IJERD – International Journal of Environmental and Rural Development (2023) 14-1

Research article
erd

Research article
Educational Material Research on the Color of Crayfish
forConversion to Edible Resources

SAKI HIGUCHI
Graduate School of Agro-Environment Science, Tokyo University of Agriculture, Tokyo, Japan

HONAMI ONO
Graduate School of Agro-Environment Science, Tokyo University of Agriculture, Tokyo, JapanHIROAKI HONDA
Technical Department, Uniflows Co., Ltd
SARVESH MASKEY
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, JapanAKIRA KUROSAWA
Faculty of Agriculture, Tokyo University of Agriculture, Tokyo, JapanTOSHIHIRO SUZUKI
Faculty of Applied Biosciences, Tokyo University of Agriculture, Tokyo, JapanNOBUYUKI TAKAHASHI
Faculty of Applied Biosciences, Tokyo University of Agriculture, Tokyo, Japan

HIROMU OKAZAWA
Faculty of Regional Environment Science, Tokyo University of Agriculture, Tokyo, Japan

KOUJI TAKEDA*
Teacher Education Course, Tokyo University of Agriculture, Tokyo, Japan
Email: k2takeda@nodai.ac.jp
Received 9 February 2023

Accepted 22 May 2023(*Corresponding Author)

Abstract The crayfish (Procambarus clarkii) is an invasive alien species consumed as a foodsource worldwide. However, there is an extremely low demand for it as a food resource inJapan, and it is typically targeted for extermination. One reason for the lack of progress inconsidering crayfish a sustainable food resource is limited education on the issue duringsecondary education programs. It is well known that astaxanthin is the pigment responsible
for the body color of common red crayfish and crustaceans such as shrimp and crabs. At thesame time, while there are ample study materials on plant pigments in the science education
curriculum in junior high and high schools in Japan, there are no such materials to ourknowledge that focus on animal pigments. Therefore, this research sought to develop teachingmaterials to deepen understanding of color as a food component of crayfish through anexperiment involving extracting and separating their pigments. Using pigments extracted fromred crayfish exoskeletons, we developed experimental teaching materials to observe
astaxanthin by employing thin-layer chromatography and high-performance liquid
chromatography. In addition, an experiment was conducted to observe the effect of different
foods on crayfish body color. For the experiment, food was developed to make crayfish bodies
white (decolorized) and orange. When the decolorized crayfish were fed norbixin or bixin,
which are components of the orange annatto pigment, norbixin tended to accumulate as orange
in the exoskeleton more than bixin based on the apparent body color change, color extraction
from images, and thin-layer chromatography. These findings are expected to be useful forⒸ ISERD

98

IJERD – International Journal of Environmental and Rural Development (2023) 14-1

science and nutrition education, supporting the development of the students’ awareness andunderstanding of crayfish as a food resource.
Keywords education, teaching material, Procambarus clarkii, pigment, food resource, SDGs

INTRODUCTION
Crayfish (Procambarus clarkii), which live in rivers, ponds, rice paddies, swamps, and other bodies
of water in the city, are a globally invasive alien species native to the United States (Gheradi, 2013).
Although the damage they cause to ecosystems is regarded as a problem, crayfish are used as a foodresource in the United States, China, and European countries (Hara, 2021). Furthermore, in the future,
their excrement and shells are expected to be used as agricultural resources such as fertilizers, as raw
materials for textiles, and as medical resources (Ifuku et al., 2004; Yihun et al., 2016).
Crayfish were introduced to Japan in 1927 as food for bullfrogs. Since then, the species’ highfertility rate and omnivorous nature have led to substantial damage to local ecosystems andagriculture. Thus, crayfish have become a well-known representative of alien species in Japan. As
part of environmental protection activities, crayfish are routinely exterminated and culled, but they
are not actively used. One reason for this is the lack of opportunities for students to learn about theusefulness of crawfish as a resource in elementary and secondary education. In elementary education,
crayfish are familiar observation materials because of their ease of care and management; however,in secondary education, they are only treated as an invasive alien species and representatives ofcrustaceans. Therefore, we thought that, in order to promote the recognition of crayfish as a foodresource, experimental materials focusing on the useful food components contained in crayfish might
be necessary. We focused on carotenoid pigments, which are a factor in crayfish body color thatchildren are likely to be interested in and have functional properties such as antioxidant effects andnutrients. In Japanese secondary education, carotenoid pigments are derived only fromphotosynthetic organisms, and their functions are rarely discussed. Among carotenoid pigments,
astaxanthin, which is found in the wild crayfish, salmon, and crustaceans (shrimp and crab) that arefamiliar to the Japanese people, is utilized in a variety of products because of its high functionality.
Although the usefulness of astaxanthin is known commercially, it has not yet been used in Japanese
secondary education. Therefore, the development of experimental teaching materials focusing on
astaxanthin using crayfish will provide new science experiment teaching materials focusing on
animal pigments and will enable students to learn about the usefulness of crayfish.
Astaxanthin is abundant in the exoskeletons of crayfish. The exoskeleton can easily extract
pigments, and the use of molting shells obtained each time the crayfish grows is useful as a type ofteaching material for collecting samples without killing the organism. Astaxanthin in the exoskeletonof crayfish exists in free and ester forms. However, the form of astaxanthin in exoskeletons derived
from molted shells is unknown. Therefore, in this study, we will examine the separation conditions
using thin-layer chromatography (TLC) and high-performance liquid chromatography (e-HPLC),
which are treated in secondary education, as teaching materials for observing the ontogeny ofastaxanthin in molting shells.
Previously, we produced crayfish that changed their body color to blue or pink by feeding
astaxanthin to genetically fixed white crayfish (Takeda, 2021). We also found that feeding anothercarotenoid pigment, annatto pigment, to white crayfish turned their bodies orange. However, the foodpigment annatto contains bixin and norbixin as components, and the difference in body color due tothese components is unknown. Wild crayfish are familiar for use as general teaching material, butsince they retain their original colors, they must be decolored. Therefore, in this study, as teachingmaterial to explore the factors involved in rearing, we prepared decolorized wild crayfish diets andconducted feeding tests of diets containing mainly bixin and norbixin. Then, the experiment willcompare the body color change for each color value and study the conditions to observe thesubstances that cause these body colors by TLC.
OBJECTIVEⒸ ISERD

99

IJERD – International Journal of Environmental and Rural Development (2023) 14-1

This research explored the development of teaching materials focusing on pigments, which are both
body color factors and nutrients in crayfish, with the aim of utilizing them in science and foodeducation to highlight their usefulness as food resources.
METHODOLOGY
Experiment Focusing on Astaxanthin Contained in Crayfish Exoskeleton and
Separation of Astaxanthin using TLC
We experimented to explore astaxanthin in crayfish exoskeletons as a body-color factor. Acetone
containing 0.01% astaxanthin (Fujifilm Wako) and krill were used as controls. It has been reported
that krill have astaxanthin and esters of astaxanthin (monoesters, diesters) (Takaichi et al., 2003).
Since the eyes of krill contain substantial amounts of these carotenoids (Maoka et al., 1985), krill
eyes were used in the experiment.
The sample for the experiment included the exoskeletons of living crayfish and molted
exoskeletons. For pigment extraction, the abdominal segment of each exoskeleton was shredded toabout 1 mm, and 100 μl of acetone was added to 0.01 g of the material. They were mixed lightly,
allowed to stand for 15 minutes, and centrifuged for 20 seconds. Each sample was spotted on a
TLC plate (TLC silica gel 60 F254 [4 × 8 cm], Sigma-Aldrich), and then the TLC was placed in thesolvent (petroleum ether: acetone = 7:3) in the deployment tank to separate the pigments.
Isolation of Astaxanthin Using e-HPLC“Kotori” (Uniflows, Japan) is an HPLC developed for educational purposes. It is smaller and cheaper
than the general HPLC. Thus, it is used in high schools in Japan. An e-HPLC was used to detectastaxanthin contained in the molted exoskeletons of crayfish. As a control, 0.001% astaxanthin(Fujifilm Wako) was dissolved in acetone, and 100 μl of acetone was added to 0.01 g of theabdominal segment as a sample. A Cadenza CD-C18 column (Φ6 × 50 mm) (manufactured by Intact
Co., Ltd.) was equilibrated with 50% methanol: THF (1:1) eluent at 600 μL/min at room temperature(25 C). Each sample of 2.7 μL was injected into the column; then, the peak of astaxanthin was
detected at 405 nm.
Creation of Bait for Decolorization and Feeding Tests
Material containing no carotenoid pigments was used to create decolorization bait. Rice powder wasused as a carbohydrate, soybean, pork bone, and fish powders were used as protein, and sardine oil
was used as a lipid. Rice powder, soybean powder, pork bone powder, fish powder, and sardine oil
were mixed at 2:3:2:2.8:0.2, and an appropriate amount of water was added. The ingredients werethen molded into a round shape, steamed for 20 minutes, and then solidified and used as feed. Four
crayfish aged about 8 weeks old were selected for a feeding test in which any changes to their bodycolor after consuming the decolorization bait were observed. The crayfish were fed 0.05 g of bait
once a day in the morning for 8 weeks. In addition, it was confirmed that there was no bait left overfrom the previous day, and any molted exoskeletons were removed, the water was changed daily
before feeding. As a control experiment, commercially available crayfish bait (Kyorin) was used
under the same conditions.
Pigment-containing bait was then created using the decolorization bait before it underwent
steaming. The bait and pigment were mixed at a ratio of 9:1, and about 0.5 ml of water per 1 g ofbait was added to assess the degree of viscosity. The pigments used were norbixin (Fujifilm Wako)
and bixin (Fujifilm Wako); they are components of annatto pigment (Scooter, 2009) used for foodcoloring. The ingredients were then molded into a round shape, steamed for 20 minutes, thensolidified. This pigment-containing bait was then used in a feeding test of crayfish decolored using
decolorization bait. Any changes to the crayfish’s body color were observed. Four crayfish were fedⒸ ISERD

100

IJERD – International Journal of Environmental and Rural Development (2023) 14-1

norbixin-containing bait, and four were fed bixin-containing bait. A piece of pigment-containing bait
was fed at a rate of 0.03 g per 0.1 g of crayfish weight in the morning once a day for 9 days. In
addition, it was confirmed that there was no bait left over from the previous day and any molted
exoskeletons were removed; the water was changed daily before feeding.
Color Extraction from an Image of Body Color Change
Pictures were taken of the subject crayfish every day under the same conditions. The red, green, andblue (RGB) values of the sixth abdominal segment were measured using a color selection software
called Spoitkun. The resulting data were expressed as the average value ± standard deviation.
Confirmation of Color Change Due to Boiling
It was important to confirm how the color would change after boiling as this is how crayfish areprepared as food. Therefore, we compared the color of the first pectoral leg of wild crayfish andcrayfish colored using norbixin before and after boiling for 15 min.
Detection of Substances Causing Body Color Using TLC
Acetone containing 0.01% norbixin (Fujifilm Wako) and 0.01% bixin (Fujifilm Wako) was used asa control, and the tail fins of crayfish were used as a sample. For each 0.01 g of tail fin mass, 50 μlof acetone was added. The solution was lightly mixed and then centrifuged for 20 seconds to extract
the pigment. After pigment extraction, each sample was quickly spotted on a TLC plate (TLC Silica
gel 60 RP-18 F254s [5×7.5 cm], Sigma-Aldrich). Then, the TLC was placed in the solvent (water:acetone 1:9) in the deployment tank to separate the pigments.
RESULTS AND DISCUSSION
Experiment Focusing on Astaxanthin Contained in Crayfish Exoskeleton and
Separation of Astaxanthin using TLC
Astaxanthin esters were mainly present in the abdominal segment of the crayfish (lane 3) as well as
the krill (lane 2), and astaxanthin was also detected (Fig. 1A). Astaxanthin was observed as a major
pigment in the abdominal segment of the molted exoskeleton (lane 4) (Fig. 1A). Although it has beenreported that astaxanthin esters and astaxanthin are the main pigments in the exoskeleton of wild
crayfish (Nakagawa et al., 1974), no research was found describing the predominance of freeform
astaxanthin in molted exoskeletons. This experiment revealed that there is a difference in the presenceof astaxanthin in crayfish exoskeletons before and after molting.
Since crustacean molting is a lifelong phenomenon, molted exoskeletons can be continuously
collected as samples for experiments. In addition, since the pigment contained is mainly astaxanthin
in the free form, it is expected that it will be easy for children to understand. Using molted
exoskeletons for experiments to observe pigments contained in the exoskeletons of crayfish isconsidered suitable.
Isolation of Astaxanthin Using e-HPLC
As with the control freeform astaxanthin, elution peaks were also confirmed in the molted
exoskeleton at 200 s–230 s (Fig. 1B). Freeform astaxanthin was confirmed to be the main pigment
contained in the molted exoskeleton using not only TLC but also e-HPLC.
“Kotori” can be used to confirm astaxanthin in a short time. Thus, it is possible to perform
multiple analyses even within a typical 50-minute class period. The device and column are very small
compared to general HPLC, and, since it is very portable, it is considered suitable for conducting
advanced experiments aimed at separating astaxanthin in various educational settings.
Ⓒ ISERD

101

IJERD – International Journal of Environmental and Rural Development (2023) 14-1

Fig. 1 Isolation of astaxanthin in wild crayfish exoskeletons A (TLC), B (e-HPLC)
Creation of Bait for Decolorization and Feeding Tests
In the feeding test using TL2-3 cm crayfish for 8 weeks, the bodies of the crayfish fed with thecommercial bait turned brown (Fig. 2A-a) while the bodies of the crayfish fed with decolorized bait
turned white (Fig. 2A-b). Body color changes to blue and white with each molt are known to occur
by feeding horse mackerel with a low amount of carotenoid pigment. However, horse mackerel bait
is likely to cause contamination and odor in the breeding water. The decolorized bait created in thisexperiment can also be used as an alternative bait, which is considered to have the advantage of easeof breeding.
In the feeding test with pigment-containing bait, the bodies of all four crayfish consuming thenorbixin-containing bait (Fig. 2B-abc) rapidly changed to orange while those consuming the bixincontaining bait gradually changed to yellow, although there were individual differences (Fig. 2Bdef).
In our previous research, genetically fixed white crayfish fed with a diet containing annatto pigment(TCI, Japan) diet turned orange. However, in that research, it was unclear whether it was norbixin or
bixin that was related to the orange body color. In this study, decolored wild crayfish turned darkorange when fed with norbixin (Fig. 2B-c) and yellow when fed with bixin (Fig. 2B-f). Thus, it wasapparent that norbixin is the main factor contributing to orange body color. Fig. 2 shows the changeover time in the crayfish with the most colored body on day 9.

Fig. 2 Body color change by feeding test A: commercial bait (Left) and decolorized bait
(Right) B: Norbixin (Left) and Bixin (Right)
Color Extraction from an Image of Body Color Change
When crayfish color is expressed as a mixture of Red (R), Green (G), and Blue (B) in 16 bits (256steps) using color extraction of images taken over time, the R value did not change significantly
for either control, bixin, or norbixin. In terms of G values, it was confirmed that the feeding periodⒸ ISERD

102

IJERD – International Journal of Environmental and Rural Development (2023) 14-1

of bixin was almost constant while norbixin was gradually declining. In addition, the B value wasalmost zero in one day for norbixin while, for bixin, it gradually decreased (Fig. 3).
In Japan industry standard color names, yellow is R ≒ G > B (255, 212, 0), and orange is R >G > B (243, 152, 0). In addition, both orange and yellow become darker orange and yellow as the B
value decreases to 0. Therefore, orange and yellow can be compared from the relative ratio of R and
G. This result supports the visual color shown in Fig. 2B. Efforts to quantify changes in the color of
the eyes seen using color extraction software will lead to activities to grasp observation events morescientifically and can be used for not only science but also STEAM education.

Fig. 3 Changes in RGB values of crayfish exoskeletons over time using
pigment-containing baits
Confirmation of Color Change Due to Boiling
Just as many crustaceans turn red when boiled, wild crayfish are red black before boiling (Fig. 4A)
but turn red when boiled (Fig. 4B). Interestingly, the first pectoral leg of a crayfish, which had turnedorange with norbixin (Fig. 4C), remained orange (Fig. 4D) when boiled. Orange has the same effecton food palatability as red (Birren, 1963). Although boiled crayfish as food have typically come only
in red, the above finding can be used to develop new colors of crayfish for food products. It is also
hoped that crayfish will be used as a teaching material for the development of crayfish as a foodsource in Japan.

Fig. 4 Change in color of the first pectoral leg before and after boiling
Detection of Causative Substances of Body Color using TLC
TLC revealed bands at the same position in norbixin (lane 1) and the tail fans colored by norbixin(lane 2) and bixin (lane 3) and the tail fans colored by bixin (lane 4) (Fig. 5). This confirmed that thepigments contained in the bait were accumulated in the body. In this experiment, a tail fan derived
from a living body was used, but the same result was obtained for the molted exoskeleton (data notshown).
By conducting this experiment in parallel with the feeding test, it is possible to use it asmultiscale experimental teaching material to detect changes in body color and substances that cause
body color.
Ⓒ ISERD

103

IJERD – International Journal of Environmental and Rural Development (2023) 14-1

Fig. 5 Separation of norbixin and bixin in TLC
CONCLUSION
Astaxanthin is contained in red crustaceans and fish. It is a factor in body color as well as an important
substance with antioxidant effects. The exoskeleton of the crayfish contains a large amount ofastaxanthin and is also useful as a teaching material because crayfish is treated in educational courses
and is a familiar creature. In addition, the development of experimental materials focusing on
astaxanthin using crayfish will be a new scientific experimental material focusing on animal
pigments and will enable students to learn about the usefulness of crayfish.
In this study, astaxanthin contained in crayfish exoskeletons was isolated using TLC and eHPLC. TLC is an experimental technique used in secondary education for the separation of pigments
derived from photosynthetic organisms. The e-HPLC is an instrument developed for educational
purposes and used in secondary schools specializing in science education. In this study, we haveclarified the experimental conditions under which astaxanthin can be detected in more detail and ina shorter time by using e-HPLC as well as TLC; these experimental methods can be implemented inschool education. Interestingly, we found that the presence of astaxanthin in the exoskeleton of wild
crayfish in living tissue differed from that derived from molting.
For wild crayfish, we developed a decolorized bait that regulated the carotenoid pigments
contained in the material and succeeded in whitening it. In addition, by feeding bait containing
norbixin and bixin, which are components of annatto pigment for food coloring, it was revealed thatbody color turned dark orange with norbixin and yellow with bixin. Furthermore, it was clarified that
norbixin turns a dark orange color in a shorter period of time than bixin from changes in appearance,
TLC, and color extraction.
The findings and experimental methods of this research can be implemented in schools and usedas new experimental materials focusing on animal pigments. Since these are teaching materials that
focus on color, it is expected they will be used in science and food education that will lead to theedible use of crayfish.
ACKNOWLEDGMENTS
We thank Mr. Kenshin Yuasa for his cooperation. This work was supported by the Tokyo NODAI
Research Program for Sustainable Agriculture and JSPS KAKENHI Grant number JP18K02989.
REFERENCES
Birren, F. 1963. Color and human appetite. Food Technology, 17, 553-555.
Ⓒ ISERD

104

IJERD – International Journal of Environmental and Rural Development (2023) 14-1

Gheradi, F. 2013. Crayfish as global invaders, Distribution, impact on ecosystem services and management
options. Freshwater Crayfish, 19 (2), 177-187, Retrieved from DOI https://doi.org/10.5869/fc.2013.v192.177
Hara, Y. 2021. Development and issues of integrated rice-aquaculture farming and research trends in China. Ejournal GEO, 16 (1), 70-86, Retrieved from URL https://researchmap.jp/hara_yuta/published_papers/31351252/attachment_file.pdf
Ifuku, S., Nogi, M., Abe, K., Yoshioka, M., Morimoto, M., Saimoto, H. and Yano, H. 2004. Preparation ofchitin nanofibers with a uniform width as α-chitin from crab shells. Biomacromolecules, 10 (6), 1584-1588,
Retrieved from DOI https://doi.org/10.1021/bm900163d
Maoka, T., Katsuyama, M. and Kaneko, N. 1985. Stereochemical investigation of carotenoids in the Antarctic
krill Euphausia superba. Bulletin of the Japanese Society of Scientific Fisheries, 51 (10), 1671-1673,
Retrieved from URL https://www.jstage.jst.go.jp/article/suisan1932/51/10/51_10_1671/_pdf
Nakagawa, H., Kayama, M., Yamada, H. and Asakawa, S. 1974. Studies on carotenoprotein in aquatic animals,
IV, Carotenoid pigments in crayfish (Procambarus clarkii). Journal of the Faculty of Fisheries and Animal
Husbandry, Hiroshima University, 13, 1-13, Retrieved from URL https://ir.lib.hiroshima-u.ac.jp/41206/files/ 35023
Scooter, M. 2009. The chemistry and analysis of annatto food colouring, A review. Food Additive andContaminants, 26 (8), 1123-1145, Retrieved from DOI https://doi.org/10.1080/02652030902942873
Takaichi, S., Matui, K., Nakamura, M., Muramatu, M. and Hanada, S. 2003. Fatty acids of astaxanthin esters
in krill determined by mild mass spectrometry. Comparative Biochemistry and Physiology, Part B
Biochemistry and Molecular Biology, 136 (2), 317-322, Retrieved from DOI https://doi.org/10.1016/S1096-4959(03)00209-4
Takeda, K., Yururi, M. and Asanuma, S. 2021. New ways of teaching science. Science Impact Ltd., 16-18.
Takeda, K., Yururi, M. Jitsuno, M., et al. 2021. An impact of biological pigments as teaching material. Open
Access Government, 406, 262-263.
Yihun, F.A., Egusa, M., Kaminaka, H., Izawa, H., Morimoto, M., Saimoto, H. and Ifuku, S. 2016.
Protein/CaCO3/chitin nanofiber complex prepared from crab shells by simple mechanical treatment and its
effect on plant growth. International Journal of Molecular Sciences, 17 (10), 1600, Retrieved from DOI
https://doi.org/10.3390/ijms17101600Ⓒ ISERD

105