Comparison of UV-B tolerance between wild-type andalbino Japanese flounder Paralichthys olivaceus

juveniles

誌名誌名 水産増殖 = The aquiculture

ISSNISSN 03714217

著者著者

Fukunishi, Y.Masuda, R.Seikai, T.Nakamura, M.Tagawa, M.Yamashita, Y.

巻/号巻/号 65巻2号

掲載ページ掲載ページ p. 149-152

発行年月発行年月 2017年6月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Aquacult. Sci. 65(2), 149-152 (2017)

Short Paper

Comparison of lN-B tolerance between wild句peand

albino Japanese flounder

Pαfαlichthys olivαceus juveniles

YuichiFu即 NISHI1・2・*,Reiji MASUDA¥

Tadahisa SEI凶 3,Mitsuo NAKAMuRA4,

Masatomo TAGAWA4 and Yoh YAMASHITA5

Abstract: Albinos in Japanese flounder Paralichthys oliva-

ceus are yellowish mutants which lack melanophores. To

reveal the釦nctionof melanophores, UV-B tolerance was

compared between wild-type (100 ± 8 mm) and albino (98

土 6mm) flounder juveniles. After 6 hours of UV-B expo-

sure (1.1 WI m2), survival was examined every 1 hour for

24 hours. Control trea佃ientwithout UV exposure was also

conducted. Albino fish in the UV treatment showed sig-

nificantly lower survival仕tan出atof wild勾pefish.τ'here

was no significant difference in survival between strains in

the control. Present research suggests that melanophores

have a UV photoprotective function in flounder juveniles.

Key words: Japanese flounder; pigmentation; chromato-

phore; melanin

Due to the increase of harmful ultraviolet-B (UV-B)

radiation (280-320 nm) reaching the earth’s surface

(Madronich et al. 1998; Herman 2010), which is asso-

ciated wi白 depletionof the ozone layer (Kerr and

McElroy 1993), the protective mechanisms against

ul仕avioletradiation in organisms have been drawing

much attention in recent years.τ'he pigment mela-

nin, which is the main component of melanophore in

the skin, absorbs ultraviolet and visible waveband in a

wide varaiety of taxonomic groups such as amphibians,

crustaceans and fishes (Lowe and GoodmanLowe 1996;

Cockell and Knowland 1999; Hessen 2002; Blaustein

and Belden 2003; Sarna and Swartz 2006). Although

several researchers have performed studies on the UV

Received 19 January 2017; Accepted 7 April 2017.

photoprotective role of melanophores in freshwater

fishes, their functions still remain controversial. Ahmed

and Setlow (1993) demonstrated that the pigment mel-

anin reduced the number of cyclobutane p戸加idine

dimers, which is the major cause of lesions produced

in DNA by ultrarviolet radiation, in the skin in platyfish,

Xiphophorus. In con仕ast,despite that melanophores of

medaka, Oryzias la均es,larvae and juveniles have the

highest absorbances at ultraviolet wavelength (below

400nm) (Armstrong et al. 2000), the wild却pemedaka

larvae which have melanophores, xanthophores and

leucophores had significantly higher UV-B induced DNA

damage and more necrosis in the epidermis compared

to血es回 insthat lack melanophores (Arms仕onget al.

2002). Furtehrmore, according to Fabacher et al. (1999),

albino medaka which lack melanophores were as tolerant

of UV-B radiation as wild-type pigmented medal王a.

In marine fish species, Moser (1981) hypothe-

sized白atUV radiation is the primary factor select-

ing for heavy melanin pigmentation in the early life

stages. According to Iρwe and Goodmanlρwe (1996),

scalloped hanimerhead shark Sphyrna lewini pups

increased their melanin concen仕ationon the skin in

response to an increase in solar radiation, leading to

the reduction of transmittance in the UV-B waveband.

Red sea bream, Pagrus major, adults that were bred in

outdoor net cages became suntanned and出eyhadmore

melanin and melanophores in the skin compared to血at

of wild fish and cultured fish shaded from exposure to

the sun (Adachi et al. 2005). Howeveζno studies have

yet to assess whether melanophores have an ul仕aviolet

photoprotection function in marine teleost宣sh.

Albino宣share mutants which inherently lack mela-

nin in the skin但yodo-Taguchiet al. 1997). In Japanese

flounder Paralichthys olivaceus, albino mutants are

available. Shikano et al. (2007) reported that the

number of melanophores on the scales of the ocular

side was remarkably lower in albino fish compared

to that in wild-type. However, albino fish had as many

xanthophores and iridophores as wild-type individu-

als (Shikano et al. 200η，resulting in a yellowish color

on血eocular side.τ'herefore, albino in Japanese

1 Maizuru Fisheries Research Station, Field Science Education and Research Center, Kyoto University, Nagahama, Maizuru, Kyoto 625-0086, Japan. 2Fisheries Research Institute, Toyama Prefectural Agricultural, Forestry & Fisheries Research Center, Takatsuka 364, Namerikawa, Toyama, 936-8536 (Present address).

3 Faculty of Marine Biosciences, Fukui Prefectural University, Obama, Fukui 917-0003, Japan. 'Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashir温mwaOiw必ce-cho,Kyoto, Kyoto 606-8502, Japan. 5 Field Science Education and Research Center, Kyoto University, Kitashirakawa Oiwake-cho, Kyoto, Kyoto 606-8502, Japan. *Corresponding author: Tel, (+81) 764-75-0036; Fax, (+81) 764-75-8116; E-mail, yuichi.fukunishi@pref.toyama.lg必 ('l.Fukunishi).

150 Y. Fukunishi, R. Masuda, T. Seikai, M. Nakamura, M. Tagawa and Y. Yamashita

flounder is distinct from pseudo-albino, or“whiten-

ing”， which frequently occurrs in hatchery production

processes.

Japanese flounder juveniles recruit to and se仕leon

shallow open sandy areas at less than 15 m depth from

early spring to summer (Minami 1982; Minami and

Tanaka 1992). Kuwahara et al (2000) reported that the

depth at which surface UV-B radiation attenuated to

1% level was 10.8±5.7m and 14.9±9.5m for 305nm

and 320 nm wave length of UV, respectively, in the

coastal waters of Sagami Bay, Japan. Therefore, they

are exposed to UV-B radiation in the natural habitat.

Wild-type Japanese flounder juveniles develop a dark

brown to olive coloration only on the ocular side which

faces the sea surface (Nakamura et al. 2010). Thus,

albino Japanese flounder juveniles are excellent mate-

rial to investigate the photoprotective function against

ultraviolet of melanophores. The aim of出isstudy was

to examine出ehypothesis that melanophores mitigate

UV-B induced damage. Survival was compared between

pigmented wild却peand albino individuals after UV-B

exposure in Japanese flounder juveniles.

Fertilized eggs of wild-type Japanese flounder were

obtained from the Miyazu Center, National Center

for Stock Enhancement, Fisheries Research Agency

of Japan, and were transported to Maizuru Fisheries

Research Station (MFRS), Kyoto University with

O珂rgenin a polyethylene bag. Fertilized eggs of albino

Japanese flounder were incubated in the Research

Center of Marine Bioresources, Fukui Prefectural

University, Obama and were sent to MFRS. The origin

of albino eggs is described in Shimada and Seikai

(2008). Wild匂rpeand albino Japanese flounder were

reared separately in the 500 l circular tanks until they

grew up to the juvenile stage. Rearing protocol of both

strains basically followed those described in detail

in Nakamura et al. (2010). The experiment was con-

ducted on 24 August 2006.τ'he age of wild-type and

albino juveniles were 134 and 143 days after hatching,

respectively. The average total length of wild-type and

albino individuals was 100 ± 8 mm (mean± SD, n = 1)

and 98 ± 6 mm (n = 12), respectively, and did not differ

significantly.

The experimental set up of UV exposure with UV-B

lights was the same as described in Fukunishi et al.

(2013) except that only one UV-B level equivalent to

the maximum irradiance observed at the sea surface in

summer in the Sea of Japan (1.1 WI m2) was used as the UV treatment in this study. A control仕eatmentwithout

UV radiation was also conducted. Juveniles were indi-

vidually stocked in the white plastic container (800 ml)

and kept in a water bath.τ'hese containers had two

holes (diameter: 35 mm) sealed with 3 mm mesh net.

τ'his arrangement allowed water to circulate in the con-

tainers and maintained sufficient O河rgenconcentration

for fish during the experiment. Fish were exposed to

UV-B radiation for 6 hours. The total UV dose during

出eexposure was 23760 J/m2. Six replicates were pro-

vided for each strain of both UV and control treatments.

Survival was assessed every hour until 24 hours after

the exposure. Death was defined as the stopping of gill

ventilation. Temperature in the water bath was kept at

21°C.

A student’sιtest was run to compare total length

between wild-type and albino individuals. Survival was

analyzed with the Kaplan-Meyer method and tested

for significance by the log-rank test. All the analyses

were run by JMP (Ver. 5.0lJ) statistical software (SAS

Institute, Cary NC, USA), and stastical differences

were declared at Pく 0.05.

In albino fish, the initial mortality was confirmed at

7 hours after the exposure and ended up 100% mortal-

ity at 17 hours after the exposure (Fig. 1). In contrast,

all individuals survived until the end of the experiment

in the control. Survival was significantly lower in the

UV treatment than in the control. In wild-type fish, 66%

of individuals survived until the end of the experiment in

the UV仕eatment,whereas no mortality was observed

in the control. There was no significant difference in

survival between UV甘ea加ientand con仕ol.Survival

was significantly different between wild-type and albino

in UV treatment but not in control.

Albino individuals showed significantly lower tol-

erance to UV-B radiation compared to wild-type

100

80

60

40

－－、 20 主ト門

(a) n=6

、、＿，

~ と 100 コυ3 80

60

40 (b) n=6

20

。0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h)

Fig. 1. Survival of wild type (solid circles) and albino (open cir-cles) juveniles of Japanese flounder, Paralichthys olivaceus. (a): UV exposure, (b): control.

UV tolerance of wild and albino Japanese flounder 151

individuals. Because仕iegenetic characteristic of albino

fish are expected to be similar to that of wild-type indi-

viduals of this population (Shikano et al. 2007), the

obserbed lower UV-B tolerance in albino宣shis at仕ib-

uted to the lack of melanophores. Thus, present results

indicate that melanophores have an ultraviolet photo-

protective function in Japanse flounder juveniles. Since

Japanse flounder juveniles inhabit in shallow sandy

areas, it is considered that they adapt to UV-B radiation

partly by developing a large number of melanophores

on血eocular side. Black sea be創nAcanthopagrus

schlegelii juveniles inhabiting in the surf zone were

found to have a higher UV tolerance and higher

number of melanophores compared to出atof red sea

bream which live in仕iedeeper areasσukunishi et

al. 2013). Harada et al. (2001), analyzing the distribu-

tion pa仕ernsof melanophores in early life stages of

Gymnogobius gobies, reported出atpelagic species

inhabiting close to the shore line tended to have large

melanophores, continuously 企om head to caudal

peduncle along the mid-dorsal line. Furthermore dis-

persion of melanophores has been observed in pelagic

larvae of northern anchovy Engraulis mordax and chub

mackerel Scomber japonicus after the UV exposure

但unteret al. 1979）.τberefore, melanophores would

play an important role to protect from UV radiation in

teleost fishes at early life stage living in shallow areas of

the ocean.

In this study, we did not investigate the causes of

death in albino fish. UV-B radiation has damaging

effects on DNA, skins, eyes and the immune systems

of fish (Zagarese and Williamson 2001; I okinen et al. 2008）.τberefore, UV-B induced mortality observed in

血isstudy may be due to the combination of仕iesemul-

tiple causes.

Contrary to our study, no significant difference was

found in UV tolerance between wild-type and albino

fish for medaka (Fabacher et al. 1999）.百ieseauthors

concluded出atit was because bo白 wild-typeand

albino fish had similar amounts of photoprotective

non-melanin substances in the outer dorsal skin layers.

Armstrong et al. (2002) reported that the wild-type

medaka exhibited higher UV-B induced DNA damage

and more epidermal necrosis白血 melanophore-lack-

ing conspecifics, although仕iecause of this difference

was unclear. In medaka, melanophores play an impor開

tant role in body color adaptation to the surrounding

environment (Sugimoto 2002). Furthermore，仕ieirtyp-

ical habitat has shades of vegetations.τbus they are

less likely to be exposed to direct UV-B radiation than

宣shesliving in open ocean.

In conclusion, we demonstrated白atmelanophores

have an ul仕avioletscreening function in Japanese

flounder. This study however does not exclude the pos・

sibility of other functions of melanophores in Japanese

flounder, such as cryptic coloration to avoid detection

by predators. As the balance of multiple functions

seems to be different depending on the species, further

study is required to elucidate whether melanophores

have the photoprotective role in marine fish inhabiting

deep areas without UV radiation.

Acknowledgemen白

We thank the staff at the Miyazu Center, National

Center for Stock Enhancement, Fisheries Research

Agency of Japan, for kindly providing仕iefertilized

eggs of Japanese flounder. All the experiments were

performed according to the guidelines of Regulation on

Animal Experimentation at Kyoto University.

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