The Developmental Stages of Paramesotriton hongkongensis

(Myers Leviton) and Its Application in The Study of

Embryology

Thesis submitted as partial fulfilment. for

the degree of Master of Philosophy

Tong Tat-mining

June, 1976

Division of Biology

Graduate School

The Chinese University of Hong Kong

Thesis Committee:

Prof. M.C. Niu

Prof. L.B. Thrower

Dr. K. W. Chiu

Dr. Y. C. Kong 1 CONTENTS

Page

CONTENT OF TABLES AND FIGURES iii iv

GENERAL INTRODUCTON 1

1. CLASSIFICATION AND KATUTAL HABITAT OF

Parmesotriton hopoklongensis

1.1 Classification and Nomenclature 7

1.2 Natural habitat 3

1.3 general behavioor 11

2.EXTERNAL FEATURES OF THE ADULT

2.1 Morphology of the adult newt

in general 17

2.2 Sexual dimorphism 20

3.REARING OF Paminesotriton hongkongensis

IN LABORATORY

3.1 Maintainance of adult newt in the

23 laboratory

3.2 Development of Prauresotriton

hongkongensis urnler laboratory

Conditions 28

4.THE DEVELOPMENTAL STAGE OF Paramesotriton

hongkongensis 2

4.1 Materials and Metbod 35

39 4.2 Results

4.3 Discussion 69

5. TUE EFFECT OF WATER MOULD ON THE

DEVELOPMENT OF Parmesotriton hongkonggensis

5.1 Materials and Method 85

5.2 Results and Discussion 86

6. THE APPLICATION OF Paramesotriton

hongkongensis IN EMBRYOLOGICAL STUDIES

6.1 Lampbrush chromosomes from the germinal

vesicle of the ovarian egg

90 6.1.1 Removal of germinal vesicle

6.1.2 Fgamination of the larpbrush

chromosomes 93

6.2 Hang drop culture of isolated

analagen

6.2. Materials 97

6.2.2 Method 98

SUMMARY 102

APPENDIX 105

ACKNOWLEDGMENTS 107

REFERENCES 108 3

Content of Tables and Figures

Page

Table 1: Description of the developmental

stages of Paramesotriton hongkong-

41-54 ensis

Table 2: Comparison among the developmental

stages of Paramesotriton hongkong-

ensis, Amblystoma mexicanum and

Triturus pyrrhogaster 65-68

Fig 1: Behaviour of the female newt at the

time of egg-laying 14

Fig 2: Arrangement of eggs on leaf 14

Fig 3: A newt with the right forelimb

bitten off 16

Fig 4a: Dorsal view of the adult newt 18

Fig 4b: Ventral view of the adult newt 18

Fig 4c: Lateral view of the adult newt 19

Fig 5: Dorsal skin 19

Fig 6: Sexual dimorphism 22 iv 4

Fig 7: A newt infected by water mould 26

Fig 8: Materials for examining the

3 developmental stages 6

Fig 9: Developmental stages of

Paramesotriton hongkongensis 55-63

Fig 10: Growth curve 64

Fig 11a: Effect of water mould on the

hatching time of Parames

hongkongensis embryos 8 7

Fig 11b: Effect of water mould on the

mortality of Paramesotriton

hongkongensis larvae 88

Fig 12: Materials and results of the

experiment on germinal vesicle 96

Fig 13: Materials and some results of the

hnanging drop culture technique 101 1 GENERAL INTRODUCTION

Amphibians have been used for a long time

as suitable material for the study of physiology,

endocrinology, anatomy and embryology. Both anurans

and urodeles are very useful, but biologists have

paid more attention to the use of anurans. The

reasons are the larger size and the higher sensiti--

t i.ty of anurans to the administration of gonadotropie

hormones. The urodeles are better for embryological

studies, both descriptive and experimental, because

they have large-sized eggs and a slower developmental

rate. The egg diameter of urodeles is often larger

than 2 mm whilst that of anurans is less than 1.5 mm.

The large sized eggs permit one to observe more

clearly the morphological changes that take place

during development. It is also easier to operate on

urodele eggs than on anuran eggs because of their

greater integrity. Many studies of experimental 2 smbryology are carried out in urodele eggs. Anuran

egge are, however, more frequently used in biochem-

ical analysis by virtue of a large n ocr of eggs

in each batch.

To-day, the most common amphibians uoed

in the laboratory, especially in the wester ccuntries,

are Xenopus laevis, Rana pipiens,R.pipiens, R.sylvatica, Bufe

amoricanue, Amblyatoma opacum,A,punctatum,A,tigrinum,

A.mexicanum, Triturus viridescens, T. granulosa.

T.pyerhogaster, T.cristatus, T. helvetcus,Taricha

toroa (Hamburger 1960 Rugh 1962 New 1966).

Xen us is no doubt an excellent embryological material.

because its breeding season is long, its sensitivity

to hormonal treatment is high and it can be sucess-

fully reared under laboratory condition, reaching

sexual maturity within two years. Maturation of eggs

is a continuous process in this species. The

limitations in the use of XeNnopus is that the eggs

are small making operation a difficult manoeuvre Furthermore, its fast developmental rate does not 3 give enough time for careful morphological observation.

By the use of urodele eggs (e.g. eggs of Triturus

pyrrhogastar), the embryologist can overcome these

limitations.

Keeping amphibians under laboratory

conditions is not easy but is nevertheless a feasible

underinking. Except the purely aquatic and the purely

tarrestrial forms, amphibians are difficult of main-

tain in a strict ecological sense. Comparatively

spaking, urodeles (especially the newts) are more

stavle than anurans when kept under laboratory codition

(Hamburger 1960; Short & Woodnott 1969).

In Hong Kong, urodeles are very rate. There

is only one local newt, Paramesotrition hongkongensis

(Lai & Ng 1972), which has proved to be very useful

in embryological studies. Yenopus laevis, which is

new becoming an international laboratory animal can 4

be reared successfully in Hong Kong. In the past

three years, a summer course for the rearing of

Xenopus under laboratory condition was sponsored by

the Biology Department of Chinese University of Hong

Kong. As a result, more than thirty secondary

schools are now maintaining this toad in their labor-

atories. If we know how to breed the local newt,

then we would have two excellent embryological materials

at hand, Conseqaontly, local embryologists would be

much better off in terms of experimental materials.

In comparison with amphibians, the use of

other groups of animals in embryology is less satis-

factory. As Brachet stated, "Very little is known

about the reptiles in the field of molecular biology.

Considerable wor has been done on chicken embryos,

but at late stages of development. The partial

cleavage and the small size of the embryos, as compared

to the huge mass of the yolk, makes the material a

difficult one for molecular embryologists interested 5

in early developm nt w The maminalian embryos, a

already mentioned, must be cultivated in vitro. sc

that only very limited amounts of material ara

available for biochemical studies" (Br ehet 1974)

In Hong Kong, teach-ling of experimental

onbryology still remains within the confined of the

university curriculum. There has ben no previous

attempt to bring this su h ject to the secondary

schools. Shortage of suitable experimental iraatarials

is aur ely one of the reasons. Nowadays theme is a

new trend in providin Eore basic] nowledge about

human development. This buowledge should ba provided

to the, Judents in eecondary schools as the basis for

a correct rderst din of sexuality. A correct con-

cept of sexuality in its biological contexc prepares

these young people for a positive attituea to fertilit

control in their adulthood. Since the davalopmantal

Patterns are sit i ar oug the vertebrate growpa

amphibian eggs are excellent materiale to demonstrate 6 the proce of hveiop nt w Uch evolves along the

same course as In human subjects

The purpose of the present investigation is

to provide a good local embryological is nrial,

Para esotriton lion- kon, ensig, that even students in

secondary schools can ha.,dle. It is also hoped to

introduce this local ne to the scientific community,

particularly the erbryologists, who may find it a

co vanient experimental model (Lau & Kong 1971).

Thus, an important objective of this investiation

is to provide a tabulation of developmental stagea

of Poramesotrition hongkongenais. The rearing methods

and some applications of this newt in embryological

experimonts are also persented Chapter 1 7

CLASSIFICATION AND NATURAL HABITAT

OF Paramesotrjton hongkongensis (Myers & Leviton)

1.1 Classification and Nomenclature

The genus, Paramesotriton, includes three

species: P. deloustali Bourret (found in north Viet

Nam),P. hongkongensis Myers & Leviton (hound in Hong

Kong island and vicinity) and P. chiriensis Gray

(found elsewhere in southern China). P. hongkongensis

and P. chinensis are closely related with some minor

differences. P . . hongkongensis has a smooth and less

tuberculated skin, short and less divergent para-

sphenoid teeth, larger red colored spots on the

aLdominal region, much stronger dorsolateral ridres

and a broaaer head and interorbital region (Myers &

Leviton 1962).

P. hongkongensis was first found in 1859

by Gray (Gray 1859) in Hong Kong island. He placed 8 this newt in the genus Cynops and called it Cynops

chinensis. Cy ops chinensis probably includes those

newts from Ningpo and Hong Kong. Later, Chang put

them into the genus Trituroides (Chang 1936). In

1939, the two species were separated by Herre (1939),

The Ningpo species was called Triturus sinensis. At

that time, thy Hong Kong species had not yet an

individual nomenclature. Romer (1951) described the

habits and life-history of the Hong Kong newt. Until

recently, Trituroides hongkongensis was the name used

for the Hong Kong newt (Myers Leviton 1962). But

the Hong Kong newt was found to resemble more closely

Paramesotriton d.eloustal.ii than Triturus (Freytag

Petzold 1961 Frey-tag 1962 Van Tien 1965), it was

therefore renamed as Paramesotriton hopgkongensi.s

The present systematic c Lassification of

Paramesotriton hongkongerlsis is as follows, according

to the classification of Noble (Noble 1954): 9 PHYlUM Chordata

SUBP11YLUt1 Vertebrata.

CLASS Amphibia

ORDER Caudata

SUBORDER Salamandroidea

FAMILY Salamandridae

GENUS Pararne sotri.ton

PEClES P. hongkongensis

1 .2 Natural habitat

The newts prefer living in cool running

streams in t tie hills at 300 metres above sea level

and higher. They are found in a number of localities

both in Hong Kong island and in New Territory

including the eastern slope of Violet Hill, Quarry

Bay, Shaukiwan, Shatin, Wang Thu Shan, Kowloon Peak,

Mount Prker (Boring 1934 Romer 1951) and Tai Mo

Shan (personal. observation). They are abundant in

the shallow water where the current is not strong. 10

When at rest they hide under the stones. The newts

usually become active at night in search of food

This special nocturnal feeding behaviour is utilised

by the poachers to the newts' disadvantage.

The population of paramesotriton hongkongensis

has been much reduced at present for three reasons

(i) Urbanization of Hong Kong

Many hills are levelled in order to

obtain more building sites. As a result, the

natural habitats of thy newts are rapidly.

diminishing.

(ii) A lot of newts have been caught and exported

This exhaustive poaching disturbs the

equilibrium of the newt population such that the

population size is reduced to a critical minimum.

(iii) Pollution by picnickers and others. 11 At present, the only place in Hong Kong where

the newts are still abundant is Ng Tung Shan ( 梧 桐 山 )

at the border where all three detrimental factors do

not apply If the Govern ent authorities cannot

establish a protection area for the remaining newts

in Tai Mo Shan, urbaoization and pollution will cer-

bainly wipe out the last breeding nucleus within 3- 5

Years.

1.3 General. Behaviour

Unlike other mphibians, cool temperature

is more suitatle for these newts. Temperature

probaly affects the activity of anterior pituitary

which in turn controls the activity of the gonads

and thus the periodicity of sexual cycle. There

seems to be an internal rhyth which can control the

Bensitivity of gonads to the secretions of

anterior pituitary (Van Oorcit 1956). The breeding

period is quite long when comparing with that of

most amuran or other urodeles. Eggs s are abundsntly 12

laid between October and January (tile coldest season

in Hong Kong). But in other months e.g. September

and Febuary, breeding also occurs (Rouler 1951).

Ovulation is a continuous la:rocoss when gravid females

are maintained in a cooled aquarium in the laboratory.

Fertilization is internal,Each egg is

probably fertilized by more than one sperm (polyspermy)

(Fankhauser & Moore 1941). After courtship, the

female newt picks up the specmatophcre and inserts it

inio the cloaca (Smith 1944; Noble 1954; Freytag

1963;Salthe 1967). The courtship patterns of

salamanders had teen fully described by S.N Salthe

(1967). The stages of oogenesis are controlled by

hormones and are affected by environmental factors

(e. g. temperature, etc.). But the final ripening of

the eggs is dependent upon the stimalos frow the,

breeding males (Porter 1972). This behaviour ensures

that the matured egg can bo fertilized. Seemingly

a firm surface is required upon which the eggs can be deposited, and aquatic plants are preferred. When 13

laying eggs, the female newt climbs up and presses

the aquatic plant together with its hind legs. (Fig 1).

The eggs are laid. between the two leavk, one after

the other in individual capsule (Romer 1951). (Fig 2).

If no ayuatic plante are present, the eggs will be

laid and attached to stones or other protruding objects.

The number of eggs was found to be less in the abeence

of suitable attaching subjects (i.e.aquatic plants).

Molting especially under unfavorabie con-

litions is common in this newt it is probably con

trolled bt bornones (Adams Richards &Kuder 1930;

Jprgenusen &Larsen 1960&1964; Kaltenbach Clark

1965) The molting frequence is not affected by age.

size or sox of the animal (Taylor & Ewer 1956) but

is rather ofren in the rapidly growing young. Unlike

bemmals, The skin of the newt is shed simultaneeusly

as a whole. The skin first breaks round the mouth

and is then sllped backwards over the trenk and eail. 14

Fig 1. Behaviour of female newt at the time

of egg-laying.

Fig 2. The arrangement of eggs on leaf. 911 ho shed bkins will be eaten by the newiwts themselves

The main purpose of molting is probably to eliminate

t4orr,e unmmutoly-tied material from epideri a1 tissue

(Adolph&(Adolph Collins& Collins 1925 1925

Tie newts have to leave water during some

period of a year. This is r Aated to the rhythmic

drop in pt-ole.ctin level (Grant Grant 1`,58). If

Llie water condition is bad, e. g. fouled, they will

also climb up and rest on the rocks above water level.

The newts will fight for food and during

c curt sh. i p. Therefore, wounded newts are often

observed. lower jaw, limbs, or part of the tail may

by bittern Off( Fig 3). Regeneration is rapid in

this newt, as in other urodeles provided the wounded

area is not infected by fungi or micro-organisms

(Thornton 1968 Vethamany--Globus Liversage 1973

Morton Liversage 1975 .Descrier 1976). 16

Fig 3. A wounded newt with right forelimb bitten off. 17 Chapter 2

EXTERNAL FEATURES OF THE ADULTS

2.1 Morphology of the adult newt in general

Parame otriton hongkongensis is deep-brown

in color with bright orange-red spots on the ventral

body surface. Along the inferior midline of the

basal part of the tail, a continuous or interrupted

orange-red color streak can be found (Fig 4). The

skin of the dorsum is thicker and carries numerous

small tubercles (Fig 5).

The head is more or less triangular in

snape but the end of the snout is not sharp. The

lower jaw is a little bit narrower than the upper.

The well developed neural arches and transverse pro-

cesses form the distinct median and dorsolateral

ridges respectively. There are 4 digits and 5 toes. 18

Fig 4a. Dorsal view of the adult newt. The median

ridge is clearly shown. There are 4 digits

and 5 toes.

Fig 4b. Ventral view of a adult female newt. Not(

the orange spots and the orange streak

along the tail. 19

Fig 4c. Lateral view of a adult female newt. The

ventral skin is more smooth than the dorsal

one.

Fig 5. The dorsal skin of Paramesotriton hongkongensis. 20

The total lenh of the adult newt is very

inconsistent. They range from 120 mm to 150 mm.

Generally speaking, when taken from the same colony,

the mature females are longer than the mature males.

Total Length (mm) Tail Length (mm)

Male 126.86 24.89 63.55 6.35

Female 140.14 4.58 69.41 2.98

From these figures, it is clear that the

length difference among the male newts is very great

Sexual dimorphism

Sexual dimorphism occurs in Paramesotriton

bongkongens (Romer 1951). The main morphological

differences between male and female newts include:

(Fig 6) 21 1) a gap between the cloaca and the beginning of tail

in the male (arrows in Fig 6a;& 6bo)

2) a swollen cloacal flap in the male due Lo more

developed abdominal and cloacal glands (Noble 1954)

(Fig 6az & 6bz)

a dark streak in the cloacal lip of the male due

to the presence of black cloacal papiliae

(Noble 1954) (Fig 6az, 6a3, 6bz & 6b3)

larger cloacal opening in the male

rough transverse lines across the cloacal lip in

the female (Fig 6a3 & 6b3)

Some minor differences, which are not always

distinguishable are sometimes observed as follown:

1) the total body length and tail length in female is

generally greater than that of males

2) the orange-red colored streak along the inferior

midline of the basal part of tail is usually ex-

tended to the end in female but it is interrupted

by brown color spots in male. 22

a1

b1

Ci

a2

b2

a3 b3

Fig 6. Sexual dimorphism in Parames r ton hong-

kongen si s. al, a2 &a3 show male characters.

bi, b2 b3 show female characters, a2 &b2

show the cloacal region of the male &female

respectively. Cl= cloacal lip. Details

see text. 23

Chapter

HEAKING OF Paramesotriton boogkonggensis in LABORATON

With the exception of ome terrestria1

(e. g. Pletodor) or aquatic groups (e. g. Pleurodeles)

of urodeles, rearing of urod.ales in the laboratory is

not cuommon (Short & Woodnott 1969), Most of the

urodeles, including the nawts, shift between an

aquatic and terrtiial mode of life in their adult-

hod,This is the main rsason why rearing of urodales

is difficult when vivarium conbditions are unfavorable,

In order to make better use. cf Parameaotriton

honphongensis in research and teaching, a successful

rearing method must be devised. Thb following

suggestions derived from experience gathjed over a

period of three year.

3.1 Haiptainance of adult newt it the laboratory

Plastic tank or agutria con be used. The 24 density of the newt should be preferably low. Up to

250 animals can be housed in a 5 sq. ft. tank with

Water le-Lei about 1 ft In the tank, provisions

that can support at least 1/6 of the newta should be

made for the newts to leave water when needed.

This is generally in forms of bricks and stone

stacked above water level. Aquatic planate are also

necessary for providing shelter and support for the

attachment of eggs. The top of the tank may ba

covered with wiro-gauige to prevent the newts from a

natural ure to mi.Erate.

Rod worms (Chi rono mus larvae) and raw meat

chunks are convenient sources of food for Partnone,ot.riton

Th.t newts move close to the prey slowly

and quitely, than it suddenly traps the preys in a

rapid leap. When two newts have caught the same worm,

each will get hold of one end and twists in opposite

d.:i. actions until the worm breaki. Before feeding,

the worms or meat must be rinsed with water to reduce

the dirt and blood so as to minimize water fouling. 25

The newts can be fed 2-3 times a week.

The newts live better in cool water (15-18°c).

The water used should be filtered through a charcoal

bed and left at least overnight. Running water is

recommended, failing which, daily exchange with fresh

water is the simplast method to reduce the fungal

infection which prevails in stagnant water.

Death is mostly the result of infection by

the water mould, Saprolegni parasitica (Scott &

O'Bier 1962; Tiffney 1936; Kanouse 1932). The

fungus rarely penetrates the intact skin, but when

the body is wounded, infection is a rapid process

(Scott 1926) (Fig 7). Therefore the infection rate

will be highe) at the time of moulting, and in

wounded areas. 26

Fig7. A newt heavily infected by Saprolegnia

parasitica. The whole dorsal surface of

the newt is covered by the mycelia of

the water mould. 27

Some changes can be observcd indicating

that the nett is not well. These symptoma are

divided arbitrarily into five atagas leading fimally

to death.

Stage 1 The newt floats on water or swims ercitadly.

Is sbeds part or all of its skiff

Stage 2 Some glippery and adhesive mucue is

sacreted.

Stage 3 The tail curls.

Stage 4 The legs are curved with toes approaching

each other.

Stage 5 Thick alippery and sticky mucuu is

secreted.

ln order to save the newt y treatmert must

be applied before stage 3. Very often, exchar with

Il e6h waiver, lowering the water level and treating

the skin of the newt with fungicide will help. The

moot effective fungicdes are mercurochro is and

ialachite green (Thomas & Fost 1972 Detwilter& 28 McKennon 1929; Powell et al 1972). Infected newts

can be dipped in malachite green solution (1:15000)

for 10-30 seconds. The dead newts must be removed

from the rearing tank immediately to prevent the

spreading of fungus.

The mortality is very high when the newts

are tranoposrted and introduced into a new tank. The

exact reason for this high mortality rate is not know.

One posaible explanation is that adaptaction of the

newts to a new environment, including water source,

is a slow process. Therefore, when suddenly intro-

duced into a new place, environmental excitatrion kills

the newts even in the absence of fungal infection.

After the hazardous period, the mortality rata will

be sharply decreased.

3.2 Development of Paramesotriton hhongkongensis under

laboratory condition------

After internal fertilization, the fertilized 29 eggs, which are surrounded by layers of je11y coats,

are shed grid attached to some solid support, e g.

stones or leaves, For a given number of ovulating

females, more eggs are obtained when water temperature

is lower, e. g. around 15 C. When aquatic plants are

present, the eggs are usually shed individually in

a linear array between two leaves. One female can

lay a marimum of 25-30 eggs in a day. There is a

great variation in the number of eggs laid sach day

In cold months, over a hundred eggs can be obtained

daily from a colony of 250-300 newts maintained in

the laboratory

The eggs are separated. daily from the

lssves by mesns of a fine forcep and put into a

container with about 5 cm deep of water.

water should be changed daily sd the con-

tainsr ahould be washed twice a week. Before changing

the water, the development of eggs must be checked.

those eggs which cannot, e. g. unfartilized 30 eggs and eggs affected by fungi, must be removed.

The frequence of spontaneous malformation, e. g.

doable-embryo, is very low. Only two of these have

1yeen observed after examlolong a few thousand eggs.

The nunther of embryos in each container should not

be wore than 15, otherwise the normal growth of the

embrycs will be affected )Rugh 1934; Adolph 1931a;

Lynn & Edelman 1936;Rirhard 1958). In anurana,

growth of tndpoles is inhibited unde, erowding con-

dition by some special-specific pioducts (Akin 1966

Rose 1959, 1960 & 1965: Rose Rost 1961; light

1967), But these product$ have not been found out

in uiode]es.

eneitivity of the ambryo to fnngal infection

soemc to be v'arible at different developmental :stages.

After the emergence of gillE, the embryos are wore

sensitive to fungal infections Gills Seem to be the

primary sites of fungal- infection. 31

Melanophores are normally well clever oped

in late embryso (Twitty 1945 & 1949). If at thi

stage no strong pigmentation occurs, the embryou

cannot develop further and will degenerate evexi

though the gross structures seem apparently normal.

The mouths of the larvae open,at the time

of hatching. But due to the pisaence of largs ancuot

of yolk in newly hatched larvae, no external food

supply is Lecessary. When the yolk is exhausted, the

larvae can he fed with Daphnia. Both over-feeding

and starvation can affect the larval growth(twitt

& DeLanney 1939; McCurdy 1939; Dempster 1933).

The adequate amount of food aepends on the number and

the aga of th larvae. When the length of larvae

reaches 2 cm, they can be and with red worms instead

of Daphois

Basides food and space, temperature and

Light ctai also affect the rate and the pattern of

nornial growth (DuShale & Hutchinson 1941; Buchanan 32

1940 Hutchinson 1939 Atlas 1935 Ryan 194! 1a

l9klb Dawes 1941 Rugh 1935). Excessive chlorine

in water can kill the larvae almost immediately.

The depth of water for rearing larvae is

best maintained at, 14-16 cm level. .1t should be:

reduced to about 10 cm as the larvae reach 35 mm in

body length. At thins time, the efficiency of osmo-

regulation beco es gradually reduced as the larvae

are approaching metamorphosis.

When metamorphosis begins, tie depth of

water should not be more than 3 cm. Provision must

be made for the larvae to leave water at this stage.

this landing behaviour is probably related to the

reduction in prolactin level (Grant Grant 1958)

the metau orphosing larvae will certainly die if they

are forced to remain in water. During metamorphosis.

the larvae will not eat.

After cornification of the skin, the yo mg 33

adult will retun to water and brgin to feed again.

The depth of water should still be maintained low

(about 5 cm) for a short period, These young adults

must not be kept together with the mature adults

otherwise they will be eaten by the latter.

The initiation of metamorphosis is promptad

byextrinsic as well as intrinsic factors Nutrition,

light, over crowding and elevated temperature can

also affect metamorphosis (Puckett 1937; D'Angelo,

Gordon & Charipper 1941; Adolph 1931b; Huxley 1929)

and probably act through the pituitary-thyoid axis

(Etkin 1968)

Like most of the other members of the

Family Salamandrdae (Porter 1972),Paramesotriton

hongkongensis reaches sexual maturity after about

three years. Chapter 4 34

THE DEVELOPMENTAL STAGES OF Paramesotriton hongkongensis

The developmental process is often divided

arbitratity into discrete stages for practical purpose

and is referred to a standardized "stage series" in

descriptive and experimental embryology (Cosner 1960).

In actual practice, one can obtain some insight into

the embryonic development of a species through

observing the changes in different stages.

The developmental stages of Amblystoma

punctatwn (Dempster 1933), ,A, mexicanum (Schrecken-

berg Jacobson 1972), Triturus pyrrhogaster (Anderson

1943 Okada Ichikawa 1947)T helveticus

(Gallien Bidand 1959), T. pestrts (Knight 1938),

T.ridescens (Hitchcock 1939), T. taeniatus

(Glucksohn 1931), ri status (Glucksohn 1931),

Pleurodele s waltii (Gallien Durocher 1957) and

.T.nlcha osa (Daniel 1937 Twitty Bodenstein

1948) had been worked out. Most of them do not 35 cover metamorphosis. The normal table of development

in Paramesotrlton spp. have never been reportted.

The present investigation is to mod out the

developmental stages of the Hong Kong newt, Para-

mesotritma hongkongensis .The evolutionary relation-

ships between the above mentioned salamanders have

been worked out by D .B. Wake and N. Ozeti (W e aS.

Ozeti 1969)

4. '1 Materials and Method.

Materials: (Fig 8)

Biological materials eggs of Paramesotriton

horgkorensis collected in

the laboratory

Technical materials:- Plastic containers (21110x7 cm3)

with about 400 141 of water

wide mouth droppers and forcep

for transfering the eggs

Petri dish 36

Fig 8. Materials used for examining the

developmental stages of

Paramesotriton hongkongensis. dis8ectig microscope with 37

ocular grid

Water used:- ordinary ta water left ovrni t

esthetic:_ LtS 222 (1:3000 solution)

Hater temperature 20° a 0 0 500

Water PH range: 7.0-8.

Method

The ,a were collected from the rearing

tank. The unfertilized and damaged eggs were discarded.

Healthy fertii i zed eggs of the same sta o were g: oucpod

together in 10 per container at 2000. A wide mowtt

dropper was u*. ed for handling. The water was changed

daily to reduce ftnn'al infect ion.

The early stages were xamin e d in half

hour to two hours intsrvaL3 while the later stages 38 (starting from tail bud stage) were examinad twice

daily. Observation was by means of the dissedting

microscop. The egg or larval size was detemined

wit: the aid of an ocular grid. The lsngths of the

late embryos were determined efter removal of the

jelly coat. Swimming larvae were examined under

general anaesthesia by MS-222

The developmental stages were dividod

according to the most prominent and easily identiriable

morphological features, e.g.gill, ear, eye, nose,

prenephric bulge, tail, body pigmentation,mouth,

Iimbs, etc..

Photograpos of he earlier stages wore

taken under dissecting microscope

The distance measured from the tip of snout

to the anus was taken as body length and that from

anus to the tip of tail as tail length. Limb length

was eaf1red from its base to the end of digit or toe. 69

4.2 Results:

There are 54 distinguishable stages in

Haramesotriton hoagkongensis. The name of each

stage is suggested by the most prominent morphological

feature first sppeared at that particular stage. By

psing the following diaguostic features,ws can

identify the main stages more easily especially in

later development.

Diagnobtic feature Stage

Blastopore groove present gastrula stage

Yolk plug present yolk plug stage

Neural fold present neurula stags

Tail bud present tail bud stage

Mslanophores in sight nelanophore stage

Emergence of gill gill emergende

Emergence of forslimt forelimb stage

Emergence of hindlimb hindlimb stage 40

For example, when one obsertes that the

forelimbs have emerged, one can gure that the

embryo has reached the forelimb stuge. But it will

be necessary to combine more than one external

feature before we can find out tne exact developmantal

stage.

The results are presented in Tabls 1. Each

identifiable developmental stage is repseated by a

photograph (Fig 9). Body length and external features

are desecribed. Each value is the mean +- standard

deviation for at ieast 10 specimens.All the agesage

and length cited refer to the beginning of each stage

The growth curve representing a composite

curve cer ved from many individual observations is

also pressated (Fig 10)

comparison among the developmental stages of

paramesotriton hongkongensis, Amblystoma mexicanum

and Triturus Dyrrhogaster is show in Table 2 11 Table I: DESCRIPTION OF THE DEVEL IP ENTAL STAGE OF

0 0.5°C Paramssotriton hongkongensis AT 20°-0.5°C

Stage 1 (One cal.) Age: Ohr Diameter: 2.9 +0.08mm.

The egg is enclosed by four layers of jelly

capsu es. Pigmentation is darker at animal

pole.Thare is often a white spot (indication

of second polar body)at animal pols.

Stage 2a (Fit at clc ava e furrow appears) age:10hr :10hr

Stage 2b (Two cells) Age: 12hr.

The cleavage fu:rrcv¢ extends from pole to polo

Gray crescent can be easly distinuiskd.

Stage 3 (Four cells) Age: l4-hr.

The first and the second furrows are

perpendicular to each other.

Stage 4 (Ei ht cells) Age: l6 1/3r.

The third cleavage furrow, is cotpeta. The

four blastomer°res at animal pole are smaller

than those at vegetal pole. They are usnally

triangular ir- appearance and with size about

0.69--0.82mm 42

Stage 5 (Sirteen cells) Ager 18%hr.

EiEht micomeres are observed frue the azims.

pole. Cuils are usually recta filar in

aliptcarance and with size about 0.66-0.79mm

Stage 6 (Mornla) Age:22hr

C1eavage becomss asynchronous. Atout 10-16

nic ltortere are cbsarved in gnimal views.

Micromre size About 0.5-0.6mm

Stage 7 (Bltstule I) Age: 1d2hr.

More than 20 complete cells are chdervsd at

ammal pole aad are round in appearance

Micromtre size sbout 0.44-0.46mm. The

boundary between cells is well derined

Stage 8 (Blastula II) age: 1d4hr.

Micronere size about 0.12-0.14 and 0.23-0.30mm.

Stage 9 (Blastula III ) Age: 2d.

Cell boundary is indistinct under dissecting

microscope. Animal pole becomes paler due

to repeated division.

Stage TO (Gastrula 1) Age: 3dlhr,,

The animal pole view is aimrlar to that in 43

stage 9, but a stall groove appears at vegetal

pole. Above this groovs liea the doraal lip

Stage 11 (Gastrula II) Age: 3d6hr.

The doraul blastopore groove benda and

becomes crescer shape.

Stagg 12 (Gastrula III) Age: 3dllhr

The lateral lips have formed. Thus the

blastopore groove is ob rvec1 as a semi-

circle. The yeliowiah yolk remaino as a

medisd streak continnous with the open end

of the semi-circle.

Stage 13 (Gastrula IV) Age: 3dl7hr.

The laterl. lips pas the equator and their

rims are appro achis. each other and results

in a horse-shos appearance,

Stage 14a (Yolk plug I) Age: 3d19hr.

Blastopore groove closes ventrally and results

in the formation of large yolk plug, YOHR

plug diameter is about 1.4mm (1/2 of tha

Of egg).

Stage 14b(Yolk plug II) age:3d2thr. 44 The diamter of youlk plug is about 1-0ma

(about ys of that of egg).

Stage 14c (Yolk plug III) Age 4ds

Yolk. plug dianeter is about 0,5mm (about

1/6 of that of egg). Pigmeted field can

bs observed ra4iuting from the yolk plug

Stage 15 (Slit blastopcre Age: 4d19hr..

The yolk plug is reduced to slit-like. Two

pigment-concentrated area (the future neural

fold) emerge from the slit on either side of

the dorsal surface. Median neural groove

is clear but is not completed aiitero-

pc sterioxly

Stage 16 (Neurula l) Age: 5d7hr.

Neural plate is clearly outlined by the

developig neural fold. Neural groove is

cler along the xedian portion. The ante for

binder of neural fold is well define.

Stage 17 (Heurula II) Age: 5d.11hr.

Neural folds are rod-iika and apparent. The

middle portion of th nural plate has

narrowed, The minimal distance becween 45

the meural folds is about 10.mm

Stage 18 (Neurula III) Age:5d14hr.

The minimal distance betwssn the neural folds

os about 0.5mm.

Stage 19 (Neurula IV) Age:5d17hr.

Neural folds touch each other at the future

hind-brain level.

Stage 20 (Nsurula V) Age:5d17hr.

Half of the total length of neural folds is

apposed togethar.

Stage 21 (Neurula VI) Age:6d2hr;Body length:3.2-0.08mm;

Height:2.7-0.08mm.

Neural folds touch each other but without

fusion. Anterior end of neural fold is much

thickened and approximately forms three brain

resion. Gill region can barely be idantified.

Eye pouch is not visible while oric region is

clearly distionuished. About 4-5 somites can

be ssen through the epidermis along and

below the neural canal. 43

Stage 22 ( Nearula VII ) Age: 6d17hr; B.L.: 3.4-0.05mm;

Height: 2.6-0.04mm.

Neural folds completely fuse, especially at

head region. The three brain levels are

identifies. Gill appears as flat plate. Eye

pouch is visible but not apparent. There

are about 6-7 somites.

Stage 23 (Pre-tail bud) Age: 6d18hr; B.L.: 3.4-0.05mm;

Height: 2.6-0.05 mm.

The embryo lies on its side. Gill plate

becomes more prominent than that in stgage 22.

About 8-9 somites can be seen.

Stage 24 (Tail bud I) Age: 6d20hr; B.L.: 3.5-0.03 mm;

Height: 2.6-0.04mm.

Mandibular arch is visible. Gill area appears

as small puch. Tail bud appears. Pronephric

bulgs is visible. About 9-10 somites can

be seen.

Stage 25 (Tail bud II) Age: 6d23hr; B.L.: 3.6-0.02mm;

Height: 2.5-0.04mm. 47

Gill pouch is apparent. Tail bud is more

prominent. More than 10 somite,i can be sean.

Stage 26 (Tail bud III) Age, 8d. B.L: 4.0 0.12mm;

Esught:2.4 0.10mm

Vitelline membrane dissolves, The roof of

rhohencephalon thins out. Tha upper portion

of the gill. pouch is grooved. representing

the first appearance of gill arches. Eye

ball i.. visible. Olfactory placode appears.

Tail bud is apparent. The number of acutes

is very confusing and is probably more than 17

Stage 27(Melanophore I) Age: 4.9 0.16mm;

Tail length: 0.7 0.06mm;

Mandibular aroh is well devaloped. Body axis

usually bons due to the increase in length.

Gill akcaea are visible. Eye cap i.a formod.

Tail separates from yolk. Innagination of

olfactory placoade occurs. Melanophores

appear sparsely baside the neural cianal.

Stomodeum. is viaibie. the, embryo shows 43 initial reaction to manipulation and other

external stimulation.

Stage 28 (Melanophore II) Age: 10d; B.L.: 5.3-0.08mm;

T.L.:0.9-0.05mm.

The mandibular arches fuse in the middle

posterior to the stomodeum. The gill arches

are quite apparent. Lens is formed. Tail

fin develops. Pronephric bulge is still

visible but is confused with the posterior

devloping forelimb bud. Melanophores

spread to the prenephric bulge level. Y-shape

bolld vessel appears at the ventral side.

Stage 29 (Melanophore III) Age: 11d; B.L.: 5.5-0.10mm;

T.L.: 1.0-0.06mm.

Gill arcdhes are apparent. Melanophores

spread to 1/3 of the body height. Heart begins

to beat. Spontameous movement occurs.

Stage 30 (Emergence of gill) Age: 12d; B.L.: 6.1-0.03mm;

T.L.: 1.6-0.12mm.

Gill emerges. Tail fin is transparent.

Melanophores spread to half of the body 49 height. Forelimb bud is pparent and with

melanophore developing on it. The ventral

blood vessel is straightened. Circulation

buginm (as indicated by the moireni nt of

blood oorpuscles inside the vental vessel.)

Stage 31 (Forolimg I) Age : 13d, B.L:6.4-0.15mm;

T.L:2.0-0.18mm;

Forelimb length:0.3-0.01mm.

Melanophores spead to 34 of the body helght.

Stomodeum has a width of about 0.6mm.

Forelimb energes.

stage 32 (Forelimb II) Age: 15d; B.L: 6.6-0.15mm;

T.L.: 2.5-0.15mm; F.L.: 0.4-0.05m.

Gill braches (1,2,2).Hindlinb bud appears.

Stage 33 (forelmmb III) Age :160; B.L.: 6.7-0.00mm;

T.L.:3.0-0.10mm;

F.L.: 0.4-0.03mm.

Operculum develops. Gill oiraulation begnns

(as revealed by the nevenent of corposeles

in the gill filamentes). Melanophores start

to invade the eye region. Stomieum is 50

about 0.8mm in Width.

Stage 34(Forelimb IV) Age: 17 B, L.: 6i 0.21mm

T.L.: 3,0+ O, 14mm

F.L.: 006 +0.0 5mm

Gill branches (2,4,4). The eye region is

black.. ry bow forms. hind Lmb bua is

apparent.

Stage 35 (Rindlimb I) Age ;18d ; B .L.: 6.840.33mm

T.L.: 3.5+0.24mm: F.L .: 1.0+0.06mm

Hindlimb length; 0.4+0.03mm

The whole body is black. The lowor jaw is

transparent in apppearance. Gill braanchss

(3,5,5).Xanophores and guanophores

develop on eye region. The ventral blood

vessel is seen not directly to beart

Forelimb shows singn of digitation. Hindlimb

emerges.

Stage 36 (Hindlimb II) Age : 19d ; B.L. :6.8+0.33mm;

T.L .: 3.9+0.24mm:F.L.: 1.1+0.06mm

H.L.:0.5+0.04mm 51 Xanophores ( as depigmented spots ) are present

in head region. Gill branches (5,7,7,)

Stonodtm is about 1.2mm in width . Two

Forelimb digits are present.

STaage 37 (Hindlimb iii) Age ; 21 d; B .L. : 7.0+0.07mm

T.L .: 4.5+0.13mm

F.L. : 1.3+0.05mm

H. L.;0.7+0.06mm

The forsal part of teh body becomes brownish

black. Melanophores rnvade lower jaw. The

Third digit will appear . Ankle farms.

Stage 38 ( Hindlimb Iv ) Age ;23d ;B .L ; 7.1+0.47mm

T.L. 4.8+0.25,F.L ;1.6+0.12mm

H.L .; 0.8+0.06mm

The whole body is brownish-black. Three

digits are resent.

Stage 39 dd(Hatchig ) Age ;25d; B .L : 7.5+ 0.57 mm

T,L .: 5.4 +0.44 mm; F, L. : 1.7+0.14mm

H.L: 0.9+0.07mm

Gill branches (7,9,9). The fcurth digit

and the second toe eill appear. Mouth opens.

About half of the body is oceupied by yollr. 52 Sudge 40 (Hindlimb VI) Age: 28d; B.L.; 7.9+0.43mm

T.L.: 5.8+0.37mm;F.L.: 1.7+0.09mm

H.L. : 1.0+0.07mm

4 digits and 2 toes are present.

Stag,e E (}find`1 i b vll) Age: 29d B a

T.L. :5.9+ 0.14mm

F, L,. :1.8+0.04mm

H.l: 1.1+0.03mm

The third to will appear;

Stage 42 (mindlimb VIII) age :31d: B. L.: 8.3+0.5mm

T.L; 6.3+0.09mm

F.L.2.2+0.12mm

H.L.:1.2+0.10mm

Xanophore spots are distributed smosg the

whole body. Gill further branohes.

3 toes are present.

Stage 43 (hindlimb IX ) Age : 34d : B .L : 8.4 + 0.05 mm

T.L. :6.9 + 0.27 m m ; F. L. : 2.3+ 0.10mm

H.L .: 1.6+0.14mm

The fourth toe will appear. 53 Stuge 44 (Hindlimb X) Age: 3cd; B. L.: 8.6 +0.+9mm

T.L. : 7.5 + 0.15 mm; F.L .: 2.7+0.15mm

H. L.: 1.9+0.11 mm

4 toes are prssent.

Stage 45 )Hindlimb XI ) Age :42d ; B. L : 9.4 + 0.05mm

T.L .: 7.6+0.05mm

F.L.: 2.8+0.10mm

H. L.: 2,1+0.11mm

The fifth toe will appsars.

Stage 46 (Hindlimb XIII)

3 toes are present. Gill branches

(9-10,14-15,14-15)

Stage 47 (Growing phase I)

Body grows in length and in width. Gill

tail fin, and limbs develop facther.

Stage 49 (Metomorphosis I)

Dege aration or fwil fin occurs.

Stage 49 (Metomorphosis II)

The branching of gill is reduced . Bod

becomes thinner. coanificat, on of skin begins

The ventral colores spots are more prominent. 54

Stage 50 (Metamorphosis III)

Cills further retrict and degenerate.

Head region becomes triangular in shape.

Stage 51 (Metamorphosis IV)

Gills almost completely degenerate. Ventral

spots take the adult form. Larva leaves

the water.

Stage 52 (Metamorphosis V)

Gills com pletely disappear.

Stage 53 ( metamorphosis VI)

Skin is heavily cornified. Dorsal and lateral

ridges appear. Skin color changes from

black to brownich-black or brown.

Stage 54 (Growing phase II)

Body grows in length and in width. Skin

is further cornified. 55

Figure 9

(pp. 55-63) Explanation of figure 9

1. One cell stage. A= animal pole V= vegetal pole

2a. First cleavage appears

2b. Two cell stage. G- gray crescent

3. Four cell stage

4A. Eighe cell stage. dorsal view

4B. Eighe cell stage. lateral view

5. Sicteen cell stage

6. Morula

7. Blastula I

8. Blastula II

9A. Blastula III. doraal view

9B. Blastuls III. lateral view

10. Glastula I D=dorsal lip

11. Glastula II

12. Glastula III

13. Glastula IV 56 FIGURE 9

v1 G 2a 2b 3

4A 4B 5 6

8 9A 9B

D

10 11 12 13

1mm 14a. Yolk plug I. V= ventral lip

14b. Yolk plug II

14c. Advance yolk plug III.

15. Slit blastopore. C= pigment concentrated area

(region of future neural fold

16A. Neurula I. dorsal view. F= future neural fold

P= neural plate

16B. Neurula I. view from the slit blastopore

17. Neurula II. N= neural fold

18. Neurula III

19. Neurula IV

20A. Neurula V. dorsal view

20B. Neurula V. lateral view

20C. Neurula V. ventral view

21A. Neurula VI.' dorsal view

21B. Neurula VI. lateral view. A= auditory pit

G= gill region

21C. Neurula VI. ventral view

22A. Neurula VLL dorsal view. S= somite 57

a

v14a 14b 14c 15

F N

P

16a 16s 17 18

19 20A 20B zoo

A,

G

F. s

21n 21a 21c 22 l mm 22B. Neurula VII. ventral view

23A. Pre-tail bud stage. dorsal view

23B. Pre-tail bud stage. lateral view. G = gill plate

23C. Pre-tail bud stage. ventral view

24A. Tail bud I. lateral view. G = gill plate

M = mandibular arch O = optic pouch

P = pronephric bulge

24B. Tail bud I. ventral view

25A. Tail bud II. dorsal view

25B. Tail bud II. lateral view

25C. Tail bud II. ventral view

26A. Tail bud III. dorsal view

26B. Tail bud III. lateral view

26C. Tail bud III. ventral view 58

G

22B 23A 23B

P

G

M

O

24B 23C 24A

25A 25B 25C

26A 26C 26B

1mm 27. Melanophore I

28. Melanophore II

29. Melanophore III. M = melanophores

30. Emergence of gill. A = anus F = forelimb bud

G = gill H = heart region

31. Forelimb I

32. Forelimb II. H = hindlimb bud

33. Forelimb III

34. Forelimb IV. dorsal view

35. Hindlimb I

36. Hindlimb II 59

27 28

G F

M

1mm AA 29 H

30

H

31

32

33 34 2mm

36 35 2mm 37. Hindlimb III

38. Hindlimb IV. ventral view

39. Hatching

40. Hindlimb VI

41A. Hindlimb VII. dorsal view

41B. Hindlimb VII. ventral view

42. Hindlimb VIII

43A. Hindlimb IX. dorsal view

43B. Hindlimb IX. ventral view 60

37 38

39 2mm

40

41A

41B 42

43B 43A 2m 44. Hindlimb X

45A. Hindlimb XI. dorsal view

45B. Hindlimb XI. ventral view

46. Hindlimb XII

47A-D. Growing phase I. A2 & D2 - lateral view 61

45A 44

45B

2mm 46

A1 A2

47

B

C

D2 D1

4mm 48. Metamorphosis I

49A. Metamorphosis II. dorsal view

49bB. Metamorphosis II. ventral view

50. Metamorphosis III

51A. Metamorphosis IV. dorso-lateral view

G = gill rudiment

51B. Metamorphosis IV. ventral view

52A. Metamorphosis V. dorsal view

52B. Metamorphosis V. ventro-lateral view

53A. Metamorphosis VI. dorsal view

D = dorsal rigde

53B. Metamorphosis VI. ventral view 62

48

49A 49B

50

G

51A 51B

52B

52A

D

53B 53A 1 54A-D. Growing phase II 63

A

B

54

C

D

1 cm 17

16

15

14

13 hatching

12

11 stage 33

10 WHOLE

9

8

7 LENGTH

6

5 (mm)

4 stage 26

3 Fif10.GrowthcuiveofFarnmesotrionhongkongeneis stage 20. 2

1

0

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

AGE (days) 64 Table 2 : Comparison among the developmental stages of Paramesotriton

hongkongensis (P), Amblystoma mexicanum (A) and

Triturus pyrrhogaster (T):

special morphological (P) (A) (T)

features stage

1 1 1 one cell

two cells 2b 2 2

four cells 3 3 3

eight cells 4 4 4

sixteen cells 5 5 5

morula 6 6 6

late cleavage 7-9 7-8 7-10 65 dorsal lip 10 9 11

crescent shape biastopore groove 11 10 12a

blastopore groove as semicircle 12 11 12b

13 12c horse--shoe shape blastopore groove

jolk plug 14a-14c 12-13 13a-13a

sllt blastopore 15 14 15

neural plate 15 15 17a

Deural icta 17-18 16 17b-18

neural folds taet at hindbrain level 19 17 19

neural folds touch each other along the whole sength 21 20 20

otic region is visit 21 24

three arain levels are idffcifisd 22 21

cptie icle is visiale 23 21 22

eandibular arch is visible 24 21 22 gill pouch is visible 24 24 22

prop phric bulge is visible 24 23 25

olfactory placode is viEible 26 26

melaLophores appear 27 34 35

Ei11 arch is visible- 27 27 24

Y-shape blood vessel appears 28 28

Frornimb bud is visible 28 40 33

heart begins to beat 29 71

gill emerges 30 37 32

forelimb emerges 31 37

gill branches 32 39 37

hind l inb budis visiblei 48 32

operculum begims to fore 33 36

melancphore invaoe eye regic

33 39

67 35 hindlimb emerges 48

36 44 2 digits

38 48 3 digits

40 hatohing 39 39

2 toes 40 51

3 toes 42 53

4toes 44 55

5toes 46 58

metamorphosis 49-54 60

68 68

4.3 Diacussion:

The eggs of Paramesctriton hongkongensis

are apprecialbly larger than many other urodelos

(Hamburger 1960)

Urodsles Egg Size(mm)

Amblystoms maculatum 2.5-3

Amblystoms tigrinum 1.5

Amblystoms opacum 2.7

Triturus viridescens 1.5

2-2.1 Trutyres pyrrhogeater

2-2.8 Tarucga tcrosus

Parabesitruton hongkongensis 2.9 0.08

Vecause of its large size, the egg of Parsmesotriton

hongkongensis should be very useful in operative

embryolofical experiments.

The normal sequence of events will now be

considered in detail with reference ot teh stages 70

showa in table 1. The data are occabionslly comparsd

wigh the leleted speciees . Triturua pyrrhogssber.

The developmental rape or pr mesotriton

hoskongansis is coparatively slow but is anill

fasuer than that of Tritnus pyerhopber (Andermon

1943) . Due to this glow developmental rave. the seq-

usnce of the appearsnce of differelt on is cah be

easily follcwed.

The ovulated egg is protected by a

vitelline rane and a jelly eapsale contuining

fear transparent layars (Selthe 1963). The cuter-

most layer is stiemy. The yitel membrane dissonves

at tail bad atage III while teh jely capsle will

be brozsn At the time of hs tchins. The jelly capsule

is found to be a good sulst ate for the grown of

water meald.

ovnlation can be indaced by injection of 71 two, to three u rode1e pituit t o tfte rt:

paritoneally (Pt..eh 1933). Ccit arciai HOG (Pr.. ie

Or on)$ which ib uued au eesof 1i to .1 iduce

ovulation in kanopus ,was not effective even at a doss

of 300 i.u. . Thersfore. artificial ions\aminat on in

nowts is not as easy as that in anurans.

The age of one cell scage is the time when

the egg is shed into water .In this experl msat, the

eggs were collected from the rearing tank every two

hours in order to determine precisely the time when

the first clavage iurrow appsared. The fist

claavage furrow appears about 10 hours after the egg

is sned. Theoretically it is better to watch the

shadding process and collent the eggs as soon at they

are lald . This is not practical because the nowts

will be distarbed too frequently and they will soo

soop laying eggs under sach cireumstances . Cou-

baqusntly, a two - hour intsy al was choseh. as the

Shortest feasible time. 72 Pigmeuts are more concentated at the

animal pole of a mature egg . It will disppear

after 10-20 hours if teh egg is not fertilimod

(Dalton 1946). when an egg is fergllized, s fertili-

zation membrane and a perivitelline space sar fommed

The fertilizel egg can float freely inside teh apace

with the heavier vegetal pol o always faaing the

earth. No snch rotation cceurs in unfertilized egge

This is oue of the citaria to distinguish butween

the fartilised eggs and the unfertilized eggs

A .small .white spot can ususlly te cbsonwel

in a fertilized egg indicating the location of

second polar body (F andkhsuser 1948). But this de-

pigmented ares is not alway easily observabls in

each egg.

The first eleavage furrow will arpear

about 10 hours after the egg is and. It does not

always cut through the anilal pole of esch egg as one 73 expeets, It may be more and less displeead to the

lateral side. This displacoemant has probably no

harmful effoct on the davelopment of the egg. From

this ,one can see that the reguletivs tivs powsr or a

newt's egg is quite grsat.

The gray crescant, which is usually

distingishable at two cell stage ( Rown 1903), way

not be obssrvad due to a hisher degree of pigmensation

in certain eggs ; hsavily pigmented or dar amlored

eggs do not show gray crescerts clsarly

The 3rd cleavage rurrew usually appears

horizontally above the egg equator cutting the four

primary blastomeren to four smaller dorsal blascomeres

and four incomplately divided venral binstomeres.

i.e. teh eight csll stuge. The synchrenisation of

sleavage will progressively loss as the develonment

advances. 74

The completion of cleavnge in Paramesotriton

hongkongehsis requires two das while that in Tritaras

ras boscater requires four foer days (Anderson 1943).

The blastula stages were traditionally

suhdivided tnto early, mid and late tlastula stagess

In order to obtain a more exact notionof blastula'

development, the blastula stages were subdivided

accerding to the midromer sise in this experiment.

There is no difference between late tlastuld

( blastula III)and early eastrula (gast ealy I) wlien

one observes them from tbhe gnimesl pole . Bat a slit

indicating the dorsal lip region can be sesn only at

the vagetal pole of gastrula I. Just like the micro-

mere size indieatss the anvancement of theblastuls

stages, the yolk plug stlge is subdividsd accoiding

to the diameter of yolk plug.

Gastrulation is a prossess tant imvolves the 75 involution of call layers. It start with the

appoararice of dorsal lip and ends up when the yolk

plug is reduced to a thin slit. The completion of

gastraltion in the Hong Kong nawt requires less

than two days while that in Triturus pyrrhogester

requires at least two days (Anderson 1943).

After the establishment of three germ leyers

(acto-,meso-,and endoderm),organ formstion

(organogenesis)begins.The first sign or organofenesis

is neurlation (neural canal formation).The otic

regiea is the first sc ily identifiable sensory

structure after neural canal formation. In Triturus

pyrrhogaster, this region can only be observed at a

later stage (per-tail bud II)(Oksda & Ichikawa 1947).

Olfactory placode is visible about 2 days later.

Eye pouch develops rapidly after it is formen at

complet neurula stage.

The development of gill (Severinghaus 1930) 76

is the most prominent mophologiesl chaoge before tfs

forelimb stages. Aftes the emergence of gill,

attention is turned to the development or llmbs.

The time for the occurrence of stomadoum

and operculum and the munber of semites are very

confusing bscause thess stauctures are not identified

until they become very prominent

The heart begins to beat in stage 29, The

rate of heart best is about 60+2 times por immto

(Copenhaver 1939). It is faster than that of adult

The langth of the embryo mcrease rapidly after the

beginning of feart beat, inlicating that the absorption

of nutriont becemes more effjcient

Melanophores appear at stage 27 . They are

derived from the neural crest cells ( Dushane 1943)

Rawles 1948 ). The stage of melanophoss anu the

aggregation of pigment colls chanse consider bly in

different stages (espaeislly in leter stages) 77 (Twitty 1945 & 1949; Barden 1942; Waring 1941).

As a result, the late embryos show a variable color-

ation pattern. At night, the larvae are nearly

transparent. This is the result of the contraction

of melanophores and is controlled by melatonin from

the pineal body (McCord & Allen 1917; Bagnara 1963

Bagnara & Hadley 1969) rather than by MSH (melano-

cyte stimulating hormone) from pituitary.

The occurrence of gill plate, eye pouch,

pronephric bulge, Y-shaped ventral blood vessel and

heart beat are quite consistant in Paramesotriton

hongkongensis and Triturus pyrrhogaster while that

of melanophore and limb buds are very different

(Okada & Ichikawa 1947).

Mouth opens at hindlimb stage V (the normal

time of hatching), but the larvae do not feed until

the reserved yolk is completely utilised.

The time of hatching may be delayed if the 78 late t mbryo c oot bra down the jell (out. Lhi

prat s probably i.isvo -Tes b oc ,s::i .i

resctiens (Carroll & Hearicuick 1974; Salthe 1963)

Thus snything that can reduce the mechanical burrior

e,g. fnngal action, can enhance the hatching time

A hatching emzyme was found in Xencpus embryos. The

presence cf this enzyme in urodale embryos is not

raportod.

The lengthe and ege of the isrege of growing

phass I after the appearance of the fifth too are

affected to a very graest extent by mnutrition.There-

fore, the langth of larae at the time of metamorphosis

is varisble. Generslly speaking, metamorphosis will

occur when the larvae reach 40-44mm in length .The

age of the larvae entering metamorphosis is about two

months and is affoctod by food and space.

The function of gill is highly radused in

metamorphosis IV. This may be one of the reasons

why the newts must leave water at this stage. As 79 previously mentioned, this landing behaviour is

cpmtrolled by hcrmones

weeks dspending on intrinsic as well as extrinsic

factores. During metamorphosis, thyroxin level is

increased while prolacin level is decrasssd

(Etkin 1968 ). Reduction of prolactin retards retards the

growth of the metamorphosing larva. Thersfore.the

length of the young adult just after matsmorphosis

is slightly reduced (to 3.6-4.0cm ))Lynn 1961;

Wld 1960 ). Degeneration of certain tissuss also Metamorphosisstageslastforabout2-3

during metamrphosis of suphibians have been rully

discussed by Frisden 91961& 1966)

As in other animals, the embryonie growth

curve in Parsmesotriton hongkongensis is S -shaped

(patch 1927). From the growth curve,we can dee that

elongation of eggs start at abcut 140 haoury ( stage 21)

after shedding. A more rapid inerease in growt

elengation of eggs start at about 140 hours (stage 21)

after shedding. A more rapid insraase in growth 80

rate is obtined after tail bud stage III (stage 26)

(192 hours) and it continues for a day. This probably

a result of tail elongation. The body axis will bend

at this stage. After this period, the growth rate

becomes more steady except for a slight rotardation

at foralimb stage III (stage 33). At this stage, gill

cirulation begins and the hindlimb bud becomes

apparent. Thece two procsoses may interfero with

the inereass in body longth.

Table 2 shows the comparison smong the

developmental stages of Parmesotriton hongkongansis,

Amblystona mexicanum and Triturus pyrrhogaster. The

embryonic developement us a Continuous prccess and the

stages into which it is separated are mainly for

convenience of study. Categories are sccording to

the appearande of certain consistant diagnostic

features. The sequance of apperance of these various

features may show discrepancy in different orgarisms

due to their genetic individuelition. Morsovar, 81

dirferent anthors hay have their own particulor

ampuraio,and their oopinions may not meot with each

cther. These would acconnt for the variaticeg in

developmetal stages among Parmesotison hoskersensis,

arlystomo eoxic thm and triturus Frhoge.

Bafore Atag 26 there is moch similarity in erternal

features among the three species,G/raoter varition

occurs at later stages.s Paranaotrich honzkogensiss

is somparatively more related to Tritrrns Pyrshogster

in termg of the sennencfe of morphogenesiss.

The most prominent difference between

Paramesotriton hogkongensis and Tritnras Pyrrhoster

is the absence of balancer in the forfmer species.

In Triturus ,balancer is used as a main feature to

dafine a developmental stage (Bodenstsein 1943;Kilros

1940). Therefore, in Paramesotriton , othere e4xternal

featuce must be used for the asigtrmont of a cetrtain

st age. 82

Pigment cells appear at stage 27 in

Paramesotriton hongkongensis, at stage 34 in Triturua

pyrrhogaster and at stage 35 in Amblystoma mexioanum.

The fonal pattern of pigmentation also varies in

the three specise, In Triturus larva, a conspicuous

streak of pigment is present along each lateral side

of the body.

Forelimb and hindlimb buds appear much

erlier in the Hong Kong newt than in the othar two

species.

The stage of hatching is quite consistent

among the three species, hut the morphology of the

embryos at the time of hatching is quite different.

In Paramesotriton hongkongensis, the highly branched

gills and 3-4 duguts can ve seen, In Amblystoma

mexicanum,forelimbs are just beginning to become

Visivble as bulges In Triturus pu\yrrhogaster.

digitation has just behun. 83

The occurrence of abnormalities is not

uncommon in living organisms. Deviations from the

present record may be dus to:

1) an intrinsic difference, i.e. a genetical difference,

in the developmental rate of the individual embryo.

2) the morphological changes within a certain stage

have a broader limit. For example, the stage

observed is blastula III. It cannot be sure

whether it is the beginning or the end of this stage.

3)the subjective record when the border-line between

two stages is difficult to distinguish.

4) the environmental effect, e. . temperature, fung l

infection, pH, aeration, etc.. 84

Chapter 5

THE EFFECT OF WATRR MOUCD ON THE

DEVELOFMINT OF Psibmesotritoq baaskong uraie

Many external factors.e.g. tsmperature,

light and space are found to affect the develcpmopg

of amphibians. The effects of these factors on the

development of Paramegotriton honfbou arsis

examined by using the mebryou of different atsgad,

following the procedure recommanded by Rugh (Rugh)

1962). In the Present investigation,only the

effect of water mould esc peed sttdinad.

The death of anuit qewt is most crser

caused by fungel inrection. The domitant epscies is

Suprolenia nacaitica (Cuher) which aicp infect fish

Saprslsgnig is an aquatic phycomete (Wolf & wolf

1947). It can be cultred in water using hemp seed as a starcar or in 2% corn meal agar, A chemically

defined medipm for the pure calture of this watar

hould was raported (Scott, Fcwcll&seymour 1563) physiological parameters that affect the growyh of 85

Saproleghia parssitica had been wotkl out by Powell

at al (Powell, Scott & Krieg 1972).

5.1 Materisls and Method

In this experimsnt, Snprolsgnia parasitica

was obtained from the infected embryo. After several

aubculture in 2% corn meal ager,the fungas with its

solidified medium was dlended. The resulting produet

was distributed among the containers in concentration

of 60g/400ml boiled water, 60g blended solidified

2% corn msal agar in 400ml boilsd water was usad in

the control group of containses. After one day,

larvae of growing phase I (18-20mm) abd stage 37

embryos werepat in botn groups of containers, Thare

were 10 embryos per container. The results ware

recorded daily, There ware no feading and chaoge of

water. However. boiled water was added to maintain

a constant watar level. 86

5.2 ReBults and Discussion

The rarulth were shown in figure 11a and

11b.

FroE figure 11a,it could be Eaen thet

saprclcania patasitica prwotes the early mtcaing of

lat Ewbryo. This promotion may be due no the

affect of wat Mould or the deatraetion or the jslls

ccat through chemical er biological means.It is

also found that no early hatching oecurs in early

ambryos, e.g. stuge 30 ,which sre infected Ey the

Fuhgue. Insted, they will bs sooner or later

"destroyel and the yolk dissolvss.

Booides Eating a later hatching tins, the

control group as soso a widor range of Latching time

This in icatas thet in normasi deve'opmsni, the

integrity of jelly cost can play a certsin rols in

determinsting the time of hgtching with S parasiting

Control

HATCHED

DA.S after inrteducing the smbryes staga 37 )into the cuitre madium 87 100

EiE 90 llb.

80

Fffeet

70 of with S.parasitica Control % S.

60

Parasitica 50 on

40 the MORTALITY

30 mortality of 20

P.

10

0 hengkonganain

8 0 1 2 3 4 5 6 7 9 10 11 12 14 13 15 16 17 19 18 laetar DAYS afisr iatroduding tha larra iate cre cu/ture msdium 88 89

Figure 11b shows that the water mould can

kill the swimming larvae but over a protracted period.

It indicates that the resistance of the healthy, i.e.

not wounded, larvae to fungal infection is quite

great. Starvation and the presenc of corn meal agar

which is a good substrate for the growth of micro-

organisms can probably explain the high mortality in

the control group. 90

Chapter 6

THE USE OF Paramesotriton hongkongensis

IN EMBRYOLOGICAL STUDIES

Urodele eggs are favorable material in

experimental embryology, e.g.transplantation,

parabiosis,induction, extirpation in early embryou.

Two simple experiments were tried on Paramesotriton

hongkongensis aggs in order to find out the suit-

ability of theses eggs for experimental attempts at

university undergraduate and secondary school level.

6.1 Lampbrush chromsomes from the germinal vesicle of

the ovarian egg:

6.1.1 Removal of the germinal vesicle

Germinal vesicle is the enlarged nucleus

of the primary oocyte in most if not all animals.

The nuclear membrane is thicker than that of most

cells, The chromosomes (of the diplotene stage) are

in a highly active, unfolded, state, There are many 91 loops extendirs irom the chromomeres of the chromesme

main axis. The whole chromssome thus shows an appear-

sace of lampbrush which wab used in ancient days for

cleaning petroleum lamps. The loops ropresent the

active syrthesizing sites. Eech loop is equal to one

DNA helix while the main axis has two (Cakkan 1963;

Gall 1963). The size of a cartain loop varies in

accordance with the activeity of the coscyre But the

number and the maximum sizes of the loops are uniqse

for the eggs of a given organism.

RNAS,mositly mRNA,are transeribed from ths

loops in a considerable amount (invidson 1968)

About 65% of these RNAS can be found in the mature

eggs(Davidson et al 1966).The function of the

stored informational RNA may be to direct the formation

of prsteins which are necssary for early embryonic

development (the cleavage of fertilized eggs.) The

chromosomes become condeased at the and of coganesis.

Lampbrush chromosomes have been fumnd in 52 the developing eggs (primary oocytes) of most but not

every organism. The chromosome soems to be lacking

in those eggs assoasiated by nurse cells In thin

case the function of the lampbrush chromosome is

taken over by the polyploidal nurse cella(Davidson

1968; Bier 1965).

In order to study the lampbrush chromosome,

the germinal vesicle must first ba removed from the

egg. The procedare can be found in Rugh's manual

(Rugh 1962). In this chapter, the procedure was

simplified to suit novices in exrarimental embryology.

The essential equipment for this experiment ars

illustrated in figure 12a.

Carsrully remove the ovary of a gravid

female and place it in 0.7% saline solution in a

petri dish (Fig 12b). Excise a large. healthy oocyte

and tranefer it to a small pstri dish filled with O7%

saline. With a pair of watchmakers forceps (#5 or

#4) and under dissecting microscope, make a Emall 93

incisin on the egg surface near the animal pole.

The action should be slow and steady. The germinal

vesicle, separately or covered with yolk platelets,

will immediately exuded into the medium. If it is

covered with yolk platelets, its location can be

recognised as an elevated region in the exuded yolk

mass (Fig 12c).

Detach the adherent yelk platelets from

the germinal vesicle by forcing the latter in and

out of an ordinary dropper. Repeat this flushing

procedure with fresh saline until the germinal vesicle

is completely free from yolk (Fig 12d). Many white

granular ksubstances are seen inside the vesicle

under the dissecting microscope.

6.1.2 Examination of the lampbrush chromosome

The clean germinal vesicle is trandferred

with a small amount (1 drop) of saline onto a clean

slide. Let the intact germinal vesicle settle near

the pariphery of the drop of saline. Then press a 94 coverslip, starting from this sids, on the drop. The

procedure is shown in the following diagrams:

dropper culture dish or petri dish

isolated germinal vesicle (G.V.)

in 0.7% saline

transfer the G.V. with

a dropper onto a slide

G.V. a drop of saline

press a coverslip on

the slide

the G.V. is broken by

the pressure and the

lampbrush chromosomes

are then forced out and

distribute throughout

the covered area 98

The lawpbrash chromonome will sorarb over

e widt area afcer the geiminal vesicle is busoted

under pressure. Cther nnelear contiats. espsaiolly

nucleoli, are also dispersed, The aria or the

cbromobomes Temain Dabroken. The slids can ds

examined without etainin. under phass-contraat

Eicrobope (Fig 12e) , AIteraerively, a drop of

diltted chromosomal stainil solution cao be uacd

The stainsd chremesomes can be examined under sr

ordinsry ligh,mieroscope. If peimanent mounts sr

deniced, the coverslip can first be detached by the

rethod amploying dry ice. Acete-orccin or iron-

nematoxylin are ofen used as chromoicmsl staias.

6.2 Hensing drop culture of igolated glagcn:

Hanging drop cuiture techninge is a simpl

tind of tisaue culture method but ip is of tdmporsry

use snly. By using this technique, some of themec

anisms about differentistion can be studied. By usin

the hanking drop technique Nin and tnithy perforasd a 96

FIGURE 12

b g.v. d

g.v.

y

y.p.

c e

Fig 12. a) materisls for the experiment on germinal vesicle.

b) ovary of Paramesotriton hongkongensis.

f = fatty tissue.

c) the germinal vesicle (g.v.) as elevation among

the yolk (y).

d) isolated germinal vesicle (g.v.).

e) lampbrush chromosome (l.c.) under phase-contrast

microscope. y.p. = yolk platelet. 400X. 97 their well xnowa experimenat in the differentistion

of gastrela eatoderm in medinm conditloned ty 4xtab

mesoderm (Niu Pwitty 1953)

Every piecs of squipment used in this

tachnique must be stelilized in autoclave, pressure

coker or simply with ow alchol. all wanipulation

are carried out under sseptic conditions.

Nsural crest of the ssrly nenrcula shage is

a good material to demoanbtrate the id yitro

differentiation of embryonic Eissues. At this stage

the fate of naural crest calls is already determined

Neural crest cells will differenliste irto manp

aifferart cell types, e. g. chromatophoras, sutonoml

ganglie, etc.

6.2.1 Materials:(Fig 13a)

bilogical materials: Paramesotriton hongkongsneis

(stage #15 or #16)enbryos 98 Technical materials: Dissecting microacope

Micro-culture slides, deprassion

slides or concave slides and

circular coverslips

Wide bore dropper

Narrow-bore dropper connected to

plastic tabing

Hair-loop

Glass-needle

A pair of watchmakers forcep (#5)

A pair of iritectcaic scissor

Petri oishes

Syracuse dishes with wax bottom

Vaseline

Sterilized water

Full strength and 10% Niu-Twitty

solution

6.2.2 Method :

Decapsulate the early neurula with the 99

scissor and watchmakers forceps in sterilized water sterilized water.

in petri dish. Then wash it in several changes of

sterilized water

Place the neurula on the syracuse dish

filled with Niu-Twitty solution. Under the

dissecting microscope, detach the rod-like neural fold

from the neurula with iridectomy scissor and cut the

detached fold with hair-loop into pieces. After

healing, the operated.embryo should be transferred back

to.10% Niu-Twitty solution for study of further

growth.

After rounding up, the neural fold is

transferredwith mouth controlled pipette into a

small drop of medium previously placed on a

coverslip. Smear a thick layer of vaseline around

the edge of the. depression directly above the drop.

Let the slide on the top of the coverslip for 1-2

days and then turn upside down for further examination. 100

Keep the culture, slide at room temperature

(22-23°C) or better 18°C. and examine it on the 3rd

day and thereafter under light microscope (Fig 13

b-d). 1 a 101

FIGURE 13

C2

M

b c Cl

d2

c d3 d1

Fig 13. a) materials for the hanging-drop technique.

b) damaged tissue does not grow explant attached

to glass. 100X.

cl) neural crest cells grow out of the explant(M). 1OOX

c2) high power micro-photograph of neural crest cell.

C = cytoplasmic extension of neural crest cell.

dl) epidermal tissue spreads as a sheet. 4OX.

d2) portion of dl. 1OOX.

d3) cilia (Ci) are observed at the edge of the

spreading epidermis. 400x. 102

SUMZARY

The purpose of the present ravestigstion is

to provide a good local embryological material of the

species Paranesotriton hongkongensis to the secondary

schools. It is a good matercal for embryological

studies because it has large lzed eggs and a moderately slow developmental re. Since the developmental

pat tees are similar arong the vertebrate groups, the

eggs of this newt an be used to domonstrate the

process of development which evolved along the same

course as in human subjects. By doing so, it is hoped

to obtaiu a correct uiderstandin of sexuality. Also,

this newt is introduced to the scientific community for

those who way find it a convenient experiment model.

Paramesotriton hongkongensis is a local newt

in Hong Kongo. It can be found in cool running streams

in the hills at 300 metres above bea leval and higher.

Unfortunabely the population of this newt is

progressively reduced due to upbanization and pollution 103

The natural habit, the morphology and the

sexual dimorphiam of Paramesotrition hongkongensis

have been fully describad in chapter 1 & 2.

In order to make easy for studing of this

newt, a rearing method is suggested. The main

trouble is the fungal infection but taking good care

and the use of fungicides would probably overcome

this problem.

There are 54 major developmental stages in

paramesotriton hongkongensis. The stages are suggested

according to the most prominent and easily identifiable

morphological features. The pattern of morphogenesis

of the Hong Kong newt is comparstively more similar

to that of Triturus pyrrhogasdter rather than

Amblystoma mexicanum.

Development can certainly be affected by

external factors. Among these factors, only the effect

of water mould (Saprolegnia parasitics) has been studied. 104

The morphological changes during development

are in fact a good text for embryonic studies.

species of Paramesotriton hongkongensis as well as

other urodeles are very usngkongensis as well

embryology. Here only two examples are cited in the

present investingation. 105 APPENDIX

DECAPSULATION TECRNRQUE

The internal features are not examinad in

this investigation,but a fixation method used to

preserve specimens for future microscopic examinstion

and be found in literatures of microscopic techique

(Gray 1958; Hamason 1972).

For the embryos of blestula to youlk plug

stages,the surrouding jelly coats must be removed

completely before fixation, otherwise the roof of

the blstocoel will collapse.

Removal of the jelly coat is not a easy

task due to the difference of pressure between the

internal and external environment. The egg will be

squezed out amd cpIlapsed if the technique is not

properly achieved. Decepsultion can be achieved by

means of a special sharp sissor or iricectomic

scissor and two pairs of watchmakere forcepa (#5 or #4) 106 (Hamburger 1960). By using the watchmakers forceps,

most of the outer jelly coats except the inner-most

one or two are removed. The remaining jelly coat(s)

can then be torn apart through a rapid cut by the

scissor as shown in the following diagrams:

(the operation must be done under water)

inner-most jelly coat a sharp egg

forcep holds

the egg in place

insert on of the blade of the

scissor into and then through

the jelly coat

close the blades and move the

scissor from the egg simult-

aneously and as rapid as possible

vitelline menbrane

egg

jelly coat ACKNOWLEDGEMENTS1 107

I would like to thank Professor L.B. Thrower

and Dr. Y.C.Kong, for their encouragement and super-

vision of this work, also for thelr patience in

correcting the first draft of this thesis. My thanks

are also due to Professor M.C.Niu in giving me

Valuable suggestions.

I also wish to thank Dr. J.E. McCosker, tha

superintendent of Steinhart Aquarium, for identifying

the newt. Finally, I would like to thank Miss P.K.

Watt, Mr. H.W. Cheng, K.M. Pang and K.S. Wong for

their technical assistance. 108 REFERENCES

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