Tokyo salamanders (Hynobius tokyoensis) may possess thecapability to distinguish between red and blue
Rina Akiyama
ABSTRACT
Color vision is a fundamental trait in many animal species that enhances survival and reproduction.
Despite the importance of this ability for survival, there have been few examples of the use of knowledge
about color vision to improve rearing environments. This study examined color vision in the endangered
Tokyo salamanders (Hynobius tokyoensis). By learning about the color vision of this creature, for whichconservation efforts are increasingly needed, this study encourages the use of knowledge about thecognitive functions of animals in captivity. The aim of the current study was to elucidate whether theTokyo salamanders have color vision. To evaluate their ability to distinguish two colors, salamanders wereplaced in a two-colored box, split evenly into red and blue sections, and their behaviors in response to thecolor differences were keenly observed. As a result, their stay in the red areas was markedly longer incomparison to the blue areas, suggesting that they appeared to exhibit a stronger affinity for the color red.
This study indicates the potential existence of color vision within Tokyo salamanders.

KEYWORDS: Tokyo salamander (Hynobius tokyoensis), color vision, salamander

INTRODUCTION
Color vision, playing a crucial role in enhancing survival and reproduction, is an essential characteristic
common to a lot of animal species. Although there is a substantial amount of research that focuses on thecolor vision of diverse animal species, there is a noticeable lack of research specifically investigating color
vision in salamanders, with the exception of the fire salamanders (Salamandra salamandra) (Rozenblit &Gollisch, 2020). Rozenbilt and Gollisch built upon the work of fire salamanders of Przyrembel andNeumeyer (1995), which estimated that fire salamanders possess trichromatic color vision, as evidenced bytheir behavioral responses to monochromatic lights with different wavelengths. However, previous studies
have not investigated the color vision of other salamanders.
Tokyo salamanders (Hynobius tokyoensis), a member of the Hynobiidae family of salamanders, havebeen endangered in recent years (IUCN SSC Amphibian Specialist Group, 2022). About half of all
salamanders in the Hynobiidae family are endemic to Japan and many are threatened with extinction.
Because of their low mobility and narrow geographic distribution, the Tokyo salamanders, which are
distributed around the Tokyo metropolitan area, are in particular danger of extinction by humanencroachment (Hayase & Yamane, 1989). Therefore, it is anticipated that a more detailed understanding of
their vision and ecological conditions will aid in their preservation of natural environments and inenhancing artificial breeding techniques.
The objective of this study was to investigate whether the Tokyo salamanders possess color vision. This
research sheds new light on the color vision of Hynobiidae, filling a gap in the literature on the color visionof salamanders in many species and contributing to their conservation efforts. Although there were nocases studied in the past, I thought Tokyo salamanders have color vision because they were more likely toapproach a red-colored smartphone, which is usually the same color as their bait.

METHODS
Study Design1

In order to confirm whether the salamanders modified their behavior in response to color stimuli, theywere placed in a black box (serving as a controlled environment) and a two-colored box (with the left halfbeing blue and the right half red). Red was chosen because salamanders have been observed to approach
the area on a daily basis, and blue was selected because it is the wavelength in the visible light spectrum
farthest from red. The duration of time they spent on each side was compared.

Animals
A pair of Tokyo salamanders, born in the spring of 2022 in Chiba, Japan, was obtained from theKannonzaki Nature Museum, Kanagawa, Japan. Each salamander was housed individually undercontrolled conditions, with a temperature of 20℃ ±4℃, appropriate nourishment, and a proper
environment in accordance with guidelines laid out by the Japanese Association of Zoos and Aquariums(2022a; 2022b). They were fed with sludge worms (Tubifex tubifex) and Propsilocerus akamusi every
other day.

Procedure
Make a Black Box and a Two-colored Box (the left half area is red and the right half area is blue) with twotransparent plastic boxes (Daiso Industries Co., 30 × 20 × 18 cm) as tanks, and black, blue, and redcolored papers (CAN DO Co., Ltd). Put the Black Box right under the LED desk light (Nitori Co. Ltd.,YP410DL) and the USB camera as no shadow exists in the box. Move a Tokyo salamander from a livingtank to the Black Box with the fish net after the water (160 ml, 20℃ ±2℃), which chlorine was removed
with WATER SUPPLE for amphibians (Axis Co.), was added to the Box. Ten minutes later, a Tokyo
salamander was moved back into the living tank, and a Black Box was rinsed in tap water. Then similarly,a Tokyo salamander was moved in a Black Box two more times. After all Black Box experiments, thesame test was done with the Two-colored Box instead of the Black Box with a Tokyo salamander. Then,the same procedure was done with another Tokyo salamander in both Boxes. As the second Tokyosalamander was sick after the first ten minutes in the Two-colored Box, the experiment was discontinued toprotect its health. The length of the Tokyo salamander stayed on the left and right sides and four areas(up-left, down-left, up-right, and down-right) in the box were counted per second. The location of theTokyo salamander was decided based on the center of their heads. The average ratio of the Tokyo
salamanders stayed on the left and right sides and was compared between the Black Box and theTwo-colored Box.

Statistical SignificanceGiven the nature of the dataset, conducting statistical tests was deemed inappropriate. This conclusion
was drawn from the fact that the identical Tokyo salamanders were used repetitively for both black box
and bi-colored box tests.

RESULTS
The primary purpose of this experiment was to determine the presence of color vision in Tokyo
salamanders by comparing the length of time they spent in a black box versus a two-colored box. It was
hypothesized that if they can recognize the difference in colors, their behavior in the two-colored box
would be different from that observed in the black box. The results of this experiment are shown in Figure
1. A striking feature of these graphs is the notably extensive high rate of the length of time in the red area.
In the Black box trials, the time spent by salamanders on either side was nearly identical, with an average
difference of merely 0.88%. However, in the two-colored box trials, the average percentage of time theyspent in the red area exceeded that in the blue region by 19.5%. This outcome suggests that Tokyosalamanders can distinguish between red and blue.

2

Figure 1 (a) the percentage of time that salamanders spent in the left and right area of the Black Box and(b) the percentage of time they spent in the red and blue area of the Two-colored Box. The mean ± SEM
The length of time spent in the four distinctive areas (up-left, down-left, up-right, and down-right) was alsocompared. Figure 2 presents the percentages of the average duration that the Tokyo salamanders spent ineach of these four areas. These findings provide evidence that the salamanders were actively moving
within the confines of the experimental boxes.

Figure 2 (a) the percentage of time salamanders spent in 4 equal size areas (up-left, down-left, up-right,down-left) of the Black Box and (b) Two-colored Box. The mean ± SEM

DISCUSSION
One of the primary goals of this project was to identify whether Tokyo salamanders possess color vision.
The current study suggests a potential for color vision in this species, as they altered behavior in responseto red and blue stimuli as compared to their behavior in the black box. This finding is consistent with thatof Przyrembel (1995) that indicated trichromatic color vision in fire salamanders.

3The color vision and cognitive abilities of salamanders have not been known until now. Therefore, it ishoped that this kind of research will advance our understanding and that conservation activities tailored totheir characteristics will flourish. For example, by knowing their color vision, it will be possible to install
breeding facilities and lighting that take into consideration the colors they can see.
However, given the small sample size, these results should be interpreted with caution, as the findings
may not represent the color vision and behavior of the entire Tokyo salamander population. Another areaof uncertainty is related to the brightness of colors utilized in the experiments. Since I did notquantitatively measure the color brightness, it remains uncertain whether the salamanders were affected bybrightness rather than the actual color wavelength.
These findings carry significant implications for furthering research in color vision, especially given thepaucity of studies in this area and the potential evidence of color vision in Tokyo salamanders. Suchinsights contribute to the refinement of experimental designs aimed at investigating color vision morecomprehensively. Future research should consider implementing experimental methods that utilize thethree primary colors of light to determine the range of light wavelengths perceivable by salamanders while
ensuring an ample sample size.

ACKNOWLEDGMENTS
I would like to thank the Research Club for the opportunity and support to conduct this research. I also
appreciate the advisors of the Life science group, particularly Mr. Okuda and Mr. Ikeda for their helpfuladvice. Last but not least, I am grateful to the Kannonzaki Nature Museum for a chance encounter withsalamanders.

REFERENCES
Hayase N & Yamane S (1989) Habitats and Reproductive Modes in Japanese Hynobiid Salamanders.
Ibaraki daigaku kyouiku gakubu kiyou (Ibaraki University Faculty of Education Minutes), 38: 85-102
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Japanese Association of Zoos and Aquariums (2022a) Tekiseisisetsu gaidorainn (The aptitude facility
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Przyrembel C, Keller B & Neumeyer C (1995) Trichromatic color vision in the salamander (Salamandrasalamandra). J Comp Physiol A. 176: 575–586
Rozenblit F & Gollisch T (2020) What the salamander eye has been telling the vision scientist’s brain.
Seminars in Cell & Developmental Biology, 106: 61-71

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