Res., Kagoshima Uoiv. 2:67-86.

Roti fer
I

I

Artemia

Control+~1-----1

Minced fish meat I
I

Zein mi cro-bounddiet

1/2 Rotifer
Type-AI
I

I
I

I Type.B

Type-CI

I

Figure 6
Series of diets used for mass production of larval red sea bream.

Experiment

Diet

H

10

20

,

,

!

Artemia

7140000 Indi.D

Kri 11

10000 9Mi need fish
meat

.

,

0

I 22000 9 I

I 96.46 108 [ndi. I

RotHerZei n-MBO

56( days)

50

.., ,

I 212.79 X 108 Indi. I

RotHerContro1

30 33 36 40

X

Type-AI 23281 9 I

Type-B

10260 9DI

I 22000 9

Type-C

Figure 7
Total amount of diets used for mass production of larval red sea bI"eam.

Table 10
Survival and growth of larval red sea bream fed on zein microbound diet.
(Survival rates in parentheses.)
No. fishTotal length
[4 mm

10 mm
Days
post-hatch

1030

Control dietZein micro- 900,000 112,000(12.4%)
bound diet3[

36

69,000 54,000*(80%)
43,000 30,000(70%)

26.3 mm37

56

15,000

6,[00
(45%)
9,000

[5,000(60%)*33 days-old.

62

Hokkaido Regional Fisheries Research Laboratory
Fisheries Agency of Japan
116 Katsura-koi, Kushiro City
Hokkaido, 085 Japan

Japan's fishery production surpassed 10 million metric tons (mt)in 1972 and amounted to 12.82 million mt in 1984 (Fig. 1). After
1977, establishment of the 2oo-nautical-mile exclusive zones reduced the production of Japan's distant fisheries. Reduction of thesefisheries was compensated by offshore and inshore fisheries, including mariculture. Recent mariculture and sea farming are supporting the development of Japan's fisheries. Sea farming is a newand profitable technology for utilizing natural productivity. Scallop
culture is large-scale and leads in the development of sea farming
fisheries (Table I). Therefore, this report briefly outlines the scallop
sowing culture in Japan.

ABSTRACT

Scallop productionin JapanSowing culture ofscallop in JapanHIROSHI ITO_

Japanese scallop, Patinopecten yessoensis, production increased
rapidly after the 1970's because of successful development of hanging and sowing culture (Figs. 2, 3). Before then, almost all production was from the natural scallop population. Over a quarterof a century, after World War IT and prior to 1968, the catch declined
to less than 22 thousand mt. The average yearly catch of 106 thousand mt in 1969-84 was nine times that of 12 thousand mt in

Recent information on sowing culture (bottom culture) of theJapanese scallop, Patinopecten yessoensis, is briefly outlined.
Scallop mariculture, sowing culture, and hanging culture,developed rapidly in the mid 1960's due to technical development of methods for mass production of scallop seeds (spatcollection and intermediate culture). Mter the mid 1970's, sowing culture production rapidly increased, mainly on the northeast Hokkaido coasts facing the Okhotsk Sea. In 1984, the production of sowing culture and wild scallops amounted to 135,239
metric tons (mt) and the hanging culture reached 73,981 mt,
for a total production of 209,220 mt. At present, most of the
production of sowing culture and wild populations is attained
around Hokkaido and Aomori Prefecture, and sowing culture
keeps the Japanese scallop industry active.

130

FISHERY AND AQUACULTURE PRODUCTION
120

110

100

.,.,

90

0
60
OTHERS IN fiSHERIES

(j)

Z

70

0

f-

U

60

c:::
f-

UJ

~

z

SHELL FISHERIES
30

OTHERS

f-

U
::J

0

0
c:::

a..

0

OTHUlS

3

SHELL
i
1965

ULTURE

,

i
1970

,

,

Y

I

I

1975

1980

E

A

R

Figure 1
Annual production of fISheries and aquaculture in Japan, 1965-84. (Source: Japan
Ministry of Agriculture, Forestry and Fisheries)

63

Table 1
Seed input (10 3 fish) of main species for sea farming in Japan, 1977-84.
(Source: Japan Fisheries Agency survey)

Year

Scallop

KurumashrimpBlue
crab

Abalone

Sea
bream

1977
1978
1979
1980
1981
1982
1983

2,139,363
1,566,655
1,699,127
1,525,333
2,127,447
1,647,327
1,607,213
1,776,130

255,515
280,075
337,229
297,843
302,138
275,402
300,584
293,620

6,917
7,870
12,171
11,519
11,212
14,997
19,523
19,972

7,015
7,143
8,462
10,56012,074
12,27918,33419,014

4,667
5,109
8,600
10,35812,044
12,866
15,61916,1761984

210000
TOTl\L
I'IOKI~I\I DO

c~

z

a

f-

a

H

0::
f-

W

:!::

~"_

Jl.A

0'-

o - nlTl-'-'""Tn-rrn-TTTT-rrrTTT-'-'lTTT,rrrr TTTTTTTT r n --I--I-TTTf rTT I T 1 TTT,--n-T I"Tr"T r I r-I 1
1910

1984

YEM:

Figure 2
Annual production of the scallop PaJinopecten yessoensis in Japan, 1910-84.

(j)

Z

~
u
iX
f-

UJ

~

o
1955

1960

lQ65

Y

1970

E

A

1975

1980

R

Figure 3
Main regions of scallop sowing culture in Hokkaido Prefecture, northern Japan.

64

~ 145°E
REGIONI~OKHOTSK

SEA

Figure 4
Annual production of the scallop PaJinopeclen yessoensis in Japan, 1955-84. (Source: Japan Ministry of Agriculture,
Forestry and Fisheries)

in Japan, 1977-84. Effects of seed input on scallop harvests havenot been accurately determined, because sowing culture harvests
are included among wild scallop fisheries in the landing statistics.
However, following are some analyses on the sowing culture around
Hokkaido.

1910-68. Historically, the scallop fishery in Japan, 1910-84, consists of six periods (modified from Tanaka 1963, Kanno and Wakui
1978, Tsubata 1982):
1910-1923 Temporary high landings, annual catch fluctuatedwith one sharp peak in 1915;
1924-1932 Stable high landings, annual average catch of 37
thousand mt;
1933-1944 Extremely high landings, average 43 thousand mt;
1945-1968 Stable low landings, catch dropped to an average
12 thousand mt with no peaks;
1970-1976 Hanging culture production rapidly increased,average 57 thousand mt;
1977-1984 Sowing and hanging culture period, with production exceeding 10 thousand mt in 1977 and the scallop industry
developing into a mass production phase, averaging 156 thousand

Seed input around Hokkaido_

A half-century has passed since the first trial in 1936 of scallop
seed sowing in the Abashiri distnct coasts of northeast Hokkaido
facing the Okhotsk Sea (Kinoshita 1949); however, nothing resulted
from the many efforts during the first 32 years. Survival of thesowed scallop population was monitored in both the Okhotsk Sea
coasts of Tokoro, Abashiri ctistrict, and Sarufutsu, Soya district,from the late 1960'~ to the early 1970's. On the Tokora coast, 23
million seed shells with one-year-old seed scallops were sowed on
the culture bottom in 1967. The sowed scallop population at theage of2 years was estimated to have a survival rate of 54.7-98.0%(Okesaku and Tanaka 1969). Additionally, seed input on Sarufutsu coast was 14-60 million shells in the years 1971-73. These threemt.

The major landings have always been around Hokkaido in northern Japan (Fig. 4). Scallops caught in Hokkaido represented 79%
of all Japanese scallop production during the period 1910-84. The
sowing culture and wild production harvested from the sea bottom
around Hokkaido contributed 86 % of the total scallop production65Effects of seed sowing

Relationships between seed input and scallop production (wild andsowing culture) around Hokkaido are shown in Figure 6. Usuallyin Hokkaido, sowed scallops are harvested at four-years-old fromone of four rotational culture grounds. Each point is dotted a coordinate location of the harvest in each year (vertical axis) in pairingwith the corresponding seed input. Dots are plotted on the assumption that every seed scallop generation would be harvested at 4-yearsold. In other words, the year of seed input is based on assumed
spat-collecting time (generating time of the scallop), traced backfour years before harvesting (horizontal axis). Harvests tend to relate
positively with the seed input. So, fitted relationships between seed
input and production are calculated (Fig. 7). A highly significant
effect on the harvest appears to result from large-scale seed input
such as in the Abashiri district; however, no apparent increase occurs
with small-scale seeding such as in the Rumoi and Thuri districts.
A large-scale seed input of more than 100 million shells might besuccessful in sowing culture. In the Soya district, the catch was
remarkably level, though seed input was relatively small. The recent scallop stock for harvesting was kept at a high level with thewild population on the Sarufutsu coast, Soya district (Tomita et al.
1982). The sowing culture contributed no more than 10% to therecent Sarufutsu scallop production. Moreover, seed sowing had
been stopped in 1980-85 because of large populations of wildscallops on this coast. Therefore, detailed results of seed sowing
were different in every locality and case. The statistical scallop
harvest records in Japan unfortunately include production from thewild population. Therefore, accurate analyses are still not possible
because of the lack of separate harvest data on sowing culture.
Statistical analyses are the primary and important step to investigate
scallop culture technology. Further researches and analyses areneeded to answer questions of effect of sowing culture on harvests.

~~oABASHIRI

10

:>

oz

Q)

o

~

x

15

5

a:

w

CO
~
~

Z

10 0

f-

~
Q..

z

TOTAL

0

w
w

(j)

5

2:i

INDEPENDENT

a:

<{
UJ

>0
1965

1975

1970

Y

E A

_

1980

R

FigUl'e 5Yearly seed input number of the scallop Patinopecten yessoensis for sowing infive main culture districts in Hokkaido, Japan.

Citations_

Hayashi, T., K. Tomita, T. Wakui, H. Ito, and M. Matsuya.
1976. Propagation of scallop by transplantation of seed in the Okhotsk Sea coastof northern Hokkaido. J. Hokkaido Fish. Exp. Stn. (Hokusuisi Geppo) 33(9):1-16 (in Jpn.).
Kanno, H., and T. Wakui.
1978. Hotate-gai bo-ga! shudan no zosei (Cultivation of mother shell population of the scallop, Patinopecten yessoensis). Suisan-gaku sel. 23:79-89 (Tokyo)(in Jpn.).
Kinoshita, T.
1949. Hotate-gai no zoshoku ni kansuru kenkyu (Studies on sea farming of thescallop, Patinopecten yessoensis). Suisan kagaku sosho 3 (Sapporo, Hokkaido),
106 p. (in Jpn.).
Okesaku, H., and S. Tanaka.
1969. Hotate-gai chi-gai no gaikai horyu koka ni tsuite (On the effects of seedsowing of the scallop, Patinopecten yessoensis, in the open sea coast). 1. Hokkaido Fish. Exp. Stn. (Hokusuis! Geppo) 26(3):32-49 (in Jpn.).
Tanaka, S.
1963. Hokkaido ni okeru hotate-gai gyogyo to kenkyu-jyo no sho-mondai (Someproblems on fishery and researches of the scallop, Patinopecten yessoensis,
around Hokkaido). Symposium at 1963 meeting, Jpn. Soc. Sci. Fish., p. 142150 (in Jpn.).
Tomita, K., K. Tajima, M. Uchida, M. Mori, and T. Wakui.
1982. On the population of scallop, Patinopecten yessoensis (JAV), in Sarufutsu, Hokkaido. J. Hokkaido Fish. Exp. Stn. (Hokusu!si Geppo) 39(6): 111-125(in Jpn.).
Tsubata, F.
1982. Mutsu-wan hotate-gai gyogyo kenkyu-shi (A history of the fisheries
researches of the scallop, Patinopecten yessoensis, in Mutsu Bay). Aomori
Prefecture, 120 p. (in Jpn.).

populations of three-year-old scallops were estimated to have survival rates of 5.5-28.9% in 1973-75 (Hayashi et al. 1976). Afterthese studies, seed sowing began to evolve on a large scale becauseof the development of mass seed production in the 1970's (Fig.
5). Yearly seed input sowed around Hokkaido amounted to 1,500
million shells in 1982, a threefold increase in the last 10 years. Mostof the seeds were sowed on the coasts of north and east Hokkaido
in the following order: Abashiri (990 million shells, 66%), Nemuro(200 million, 13%), and Soya (180 million, 12%). Of the 1,500
shells planted around Hokkaido, 920 million were produced locally by independent intermediate culture and 590 million by introduction of seed produced in other areas. Seed production in the Abashiri
district is almost self-sufficient because the brackish lakes of Saroma
and Notoro are good for seed production. In the other districts,
almost all seed is dependent on importation from other areas. Seed
supply has been chronically insufficient, because of recent excessive
demand. To eliminate seed deficiency, a new technology of offshore spat collection and intermediate culture on the open sea coastis developing rapidly.

66

TOTAL'll'811'8279..--e'80

SOYA,.

79

'8~21

l'77

'79~'B0'78,?

v>

7 t76'~7~

:z:

.....
0

7:

~

...
...

..... 10

<>=

1

4

~'78

ABASHIRI,.

'8r~'80

/

·'76

76

~

')P
/

<>=

=
=

~

740'

""

'75
'81 ~tt!NEMURL'
•
'82

/~~80
79

!::i

~~
). \'78\ Ii IBURI

:z:

3:
10 3
0

/'75,

v>

"74

'80

Cl

:z:
CI:
Cl

-'

3:

....
0

'79

:z:
0

.....
=
Cl

10 2

'78

'76

~

0

<>=

CC-

o

'74 ..

-'-'RUMOI it

CI:

~

v>

'75

10
106
YEARLY

SEED

INPUT

NUMBERINDIVIDUALSFigure 6
Relationship between seed input and production (wild and sowing culture) of the scallop Patinopecten yessoensis in five main culture districts around Hokkaido, Japan, 1974-82. Seed input numbers represent corresponding
values in assumed previous spat-<:ollecting time (generating time of the scallop), traced back 4 years before
harvesting.

67

P:HI14M2/F:ABASHIRI/ABASHIRI REGION/1974-19821111
P:HI14M21F:TOTALITOTAL OF HOKKAID0/1976-19821111
Y= SCALLOP PRODUCTION (*10A3 METRIC T.) I X = SEED INPUT
100.000A
ABASHIRI•.

TA 0.923 TB 2.102~OF

5 REGIONS•
I. 000 '--------~-----._--.---.--~__,_____._--"T----,

100.000

1000.000
SEED 1t-f'UT (*10A6 IND.)
Y=B*XAA A: 2.686061346423545
B: 1.74874455324604D-06 R:0.964 N: 9 T 9.602
Y=B*XAA A: 2.098467043293528
B: 4.8773916823231520-05 R:0.848 N: 7 T 3.581
P:HI14M2/F:SOYA/SOYA REGION/1974-19821111
P:HII4M21F:TOTALITOTAL OF HOKKAID0/1976-19821111
Y= SCALLOP PRODUCTION (*10A3 METRIC T.) I X= SEED INPUT
100.000

B
SOYA•

TA I. 000 TB 4.432

/

o

TOTAL

I. 000 l...---~-~""-""-""-"--'-'..-r-----.,.--~~~--.,.-~--.-,

10.0001000.000
SEED INPUT (*10A6 IND.)
Y=B*XAA A: 1.417646609723416
B: 2.0205238415659520-02 R:0.950 N: 9 T 8.016
Y=B*XAA A: 2.098467043293528
B: 4.8773916823231520-05 R:0.848 N: 7 T 3.581
P:HII4M21F:NEMUROINEMURO REGION/1975-19821111
P:HII4M21F:TOTALITOTAL OF HOKKA:DO/1976-19821111
Y = SCALLOP PRODUCTION (*10A3 METRIC T.) I X = SEED INPUT
100.000

C

TA 2.264 TB I. 624/
NEMUROTOTAL

O. 100 '----~-~~~--ro~o~o ~oTI---"---"--~~~"""""-""
1O. 000
I00'0: 000
SEED INPUT (*10A6 IND.)
Y=B*XAA A: .7959288467692902
B: 7. 31 639591 87501 99D-02 R:0.884 N: 8 T 4.629
Y=B*XAA A: 2.09846704$293528
B: 4.8773916823231520-05 R:0.848 N: 7 T 3.581
Figure 7
Fitted relationship between seed input and production (wild and sowing culture) of the scaUop Palinopecten yessoensis
in five main culture districts (A-E) in Hokkaido, Japan. Seed input numbers represent corresponding values in assumed
previous spat-coUecting time (generatm.: time of tbe scaUop), traced back 4 years before barvesting.

68

P:HII4M2/F:IBL~I/IBURI REGION/1976-19821111
P:HII4M2/F:TOTAL/TOTAL OF HOKKAID0/1976-19821111
Y = SCALLOP PRODUCTION <*IOA3 METRIC T.) I X= SEED INPUT
100.000oTA 0.550 TB 0.129

/
o

IBURI

TOTAL

. .

~/

O. 100 '------~~-~~~~-.-----.--~--.---.-~..---,-_r_,
10.0001000.000
SEED INPUT <*lQA6 IND.)
Y=B*XAA A: 1.167935070085008
B: 9. 944002521 736505D-03 R:0.320 N: 7 T 0.756
Y=B*XAA A: 2.098467043293528
B: 4.8773916823231520-05 R:0.848 N: 7 T 3.581
P:HI14M2/F:RUMOIIRUMOI REGION/1974-19821111
P:HI14M2/F:TOTALITOTAL OF Ha<KAIDO/1976-19821111
Y = SCALLOP PRODUCTION <*1 QA3 METRIC T. ) I X= SEED Ir\PUT
100.000E
RUMOI

TA 0.627 TB 2.045

~
TOTAL

0.01 0 '-----~~~~...,...,..,-~~~~~,.....,------.-~,......,..~~
1. 000I000. 000
SEED INPUT <*1 QA6 IND.)
Y=B*XAA A: .6904272872067725
B: 2.3386492646986750-02 R:0.489 N: 9 T 1.484
Y=B*XAA A: 2.098467043293528
B: 4.8773916823231520-05 R:0.848 N: 7 T 3.581

69