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�#�%i###�#� �#�#b###�#� I#�Achieving Natural Coloration in Fish Under
Culture

Harry Ako,\* University of Hawai�i, Molecular Biosciences and Biosystems

Engineering College of Tropical Agriculture and Human Resources 1955
East-West

Road, Room 511, Honolulu, HI 96822 USA \*Tel: (808) 956-2012; Fax: (808)
956-3542

e-mail: hako@hawaii.edu Clyde S. Tamaru, University of Hawai�i, Sea
Grant

Extension Service School of Ocean and Earth Science and Technology 2525
Correa

Road, HIG 205, Honolulu, HI 96822 USA Lena Asano, University of Hawai�i,

Molecular Biosciences and Biosystems Engineering College of Tropical
Agriculture

and Human Resources 1955 East-West Road, Room 511, Honolulu, HI 96822
USA Becky

Yuen Palaau Prawn and Fish Company P.O. Box 978, Kaunakakai, HI 96748
USA and

Michael Yamamoto 3461-A Akaka Place, Honolulu, HI 96822 USA Abstract
Fish that

are colored in nature often acquire faded coloration under intensive
culture

conditions. Experiments adding top-coated algae to the diets of
ornamental fish

have resulted in color enhancement. Freshwater red velvet swordtails
Xiphophorus

helleri, rainbowfish Pseudomugil furcatus, and topaz cichlids Cichlasoma
myrnae

became significantly more intensely colored when fed a diet containing
1.5-2.0%

of a carotenoid-rich strain of Spirulina platensis and 1.0% of a
specially grown

Haematococcus pluvialis for 3 wk. Though color enhancement was apparent
after

only a wk, when the fish consumed these doses of algae, lower doses
(0.5% and

0.4%, respectively) were not significantly different for kissing
gouramis

Helostoma temmincki, 24 K mollies Pachouli latipinna, and rosy barbs
Barbus

chunkiness, were examined after the 3-wk feeding period. Both treatments
were

significantly more effective than control treatments with no added
carotenoid,

and better than treatments with traditional carotenoid sources. Color

enhancement appeared to occur via natural carotenoid receptors. Thus,
color

intensity diminished when fish were stressed, coloration appeared only
in males

in species where only the males are normally colored, and between rosy
barbs and

topaz cichlids color enhancement was environment-sensitive. Topaz
cichlid color

developed only after the aquaria were divided into territories and rosy
barb

color intensified when floating substrate was present. It is concluded
that

ornamental fishes are good models for color enhancement through diet and
that

this enhancement may be achieved using products made by marine
biotechnology

companies. Introduction The fledgling freshwater ornamental fish
industry in

Hawai�i has experienced the problem of faded coloration in fish,
especially

those grown in clear water. These fish are traditionally rejected by
ornamental

fish wholesalers, leaving the growers with fish that can�t be sold or
fish sold

at a very reduced price. For this reason it was decided to investigate
fish

coloration. The current work was based on prior studies using cultured
clownfish

Amphiprion ocellaris and Premnas biaculeatus which only achieved color
patterns

and intensities seen in fish in the wild when treated for 2 wk by
incorporating

astaxanthin into the diet (Ako and Tamaru 1999). Several species of fish
at the

Waikiki Aquarium and Sea Life Park on O�ahu, Hawai�i, USA, responded
similarly,

and maintenance of the bright coloration appears achievable with
incorporation

of 25 mg/kg astaxanthin into the various diets on a continual basis.
However,

chemically synthesized astaxanthin is a different stereoisomer from
natural

astaxanthin (Ako and Tamaru 1999) and is difficult for the small grower
to

incorporate into fish diets. Fortunately, carotenoid sources were
available from

a marine biotechnology company in Hawai�i in the form of carotenoid-rich
strains

of Spirulina platensis and specially grown Haematococcus pluvialis.

Incorporation of these algae into the diets of ornamental fish is the
subject of

this report. Materials and Methods All fish were treated (fed enhanced
diets) in

clear water aquaria equipped with biofilters. They were fed three
times/d with

meal sizes adjusted so that very few feed particles (\<5% of the applied
dose)

remained in the aquarium after 10 min. All treatment periods were a
duration of

3 wk. Treatment algae containing carotenoids were obtained from
Cyanotech

Corporation (Keahole, Hawai�i, USA) and were added to the high
palatability

salmon fry feeds we are recommending to ornamental fish farmers (Tamaru
and Ako

1997; Ako et al. 1997; Tamaru et al. 1998; Ako et al. 1999). The algae
easily

adhered to the 14% lipid feed particles. Swordtail fish of the red
velvet

variety were obtained from grazed down (clear water) ponds. They were
treated

with the feed described above � 1.5% Spirulina and 1% Haematococcus.
Rainbowfish

were obtained from clear water, indoor aquaria and treated with the feed

containing 2% Spirulina and 1% Haematococcus. Control fish of each
species were

fed a flake feed which reported no source of carotenoids in the listed

ingredients. Topaz cichlids were obtained as juveniles and were reared
in the

same way as the rainbowfish. Treated cichlids were fed as described
above but

control fish animals were fed a commercial cichlid feed containing
alfalfa meal

as the carotenoid source. Among the cichlids, no color changes were
observed

after 3 wk and the experiment was continued for an additional 3 wk after
bottles

were put into the tanks to provide territories. The rosy barbs used in
the

experiment were obtained from a grower who had reared the fish in green
water.

Because the fish has bright coloration when obtained, they were held for
1 mo.

in shaded, algae-free aquaria before the trials began. The rosy barbs
were

treated with one of four diets: a flake with no added carotenoid source,
a flake

with traditional carotenoids, a flake incorporating 0.5% Spirulina and
0.4%

Haematococcus, and a flake with 1.5% Spirulina and 1.0% Haematococcus.
Kissing

gouramis and 24K mollies were also obtained from growers but were from
shaded

aquaria containing no algae and they received the same diets as the rosy
barbs.

Color was judged by test panels of persons randomly recruited from
around the

biochemistry laboratory at the University of Hawai�i. The treatments
were not

revealed to the individuals who were asked to rank the fish according to

intensity of color. Color ranking was by a score of 1-4 (one being the
lowest)

for fish with four treatment groups. Ties were allowed but did not often
occur.

Color rankings were either one or zero for fish of two treatment groups
with the

score of one relating to more intensely colored fish. Scores were
subjected to

ANOVA or t-test. In all cases, pairs of control and treatment fishes
were

photographed and can be seen at http://www.sandersbshrimp.com. Results
Panel

members clearly identified the most intensely colored swordtails,
rainbowfish

and topaz cichlids. Those treated with Spirulina and Haematococcus
(biotech

algae) received significantly higher scores than control fish fed no
carotenoid

(Table 1). Table 1. Mean scores of treated and control fish. Different

alphabetical designations indicate significantly (P\<0.05) different
scores.

Number of SCORES Fish panelists Control Experimental Swordtails 8 0.0b
1.0a

Rainbowfish 15 0.1b 0.9a Topaz cichlids 13 0.0b 1.0a While the panelists

convened after the 3-wk treatments, differences were already noticeable
between

treated and control swordtails after only 1 wk. It should be emphasized
that,

unlike some of the other fishes tested, only male rainbowfish and topaz
cichlids

acquired color. Rosy barbs fed the Spirulina and Haematococcus were
judged to be

significantly (P\<0.05) more intensely colored than rosy barbs given
feed

containing no carotenoids (control) or those given control feeds
containing

traditional carotenoid sources (traditional) (Table 2). There was a dose
effect

of low (low biotech) or medium (med biotech) levels of Spirulina and

Haematococcus in the feeds but the effect was opposite of expectations.
The 24K

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(P\<0.05) more

intensely colored than those fed no carotenoids or traditional sources
of

carotenoid though there was no detectable dose effect among fish fed
Spirulina

and Haematococcus. Kissing gouramis fed carotenoids were significantly
(P\<0.05)

more intensely colored than those fed no carotenoids. Table 2. Mean
scores of

treated and control fish. Different alphabetical designations indicate

significant (P\<0.05) differences. Number of SCORES Fish panelists
Control

Traditional Low Med biotech biotech Rosy barbs 16 1.7d 1.9d 3.9a 3.0b
24K

mollies 16 1.9c 1.4c 2.8a 3.6a Kissing gouramis 16 1.0cd 3.0a 2.6a 2.8a

Discussion The treatments reported here are efficacious. Since they use
natural

algae that mimic the absorption of carotenoid that occurs in the wild,
they

should be more acceptable to consumers who may have concerns about the
use of

chemicals or hormones to enhance color. A �cocktail� of algae supplying
natural

stereoisomers of b-carotene, zeaxanthin, lutein, canthaxanthin, and
astaxanthin

was used in the current work. The cocktail approach was taken because
fish

sometimes seem to metabolize carotenoids before depositing them onto
natural

receptors in the skin in a species dependent way (Miki et al. 1985;
Matsuno et

al. 1985; Katsuyama et al. 1987; Katsuyama and Matsuno 1988). This was
observed

in preliminary results whereby the blue-green fluorescent colors in
discus fish

Symphysodon var. seemed to be enhanced by feeding sources of b-carotene
and red

colors seemed to be enhanced by feeding sources of canthaxanthin and

astaxanthin. However, the red color of red swordtails and tinfoil barbs
Barbus

schwanenfeldi seemed to be enhanced by b-carotene sources. The test
panels used

to judge color intensity in the current work were modeled after panels
used to

judge taste and texture of tropical fruits. Panelists were blinded as to

treatments and did not interact with those who recorded results, as the
latter

stood physically behind the panel members. Chemical extraction (Miki et
al.

1985; Matsuno et al. 1985; Katsuyama et al. 1987; Katsuyama and Matsuno
1988)

and fiber optic reflectance spectrophotometry (Wallat and Lazur 1999)
are other

methods of quantifying color intensity. The test panel method, however,
offers

the advantages of low cost and convenience but color photographs (which
are

expensive to produce) have an immediate and long-lasting impact.
Photographs of

control and experimental swordtails and rainbowfish may be viewed on the

Internet at http://www.sandersbshrimp.com. The current work should be of

immediate interest to pet feed manufacturers who service customers who
keep

their fish in clear water aquaria. The coloration of pet fish will fade
if the

fish are not provided with carotenoids. Traditionally, manufacturers
have used

complex formulations which may result in reduced palatability when
introducing

carotenoids into their feeds. The current work may also be of interest
to

ornamental fish feeds manufacturers who service growers with grazed down
or

otherwise clearwater systems, but it should be noted that ornamental
fish

growers do not use large amounts of feed because their fish are small.
The

largest market for the enhancement formulas may be food fish growers who
wish to

color the flesh of their fish or who grow fish such as the red sea bream
that

lose their coloration under culture. The algae described here work well
to

provide color and also offer advantages in application. They may be used
to

topcoat other feeds, which is appealing in simplicity, reducing losses
during

extrusion. Because the algae used in this study are natural products,
their use

would negate possible hesitation of the public to consume food fish
grown on

chemically produced algae and satisfy those who want to feed more
�organic�

feeds to their pets. Literature Cited Ako, H., L. Asano, M. Fukada and
C.S.

Tamaru. 1999. Culture of the rainbowfish Pseudomugil furcatis and the
use of a

Hawaii-specific feed, Tropical Gold. I�a O Hawai�i. 2: 1-9. Ako, H. and
C.S.

Tamaru. 1999. Are feeds for food fish practical for aquarium fish?

In#ter#na#tion#al Aquafeed. 2: 30-36. Ako, H., D. Tagomori, K. Lum, C.S.
Tamaru

and R. Spencer. 1997. Palatability of dif#fer#ent feeds using the
swordtail,

Xiphophorus helleri. I�a O Hawai�i. 11: 4-5. Katsuyama, M., T. Komori
and T.

Matsuno. 1987. Metabolism of three ste#re#oi#so#mers of astaxanthin in
the fish,

rainbow trout and tilapia. Comp. Biochem. Physiol. 86B: 1-5. Katsuyama,
M. and

T. Matsuno. 1988. Car#o#tenoid and vitamin A\*, and metabolism of
car#o#tenoids,

b-carotene, canthaxanthin, astaxanthin, zeaxanthin, lutein, and
tunaxanthin in

tilapia Tilapia nilotica. Comp. Biochem. Physiol. 90B: 131-139. Matsuno,
T., M.

Katsuyama, T. Maoka, T. Hirono and T. Komori. 1985. Reductive
met#a#bol#ic

pathways of carotenoids in fish (3S, 3�S)-astaxanthin to tunaxanthin A,
B and C.

Comp. Biochem. Physiol. 80B: 779-789 Miki, W., K. Yamaguchi, S. Konosu,
T.

Takane, M. Satake, T. Fujita, H. Kuwabara, S. Shimeno and M. Takeda.
1985.

Origin of tunaxanthin in the integument of yel#low#tail (Seriola

quinqueradiata). Comp. Biochem. Physiol. 80B: 195-201. Tamaru, C.S. and
H. Ako.

1997\. Growth of the angelfish Pterophyllum scalare using var#i#ous
commercial

feeds. I�a O Hawai�i, 4: 1-8. Tamaru, C.S., H. Ako, R. Paguirigan and C.
Chun.

1998\. Growth of guppies, Poecilia reticulata, using various commercial
feeds.

Makai. 20: 3. Wallat, G.K. and A.M Lazur. 1999. Diet source effects on
skin

color development and growth rate in juvenile goldfish Carassius auratus
Red

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