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Metamorphosis Inhibition: An Alternative Rearing Protocol for the Newt,Cynops pyrrhogasterAuthor(s): Chikafumi Chiba, Shouta Yamada, Hibiki Tanaka, Maiko Inae-Chiba, Tomoya Miura,Martin Miguel Casco-Robles, Taro Yoshikawa, Wataru Inami, Aki Mizuno, MD. Rafiqul Islam,Wenje Han, Hirofumi Yasumuro, Mikiko Matsumoto and Miyako TakayanagiSource: Zoological Science, 29(5):293-298. 2012.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zsj.29.293URL: http://www.bioone.org/doi/full/10.2108/zsj.29.293

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2012 Zoological Society of JapanZOOLOGICAL SCIENCE 29: 293–298 (2012)

Metamorphosis Inhibition: An Alternative Rearing Protocol

for the Newt, Cynops pyrrhogaster

Chikafumi Chiba1*, Shouta Yamada2, Hibiki Tanaka2, Maiko Inae-Chiba1,

Tomoya Miura2, Martin Miguel Casco-Robles2, Taro Yoshikawa2,

Wataru Inami2, Aki Mizuno2, MD. Rafiqul Islam2, Wenje Han2,

Hirofumi Yasumuro2, Mikiko Matsumoto2,

and Miyako Takayanagi2

1Faculty of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1,

Tsukuba, Ibaraki 305-8572, Japan2Graduate School of Life and Environmental Sciences, University of Tsukuba,

Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan

The newt is an indispensable model animal, of particular utility for regeneration studies. Recently,

a high-throughput transgenic protocol was established for the Japanese common newt, Cynops pyrrhogaster. For studies of regeneration, metamorphosed animals may be favorable; however, for

this species, there is no efficient protocol for maintaining juveniles after metamorphosis in the lab-

oratory. In these animals, survival drops drastically after metamorphosis as their foraging behav-

iour changes to adapt to a terrestrial habitat, making feeding in the laboratory with live or moving

foods more difficult. To elevate the efficiency of laboratory rearing of this species, we examined

metamorphosis inhibition (MI) protocols to bypass the period (four months to two years after hatch-

ing) in which the animal feeds exclusively on moving foods. We found that ~30% of animals sur-

vived after 2-year MI, and that the survivors continuously grew, only with static food while

maintaining their larval form and foraging behaviour in 0.02% thiourea (TU) aqueous solution, then

metamorphosed when returned to a standard rearing solution even after 2-year-MI. The morphology

and foraging behavior (feeding on static foods in water) of these metamorphosed newts resembled

that of normally developed adult newts. Furthermore, they were able to fully regenerate amputated

limbs, suggesting regenerative capacity is preserved in these animals. Thus, controlling metamor-

phosis with TU allows newts to be reared with the same static food under aqueous conditions, pro-

viding an alternative rearing protocol that offers the advantage of bypassing the critical period and

obtaining animals that have grown sufficiently for use in regeneration studies.

Key words: newt, rearing, metamorphosis, thiourea, regeneration

INTRODUCTION

The newt has been recognized as a useful model animal

for over two centuries and across a broad spectrum of life

science fields (Bonnet, 1781; Collucci, 1891; Eguchi et al.,

2011; Eto et al., 2009; Inoue and Nakatani, 2010; Kikuyama

et al., 1995; Kurahashi, 1990; Mitashov, 1996; Okada, 1991;

Spemann and Mangold, 1924; Taguchi et al., 1989; Takano

et al., 2011; Watanabe et al., 2010; Wolff, 1895; Yoshikawa

et al., 2012). This animal is particularly valuable for studies

of regeneration due to its remarkable regenerative ability;

newts can regenerate various body parts, including eye tis-

sues such as the retina and lens, parts of the brain, heart

and jaws, limbs and the tail. This regeneration is mediated

by dedifferentiation or transdifferentiation of somatic cells at

the site of injury, making this process relevant to a number of

important issues, such as the involvement of stem cells in

wound healing, reprogramming, differentiation, patterning, and

the restoration of physiological function (Brockes and Kumar,

2002; Chiba and Mitashov, 2007; Singh et al., 2010; Stocum

and Cameron, 2011; Tsonis and Del Rio-Tsonis, 2004).

Recently, to address these issues, we established a high-

throughput transgenic protocol for the Japanese common

newt, Cynops pyrrhogaster (Casco-Robles et al., 2010, 2011).

Metamorphosed or adult animals may be ideal for the

study of regeneration. However, for C. pyrrhogaster there is

no efficient protocol to maintain metamorphosed juveniles in

the laboratory. Swimming larvae of this species can be read-

ily reared by feeding them live brine shrimp larvae and fro-

zen mosquito (Chironomidae) larvae, called akamushi in

Japanese (Kyorin Co., Ltd; Casco-Robles et al., 2011).

However, once they move to land after metamorphosis (4–

5 months after hatching), rearing becomes harder as their

foraging behavior drastically changes; they prefer moving

* Corresponding author. Tel. : +81-29-853-4667;

Fax : +81-29-853-6614;

E-mail: chichiba@biol.tsukuba.ac.jp

Supplemental material for this article is available online.

doi:10.2108/zsj.29.293

C. Chiba et al.294

foods on land or in midair, while losing interest in static

foods. Moving foods, such as live cricket larvae or small

earthworms, therefore need to be prepared, or alternatively,

dead foods such as akamushi or a small piece of meat must

be dangled in front of their eyes, one by one. Such time-

consuming feeding is often responsible for a drop in survival

(or loss of entire populations), limiting the number of animals

that can be kept in the laboratory. Once the animals have

grown to ~5 cm (1–2 years after hatching), rearing becomes

easier as they are once again able to eat static foods in the

aquatic habitat; thereafter, they can be fed only with

akamushi beyond the sexually matured age (it takes at least

3 years) for their remaining lifetime (the average life span is

estimated as > 15 years). Thus, if a way could be found to

bypass the critical period (four months to two years after

hatching) in which the animal exclusively eats moving foods,

rearing efficiencies might be improved dramatically, allowing

researchers to maintain larger numbers of transgenic ani-

mals in the laboratory.

Amphibian metamorphosis has been thought to be a

change of both behavior and body system to adapt to a

terrestrial habitat (Brown and Cai, 2007). If swimming newt

larvae are reared under a condition of metamorphosis inhi-

bition (MI), they may thus retain their foraging behavior as

well as their larval form, allowing them to grow while being

fed the same static foods in aqueous conditions. Interest-

ingly, in urodele amphibians, neoteny has been reported, as

in axolotl (Ambystoma mexicanum), in every family (Matsui,

1996; Wakahara, 1996). In addition, it is known that when

the thyroid hormone in swimming larvae is artificially inhib-

ited by rearing them in an aqueous solution containing goni-

trogens such as thiourea (TU) or sodium perchlorate (SPC),

they do not metamorphose, but continue to grow while

retaining their larval form (Wakahara, 1994). Therefore, in

the current study, we examined whether MI can be an effec-

tive means to bypass the critical period, allowing us to

obtain adult newts to conduct a regeneration study.

MATERIALS AND METHODS

Animals

Adult C. pyrrhogaster newts were purchased from Mr. Kazuo

Ohuchi (Misato, Saitama, Japan) and Tsuchiura Kanshougyo

ADOKAN (Tsuchiura, Ibaraki, Japan), and housed in polyethylene

containers with water at 18°C under natural light before use. Fertil-

ized eggs were obtained from female newts (~12 cm, total body

length) as described previously (Casco-Robles et al., 2010, 2011).

Embryonic–larval development and limb regeneration stages were

determined according to Okada and Ichikawa (1947) and Brockes

and Kumar (2002), respectively. The original research reported

herein was performed under the guidelines established by the

University of Tsukuba animal use and care committee.

Rearing

Diluted (0.1x) Holtfreter’s solution was used as the standard

rearing (SR) solution (Casco-Robles et al., 2011). For MI, 0.002–

0.02% (w/v) TU (208-01205, Wako, Osaka, Japan) or 0.0002–

0.04% (w/v) SPC (MKBB3051, Sigma-Aldrich, St. Louis, MO63103,

USA) was dissolved in SR solution.

Fertilized eggs were collected into plastic cups or containers,

labeled with the egg-collection date, filled with SR solution and kept

in an air incubator (M-200, TAITEC, Saitama, Japan) at 22°C [light-

dark (LD) cycle = 12 h:12 h]. At ~3 weeks after egg-collection, lar-

vae (stage 42) were hatched (Fig. 1A). They were transferred into

6-well plates (Falcon 5302, Becton Dickinson, NJ07417, USA) filled

with SR solution (~2 ml) at one larva per well. From stages 45–47,

larvae (10–11 cm, total body length) were fed with live brine shrimp

larvae (A&A Marine LLC, Salt Lake City, Utah, USA) twice a week

and the rearing solution was kept clean as described previously

(Casco-Robles et al., 2011). When the larvae grew up to 2.5 cm,

they were transferred into plastic cups (bottom diameter: 6 cm; lid

diameter: 8 cm; height: 4 cm) filled with SR solution (~50 ml) at one

larva per cup. The cups were gathered into a plastic container and

placed together in a rearing room (18–24°C; LD cycle = 12 h:12 h;

see Supplementary Fig. S1). When the larvae grew larger than

2.5 cm, the food was shifted to Akamushi (Kyorin Co., Ltd, Hyogo,

Japan). At three months after hatching, most larvae had grown up

to ~3 cm (Fig. 1B). For MI, at this time point, the rearing solution

was changed to a gonitrogen-containing 0.1x Holtfreter’s solution

(Fig. 1). Thereafter, the animals were reared as before with

akamushi under the MI condition for two years. During this period,

depending on the size of animals, larger rearing cups were used.

After two years, they were transferred into a small aquarium tank

(width 15 cm × depth 8 cm × height 9 cm), in which SR solution was

filled up to ~2 cm and a piece of sponge was placed therein, serving

as land, at one newt per tank and allowed to metamorphose in the

incubator (M-200, TAITEC; 22°C; LD cycle = 12 h:12 h). During and

after metamorphosis, the animals were fed with akamushi only, as

done for the normal adult newts (see Results).

Surgery

Limb regeneration was tested to evaluate the regenerative abil-

ity of newts which had undergone metamorphosis following 2-year

MI; a forelimb of the animal was amputated between the wrist and

the elbow after anesthetized in 0.1% FA100 (4-allyl-2-methoxyphenol;

Tanabe, Osaka, Japan) for 2 h; the operated animals were placed

in a moist container (width 20 cm × depth 15 cm × height 6 cm; a

paper towel lightly wet with distilled water was placed on the bot-

tom) and allowed to recover in the same incubator. After the wound

had healed (~10 days after surgery), the animals were returned into

the original aquarium tank and reared as before in the incubator.

RESULTS AND DISCUSSION

Thiourea inhibits metamorphosis of C. pyrrhogasternewt

Firstly, we examined the optimal concentration of TU for

MI of C. pyrrhogaster newt (Table 1); Swimming larvae

(2.5–3.5 cm, total body length) were transferred into TU-

containing 0.1x Holtfreter’s solution at three months after

hatching. Survival and metamorphosis in the subsequent

three months, in which the animals normally metamorphose

when reared in SR solution (Fig. 1A–D), were examined. In

fact, in SR solution (i.e., 0% TU solution), most survivors

(95%) completed metamorphosis in this period (Table 1). TU

at concentrations up to 0.01% was unlikely to have affected

metamorphosis whereas higher concentrations of TU

resulted in a lower survival rate (90% at 0.002% TU; 60% at

0.01% TU). However, intriguingly, in 0.02% TU solution the

survival rate was dramatically improved (to 90% in this case)

and no survivors showed any indication of metamorphosis

for longer than three months (see below). In parallel with this

experiment, we tested another gonitrogen SPC at concen-

trations up to 0.04%, which was used for MI of a salamander

Hynobius retardatus (Wakahara, 1994); however, this dose

was unlikely to affect either survival or metamorphosis of C.

pyrrhogaster newt (data not shown); this may be due to a

species difference in the sensitivity to this chemical. There-

fore, we decided to use 0.02% TU solution in the following

MI Protocol for Rearing Newt 295

newt MI experiments.

Newts can grow under MI condi-

tion

We attempted a long-term rear-

ing of newts under MI condition

using two batches of fertilized eggs:

Group-1 (1010 eggs) was obtained

on 8–23 July, 2008 from 32 females

that had been caught in Chiba pre-

fecture in the previous mating sea-

son (November to December in

2007); Group-2 (273 eggs) was

obtained on 2–20 February, 2009

from four females that had been

caught in Fukushima prefecture in

November to December of 2008.

The number of hatched larvae was

589 (58.3% of fertilized eggs) in

Group-1 and 252 (92.3%) in Group-

2. Three months after hatching,

swimming larvae (2.5–3.5 cm in

Group-1; 3.0–3.5 cm in Group-2)

were transferred into 0.02% TU

solution (Fig. 1B). Under this MI

condition, the animals kept their lar-

val form – as characterized by the

gills and the dorsal tailfin – for 2

years (Fig. 1E, F).

The survival rate and total body

length of animals were monitored

for ~2 years after hatching (Fig. 2).

In both groups, the number of survi-

vors gradually decreased with rear-

ing time (Fig. 2A): in Group-1, 154 (26.1%) at 382 days, 77

(13.1%) at 720 days; in Group-2, 88 (34.9%) at 344 days,

75 (29.8%) at 759 days. On the other hand, total body length

increased significantly (Fig. 2B, C): in Group-1, 3.5 ± 0.1 cm

(mean ± SEM; range: 2.0–5.5 cm) at 382 days, 5.8 ± 0.1 cm

(range: 3.5–8.5 cm) at 720 days, P < 0.00001 (Mann-

Fig. 1. Growth of newts under different rearing conditions. (A–D) Standard condition. Animals were reared in 0.1x Holtfreter’s solution from

the one-cell stage embryo (fertilized egg) to the juvenile stage beyond metamorphosis. Photographs show swimming larva immediately after

hatching (~3 weeks; A), with ~3 cm in total body length (three months; B), immediately before metamorphosis (4–5 months; C), and a juvenile

after metamorphosis (six months; D). (E, F) MI condition. Swimming larvae (2.5–3.5 cm, total body length) which had been reared for 3 months

under the standard rearing condition (see B) were transferred into 0.1x Holtfreter’s solution containing 0.02% TU to inhibit their metamorphosis.

Under this condition, the animals lived underwater while retaining their larval form beyond two years. Photographs show the larval form (char-

acterized by the gills and the dorsal tailfin) of 1-year-old (E) and 2-years-old (F) newts. Arrow: the gill. Arrowhead: the dorsal tailfin. Scale bar:

1 cm.

Fig. 2. Survival and growth of newts under MI conditions. (A) Changes in the survival rate (%)

after hatching (defined as day-0). The values were calculated against the number of hatched lar-

vae. Data from two experimental groups are shown in different symbols. Dotted lines indicate

365 days and 730 days after hatching. (B, C) Changes in the total body length. In both Group-1

(B) and Group-2 (C), the frequency distribution of the total body length in 1 year-old newts had

shifted obviously toward larger values in 2-year-old newts; the mean value of total body length

(Group-1, 57.8 ± 1.5 mm, n = 77; Group-2, 54.7 ± 0.8 mm, n = 75) in 2-year-old newts was signif-

icantly larger than that that (Group-1, 34.9 ± 0.7 mm, n = 154; Group-2, 42.8 ± 0.4 mm, n = 88) of

1-year-old newts (in both groups, P < 0.0001 in Mann-Whitney’s U-test).

C. Chiba et al.296

Whitney’s U test); in Group-2, 4.3 ± 0.0 cm (range: 3.1–5.0

cm) at 344 days, 5.5 ± 0.1 cm (range: 3.0–7.0 cm) at 759

days, P < 0.00001 (Mann-Whitney’s U test). These results

demonstrate that under this MI condition, C. pyrrhogaster

newt can grow while maintaining their larval form for at least

two years.

In the current study, hatching and survival rates of ani-

mals were evidently higher in Group-2. In addition, growth of

animals was also better in this batch: at ~1 year after hatch-

ing, the total body length of larvae in Group-2 was signifi-

cantly larger (P < 0.00001, Mann-Whitney’s U test) than that

in Group-1, although the statistical difference became insig-

nificant during the succeeding 1-

year rearing. Furthermore, morpho-

logical abnormalities, such as a

curved spine or missing toes, were

less frequent in Group-2 (16% of

survivors at 759 days) than in

Group-1 (32.5% of survivors at 720

days). Such differences between

batches may be attributed, at least

in part, to the difference in the egg-

collection season (Group-1, July;

Group-2, February). In this species it

is known empirically that the quality of

fertilized eggs declines in summer

(July-September) as evaluated by

early embryonic development (also

see Casco-Robles et al., 2010, 2011).

Larval form newts are capable of

metamorphosis even after 2-year-

MI

Subsequently, we examined

whether the newts that underwent

MI for two years retained the ability

to metamorphose; 2-years-old larval

form newts with no apparent mor-

phological abnormalities in Group-1

(52 in total) were transferred into SR

solution, and allowed to metamor-

phose at 22°C (Figs. 3, 4). The first

indication of metamorphosis was

evident in their behavior in week-1;

specifically, the animals started to

refuse food. At ~10 days, the first

ecdysis occurred, and then the

smooth and pale-brown larval skin

gradually changed to a rugged and

dark-brown one (Fig. 4A). At weeks

2–3, gill- and tailfin-resorption

became recognizable (Figs. 3C, 4A,

C), and some animals started to rest

on land despite still having gills (Fig.

3B). At weeks 5–6, gill- and tailfin-

resorption was complete (Fig. 4A,

C), and nearly all the animals (90%)

had transferred to land. These ani-

mals spent most of their time on

land, but did not show curiosity,

even to moving foods in midair.

Table 1. Dose-dependent inhibition of metamorphosis by thiourea

(TU).

TU

concentration

% (w/v)

TotalNo

metamorphosisMetamorphosis Dead

0 19 (100%) 0 (0%) 18 (95%) 1 (5%)

0.002 10 (100%) 0 (0%) 9 (90%) 1 (10%)

0.01 10 (100%) 0 (0%) 6 (60%) 4 (40%)

0.02 10 (100%) 9 (90%) 0 (0%) 1 (10%)

Fertilized eggs were collected from one female newt in February.

Fig. 3. Metamorphosis induction after 2-year-MI. (A1, 2) A 2-year-old larval form newt before

metamorphosis induction. See Supplementary File S1 for living animals. (B1, 2) 17 days after

metamorphosis induction. A larval form newt still retaining its gills (white arrowhead) started to go

and rest on land (a piece of sponge). (C1–4) Dorsal views of a newt during metamorphosis.

Arrowhead: gill. Scale bars: 1 cm.

MI Protocol for Rearing Newt 297

Within 1–2 weeks after moving to land, the animals started

to move back and forth between land and water, and again

to eat akamushi placed statically in water. Within 11–12

weeks after the induction of metamorphosis, the color pat-

tern of the belly skin became vivid and sharp (Fig. 4B). By

this stage, all the animals had moved to land and resembled

normal adult newts both in morphol-

ogy and behavior.

Newts that underwent metamor-

phosis following 2-year-MI retained

their regeneration capacity

To evaluate whether the combi-

nation of 2-year-MI and following

metamorphosis induction affects the

regenerative ability of newt, we

examined limb regeneration of the

metamorphosed newts that had

undergone 2-year-MI. When the

forelimb was amputated at a region

between the elbow and the wrist, it

regenerated completely in ~3

months, as in normal limb regenera-

tion (Fig. 5); the wound of the ampu-

tated limb closed within a week

(Wound healing stage; Fig. 5A, B);

at ~2 weeks after amputation, a bud

(blastema) appeared on the end of

the limb (De-differentiation stage;

Fig. 5C, C’); it grew gradually and at

~1 month after amputation it

reached a medium size (Medium

bud stage; Fig. 5D, D’); in the sev-

eral succeeding days, the bud

became flat (Palette stage; Fig. 5E,

E’); at ~45 days after amputation,

four toes emerged (Early differentia-

tion stage; Fig. 5F, F’); at ~70 days

after amputation, the limb had

Fig. 4. Morphological changes of 2-year-old larval form newts after metamorphosis induction.

Identical animals were traced for a series of photographs. (A1–5) Head region. The pale-brown

skin changed gradually to a rugged and dark-brown skin. Gill-resorption was recognized from ~2

weeks (A1) and completed around ~6 weeks (A5) in this case (arrows). (B1–5) Belly. The black

and orange mosaic pattern of the skin color gradually became vivid and sharp. It took ~11 weeks

(B5) in this case. (C1–4) Tail. Resorption of the dorsal tailfin was recognized from ~2 weeks (C2)

and completed around ~6 weeks (C4) in this case (arrowheads). Scale bars: 1 cm.

Fig. 5. Limb regeneration of a metamorphosed newt which had undergone 2-year-MI. (A–G) Dorsal views of a regenerating limb. A forelimb

was amputated between the elbow and the wrist (arrow). The number indicates the day after amputation. (C’–G’) Magnified images of the

regenerating limb shown in the panel C–G. Scale bar: 50 mm (A–G); 12.5 mm (C’–G’).

C. Chiba et al.298

almost regenerated (Fig. 5G, G’). Such a time course of limb

regeneration was in the range of that observed in normally

developed adult newts (caught in the field) with the same

total body length (data not shown), suggesting that the

regenerative ability of the newt was preserved even after

metamorphosis following 2-year-MI.

Conclusions and perspectives

We demonstrated that controlling metamorphosis with

TU allows us to rear C. pyrrhogaster newt with the same

static food under aqueous conditions for at least two years,

providing an alternative rearing protocol that makes it possi-

ble to bypass the critical period in which the survival of the

animals in the laboratory ordinarily drops significantly. In the

current study, we used akamushi (mosquito larvae) as a

static food. However various foods, for example those com-

mercially available for the feeding of Xenopus larvae or axo-

lotl, should be able to substitute for akamushi. Therefore,

this protocol could make storage of transgenic newts in the

laboratory easier, allowing laboratories with no special facili-

ties for keeping live foods to initiate studies with transgenic

newts. We have successfully screened, in the current trial, a

line of newts that are tolerant to long-term rearing under MI

condition. We continue to rear the metamorphosed juvenile

newts as a founder (F0) of the next generation (F1). Hopefully

this line of newts will serve as laboratory newts for transgen-

esis that have higher survival rate under the current rearing

condition. We want to obtain fertilized eggs for F1 in the future

by allowing F0 to mate under a semi-natural conditions, as

described in our previous study (Casco-Robles, 2010).

ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid for Challenging

Exploratory Research (20650060) and a Grant-in-Aid for Scientific

Research (B) (21300150) from the Japan Society for the Promotion

of Science (JSPS), a Grant-in-Aid for Scientific Research on Inno-

vative Areas (23124502) from the Ministry of Education, Culture,

Sports, Science and Technology (MEXT).

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(Received December 2, 2011 / Accepted January 4, 2012)