Embryonic Development of Attacus atlas L. (Lepidoptera: Saturniidae)
EMBRYONIC DEVELOPMENT OF Attacus atlas L.
(LEPIDOPTERA: SATURNIIDAE)
YUSTINA YUSUF
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATIC AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2009
ABSTRACT
YUSTINA YUSUF. Embryonic Development of Attacus atlas L. (Lepidoptera; Saturniidae).
Supervised by DEDY DURYADI SOLIHIN and ARIEF BOEDIONO.
Attacus atlas was one of wild silkworm species that has been developed in Indonesia as an
alternative silk source to domestic silkworm Bombyx mori. A. atlas was spread in tropical and
subtropical rain forest and can be found almost in entire Indonesian islands. It has complete
metamorphosis stage, including egg (embryo), larvae, pupa, and imago (adult). Embryogenesis
was an important stage to know the physiology characteristic of insect such as diapauses. The
research aimed to know the embryogenesis of A. atlas. The observation of embryonic development
was done in two ways, observation within the eggshell and without the eggshell. The
embryogenesis in A. atlas was done in seven days. Morphological observation on A. atlas embryo
can be divided as pre-organogenesis and organogenesis stage. Pre-organogenesis stage occurred in
first to fourth day after oviposition. Organogenesis stage can be observed in fifth to seventh day
after oviposition. In the fifth day, body segmentation was completed. In the sixth day, cephal
section and patterning of appendages was completed. Besides, the pigmentation of cephal also
occurred in sixth day. In the seventh day, the pigmentation of entire body was completed and
embryo was ready to hatch. In the eight day after oviposition embryo was hatching by cracking the
eggshell.
Key words: Attacus atlas, embryogenesis, development.
ABSTRAK
YUSTINA YUSUF. Perkembangan Embrio Attacus atlas L. (Lepidoptera; Saturniidae).
Dibimbing oleh DEDY DURYADI SOLIHIN and ARIEF BOEDIONO.
Attacus atlas adalah salah satu species ulat sutera liar yang dikembangkan di Indonesia
sebagai alternatif penghasil sutera dari ulat sutera domestik Bombyx mori. A. atlas tersebar di
daerah tropis dan dapat ditemukan di seluruh wilayah Indonesia. A. atlas memiliki metamorfosis
sempurna mencakup fase telur, larva, pupa, dan imago. Perkembangan embrio adalah tahapan
penting untuk mengetahui karakteristik fisiologi serangga, seperti diapause. Penelitian ini
bertujuan untuk mengetahui perkembangan embrio A. atlas. Pengamatan perkembangan embrio
dilakukan dengan dua cara, yaitu pengamatan embrio dalam cangkang telur dan tanpa cangkang
telur. Tahapan perkembangan embrio pada A. atlas terjadi selama 7 hari. Pengamatan terhadap
perkembangan morfologi embrio A. atlas hanya dapat dibagi menjadi tahap, pre-organogenesis
dan organogenesis. Tahap pre-organogenesis terjadi pada 1 sampai 4 hari setelah oviposisi dan
tahap organogenesis dapat diamati pada hari ke-5 sampai 7. Pembentukan segmentasi tubuh telah
selesai pada hari ke-5 setelah oviposisi. Perkembangan embelan dan segmentasi kepala telah
selesai pada hari ke-6 setelah oviposisi, pigmentasi pada kepala juga terjadi di hari yang sama.
Pigmentasi keseluruhan tubuh terjadi pada hari ke-7 setelah oviposisi. Larva menetas pada hari ke8 setelah oviposisi dengan memecah kulit telur.
Kata kunci: Attacus atlas, perkembangan embrio, perkembangan
EMBRYONIC DEVELOPMENT OF Attacus atlas L.
(LEPIDOPTERA: SATURNIIDAE)
YUSTINA YUSUF
Skripsi
In partial fulfillment of the requirement for the Sarjana of Science
In Department of Biology
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATIC AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2009
Title : Embryonic Development of Attacus atlas L. (Lepidoptera: Saturniidae)
Name : Yustina Yusuf
NRP : G34104072
Approved by,
Supervisor I
Supervisor II
Dr. Ir. Dedy Duryadi Solihin, DEA
NIP 19561102 198403 1 003
Prof. Dr. drh. Arief Boediono
NIP 19640305 198803 1 002
Endoresed by,
Dean of Faculty of Mathematic and Natural Sciences
Bogor Agricultural University
Dr. drh. Hasim, DEA
NIP 19610328 198601 1 002
Graduation Date:
Acknowledgments
All praise and gratitude to Allah SWT the Almighty for His bless and mercy to all human
kind so that this minithesis could be written.
I would like to send the acknowledgment to Dr. Ir. Dedy Duryadi Solihin, DEA and Prof.
Dr. drh. Arief Boediono as their advices, knowledge, and supports during the research. Thanks
also to Dr. Anja Meryandini for the help and suggestion in my skripsi. Deepest thanks to Mama,
Papa, Teteh, and Bibi for their support, love, and sacrifices. Deepest appreciation to Mrs. Hara and
Goodwill Committee, Australia-New Zealand Association, PT. Hutchincon, and dr. Charter for the
trust, motivation, and financially support during my last two years.
Thanks to Ibu Endang Sri Ratna, Pak Heri, Pak Thomas, Mbak Tini, all staff of Department
Biology for the help and suggestion. Special thanks to Disti and family, Ifa, Iday, Achid, Bu Septi,
Mbak Andri, Kiki, Eti, Desi, Lia, Indri, Dyna, Riana, Pampam, Mas Andik, all Friends in Biology
41 and Bio-moleculer Laboratory for the support, cheerfulness, and friendship that they given to
me this far.
Hopefully this skripsi could be useful.
Bogor, Juni 2009
Yustina Yusuf
Curriculum Vitae
Author was born in Sukabumi, August 21st 1986 as the last child of Muhammad Yusuf
Wahyudin and Titi Kurniati.
Author was graduated from MA Husnul Khotimah Kuningan in 2004 and accepted in
Department of Biology, Faculty of Mathematic and Natural Science, Bogor Agricultural
University (IPB) through Seleksi Penerimaan Mahasiswa Baru (SPMB) in the same year.
During the college, author assisted the practical class for Biology in 2007 to 2009, Plant
Anatomy in 2007 and 2008, General Botany in 2008, Environmental Science in 2008, and Micro
technique in 2008. Author also was the active member of Himabio for 2005 to 2007, Department
of Science BEM MIPA 2006-2007, and Public Relation for Pesta Sains Nasional 2006.
Author did the field work entitled Processing and Use of Slaughtery Waste in PT. Sumber
Prima Anugerah Abadi at 2008. Author was the recipient of PPA scholarship in 2005-2006 and
Goodwill International Leadership Scholarship Program for 2007 to 2009.
List of Contents
Pages
List of Tables .......................................................................................................................... viii
List of Figures ........................................................................................................................ viii
INTRODUCTION ...................................................................................................................... 1
Background ..................................................................................................................... 1
Objective ......................................................................................................................... 2
MATERIALS AND EQUIPMENTS
Time and Place ................................................................................................................
Materials .........................................................................................................................
Equipments .....................................................................................................................
Methods ...........................................................................................................................
Moth copulation and egg selection ............................................................................
Observation of embryonic development .....................................................................
Observation of embryonic development within the eggshell ..................................
Observation of embryonic development without the eggshell ................................
Temperature effect on embryogenesis .......................................................................
1
2
2
2
2
2
2
2
2
RESULT
Pre-organogenesis stages ................................................................................................. 3
Organogenesis stages ....................................................................................................... 3
Hatching ........................................................................................................................... 3
Temperature effect on embryogenesis .............................................................................. 3
DISCUSSION
Pre-organogenesis stages .................................................................................................
Organogenesis stages .......................................................................................................
Hatching ..........................................................................................................................
Temperature effect on embryogenesis ..............................................................................
5
6
6
6
CONCLUSION .......................................................................................................................... 6
SUGGESTION ........................................................................................................................... 7
REFERENCES ........................................................................................................................... 7
APPENDICES............................................................................................................................ 8
List of Tables
Pages
1 Copulation time of A. atlas .................................................................................................... 2
2 Total number of egg produced by A. atlas couple.................................................................... 2
3 Percentage of egg hatching .................................................................................................... 5
4 Seven days old egg preserved in 10oC for 1 week ................................................................. 5
5 Seven days old egg preserved in 10oC for 2 weeks ................................................................. 5
6 Seven days old egg preserved in 10oC for 3 weeks ................................................................. 5
7 Seven days old egg preserved in 10oC for 4 weeks ................................................................. 5
List of Figures
Pages
1 The developmental stage of A. atlas ...................................................................................... 1
2 A. atlas having copulation ..................................................................................................... 4
3 The embryonic development of A. atlas (1 to 4 day old) within the eggshell after being
immersed by NaOH 5 M overnight ...................................................................................... 4
4 The embryonic development of A. atlas (5 to 7 day old) ........................................................ 4
5 The first day larva (instar 1) .................................................................................................. 5
6 Cephal section of A. atlas embryo ......................................................................................... 6
1
INTRODUCTION
Background
The sericulture of silkworm in Indonesia
has begun since 10th century (Atmosoedarjo et
al. 2000). So far, the cocoons of domestic
silkworm Bombyx mori have been used as the
source of silk fiber. According to BKPPMD
(Bureau of Local Region Promotion and
Infestation Coordination) (2006), almost 70%
of raw silk material (cocoons) has been
imported from China and India. Meanwhile
Indonesia has many species of wild silkworm
that can produce silk fiber. Therefore, the
Yogyakarta royal family initiated the wild silk
moth development project in Indonesia in
1994. The project aimed to introduce new
material of wild silk as an alternative to the
domestic silk. The wild silk worms in
Indonesia, on which the project focused, are
Attacus atlas and Cricula trifenestrata
(Nurmalitasari & Kuroda 2002).
Based on its taxonomy A. atlas is
classified into kingdom Animalia, phylum
Arthropoda, subphylum Atelocherata, class
Hexapoda (insecta), order Lepidoptera,
superfamily
Bombycoidea,
family
Saturniidae, subfamily Saturniinae, genus
Attacus, species A. atlas L. (Triplehorn &
Johnson 2005). It is known as giant silk moth
or “si rama-rama” and it is considered to be
the largest moth in Asia with the wingspans
about 250 mm. A. atlas is primarily found in
tropical forest and spread around South East
Asia, Southern China, and India. It can be
found almost in entire Indonesian islands
(Peigler 1989).
A. atlas has a perfect metamorphosis stage
that included egg (embryo), larval, pupae, and
adult stage (Figure 1). According to its annual
breeding, A. atlas is polyvoltine, which means
that it has more than two generation in a year
while B. mori has two characteristic
polyvoltine and bivoltine. Bivoltine here
means that it has only two generation in a
year. Therefore, A. atlas can be found all over
year (Chapman 1971). The larva of A. atlas is
polyphage that means it has more than one
host plant, such as cinchona, cinnamon, tea,
sour soup, and avocado (Kalshoven 1981).
The adult female lay about 200 to 250 eggs
that oviposited in 3 to 5 days in stage and will
hatch 8 days after oviposition. A. atlas eggs
are oval in shape, 0.008 gram in weight, 2 mm
in length, and 1.5 mm in width (Peigler 1989).
a
b
c
d
Figure 1 The developmental stage of A. atlas. Egg (embryo)
(a); larva (b); Pupae (c); imago (adult) (d).
Insect embryogenesis is an interesting
field of study. The embryonic development of
insect occurs in the egg directly after
fertilization
ended.
The
stage
of
embryogenesis in each insect can be different,
depends on temperature of environment and
species characteristics of development
(Yamashita & Hasegawa 1985). For example,
B. mori (Bombycidae) has 17 developmental
stage in 9 days (Tazima 1978) while another
Lepidoptera
Nemophora
albiantennella
(Adelidae) has 12 developmental stage in 7
days (Kobayashi 1997).
Observation of insect embryogenesis is
important in order to know the characteristic
of each embryonic stage. One of the important
physiological characteristic that is used to be
studied at the embryonic stage is diapauses.
Diapause is a condition in which animal’s cell
growth and development are reversibly
stopped or slowed (Yamashita & Hasegawa
1985). In sericulture this condition was very
useful. Because, by stopping the development
the egg hatching also was postponed. These
diapauses egg could be used as egg stock for
further sericulture program. Although much of
insect embryology is still a mystery, there are
remarkable progresses in some insect like B.
mori.
Nevertheless,
the
embryonic
development of A. atlas has not been observed
yet.
2
Objective
The research aims to know the embryonic
development of Attacus atlas in order to know
the possibility of artificial hibernation and
accurate time to postpone hatching.
MATERIALS AND METHODS
Materials and Equipments
Materials that were used for this research
were eggs A. atlas, NaOH 5M, glycerol 70%,
lacto phenol, and alcohol 70%. Equipments
that were used for the research were tweezers,
ependorf
tube,
serum
bottle,
stereomicroscope, digital camera Nikon
COOLPIX S210, petridish, and pipette.
Time and Place
This research was conducted in April 2008
to March 2009 in Laboratory of Molecular
Biology Research Center for Bioresources and
Biotechnology (PPSHB) and Laboratory of
Embryology Faculty of Veterinary Medicine,
Bogor Agricultural University (IPB).
Methods
Moth copulation and egg collection
A couple of A. atlas moth was isolated
from the population and separated into
different cage to have breeding. After
copulation ended, eggs from each couples was
collected in petridish. Egg collection was done
everyday at 11 to 12 am. From 13 couples that
has been breed, 4 couples produced the
highest amount of egg. The fertile egg from
these 4 couples then was used for observation
of embryogenesis and treatment of
temperature effect on embryogenesis.
Observation of embryonic development
The embryonic development of A. atlas
was observed in 7 days. The observation was
done everyday (every 24 hours) from 1 day
old to 7 day old after oviposition. It was done
in two ways, observation within egg shell and
observation without eggshell.
Observation of embryonic development
within the eggshell. Ten eggs by the age of 1
to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 70 eggs.
Those eggs then were immersed in NaOH 5 M
overnight and moved into glycerol 70% to be
stored. The egg then was observed by using
stereomicroscope.
Observation of embryonic development
without the eggshell. Five eggs by the age of
5 to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 15 eggs.
Those eggs were opened by cracking the
eggshell. The embryo then was taken and
moved into alcohol 70%. After that, the
embryo was moved into lacto phenol
overnight to be cleared and stored in glycerol
70%. The embryo then observed by using
stereomicroscope.
Temperature effect on embryogenesis
The egg was preserved in 5oC (in
refrigerator) to know temperature effect on
embryogenesis. The egg prese
rvation was done in three treatment,
preservation without medium, preservation in
sucrose 0.5 M, and preservation in NaCl
0.89%. Ten eggs were used for each
treatment. Preservation was done in 1, 2, 3,
and 4 weeks. Total eggs that have been used
are 120 eggs. After preservation, eggs then
were placed in the breeding room.
RESULT
A. atlas got copulation overnight (Figure 2).
The copulation begun at night, about 10 pm to
2 am and it ended at 8 am to 11 am (Table 1).
Female A. atlas produced about 326 to 462
eggs in five days (Table 2). The period of
embryonic stage (egg) was about 7 days in
room temperature (26 to 31oC) (Appendix 1).
Table 1 Copulation time of A. atlas
Copulation time
Couple
Begin
Ended
1
09.57 pm
08.44 am
2
01.07 am
09.38 am
3
01.07 am
10.55 am
4
01.15 am
10.13 am
Table 2 Total number of egg produced by A. atlas couple
Egg produced by couple A. atlas
Couple
Total
number
of egg
1
Day
1
119
Day
2
92
Day
3
91
Day
4
33
Day
5
7
2
171
63
39
23
30
326
3
47
153
70
39
38
347
4
124
214
64
35
25
462
Mean
115.3
130.5
66
32.5
25
369.3
342
3
Eggs that were used for observation of
embryogenesis were selected from: day 1 of
1st couple (119), day 1 of 2nd couple (171),
day 2 of 3rd couple (153), and day 2 of 4th
couple (214). Meanwhile, eggs that were used
for the treatment of temperature effect on
embryogenesis were selected from: day 2 of
couple1 (91), day 2 of couple 2 (63), day 1 of
couple 3 (47), and day 1 of couple 4 (124).
Pre-organogenesis stages.
The 1st day egg after oviposition of A.
atlas showed the large number of yolk
fulfilling the entire egg. The 2nd, 3rd, and 4th
day eggs also showed the same thing. In these
days, the embryo could not be seen because it
was covered by a large amount of yolk
(Figure 3).
Organogenesis stages.
In the 5th day, embryo within eggshell that
was immersed by NaOH 0.5M showed that
the embryo was migrated to the peripheral
surface of egg (surrounding the yolk). The 5th
day embryo was 3.5 to 4 mm in length. It has
reached the organogenesis stage and
performed 11 segments. In the 6th day old
embryo within eggshell, it can be seen that the
amount of yolk was decreasing while the body
size of embryo increasing. The 6th day embryo
reached 4.5 to 5.5 mm in length and it showed
the complete patterning of segmentation and
appendages.
Table 4 Seven day old A. atlas egg preserved in 10oC
for 1week
The 7 th day old embryo within the eggshell
showed the smaller amount of yolk than
before. 7th day old embryo without eggshell
showed that it has 5.5 to 6 mm length. After 7
days from oviposition, the formation of
embryo is completed, as shown in Figure 4.
Hatching
The larva hatched in 8 to 10 days after
oviposition (Table 3). It has light green color
with seta in entire body. The body length was
about 6 to 7 mm, it has 3 thoracic legs and 5
prolegs (abdominal legs) (Figure 5). A. atlas
hatched by cracking the eggshell.
Table 3 Percentage of egg hatching
% Hatching
Couple
Day 8
Day 9
Day 10
1
97%
0
2%
2
95%
2%
1%
3
90%
3%
0
4
100%
0
0
Mean
96%
1%
1%
Temperature effect on embryogenesis
The cold temperature treatment caused the
unsuccessful hatching to A. atlas embryo. The
control eggs for 1 week preservation hatched
97 %, for 2 weeks preservation hatched 93%,
for 3 weeks preservation hatched 89%, while
for 4 weeks preservation hatched 100% (Table
4 to 7).
Table 6 Seven day old A. atlas egg preserved in 10oC
for 3 weeks
Preservation time
0 week
(control)
1 week
C
S
Preservation time
Treatment
0
97%
N
0 week
(control)
C
0
S
0
N
2 weeks
0
93%
0
0
C : Cold treatment without medium
S : in medium sucrose 0.5 M
N : in medium NaCl 0.89%
Table 5 Seven day old A. atlas egg preserved in 10oC
for 2 weeks
Table 7 Seven day old A. atlas egg preserved in 10oC
for 4 weeks
Preservation time
Treatment
0 week
(control)
C
S
N
89%
3 weeks
Preservation time
Treatment
0
C
0
S
0
N
0 week
(control)
4 weeks
100%
0
0
0
4
Figure 2 A. atlas having copulation. Female
A. atlas (♀) was hanging on the cage,
while the male (♂) was hanging on
female’s body.
a
Figure 3 The embryonic development of A. atlas.
(1 to 4 day old) within the eggshell after
being immersed by NaOH 5 M
overnight. mcp: micropyle.
b
c
d
e
f
Figure 4 The embryonic development of A. atlas (5 to 7 day old). 5th day embryo within eggshell (a); 5th day
embryo without eggshell (b); 6th day embryo within eggshell (c); 6th day embryo without eggshell (d);
7th day embryo within eggshell (e); 7th day embryo without eggshell (f); ant: anterior; cep: cephal; post:
posterior; prl: prolegs; trl: thoracic leg; T1-T3: thoracic segments; A1-A8: abdominal segments
5
Figure 5 The first day larva (instar 1). ant: anterior;
cep: cephal; post: posterior; prl: prolegs;
trl: thoracic legs.
DISCUSSION
Insect embryogenesis occurs inside the
egg directly after the fertilization. It is divided
into four main processes, cleavage (formation
of blastoderm), gastrulation, differentiation,
and growth (Tazima 1978; Sander et al 1985).
In the stereomicroscopic observation of A.
atlas embryo, the only embryogenic stages
that can be seen are pre-organogenesis stage
and organogenesis stage.
Pre-organogenesis stages.
The 1st to 4th day old embryo of A. atlas
didn’t show a specific difference that could
marked it into one of embryo developmental
stage. The large amount of yolk covered the
zygote, so that the stereomicroscopic
observation could not be performed. The 1st to
4th day old embryo only can be categorized as
the pre-organogenesis stage. Oppenheimer
(1944)
mentioned
that
insect
has
centrolecithal egg with a large amount of
yolk. In the eggs with highly concentrated
regions of yolk, cleavage occurs only in the
non-yolky cytoplasm. The cleavage begins
with mitotic division of zygote nuclei (without
cytokinesis) in yolk that occurred in several
cycle. This process performs hundred nuclei
that often called as energids. The energids
then migrated from the yolk to the peripheral
surface of the egg and continued the mitotic
division. This division performs one-cell-thick
layer of cells surrounding the yolk in the
peripheral surface that is called blastoderm,
while the central yolky part enclosed by
blastoderm is known as yolk sac or yolk
system (Tazima 1978). In this time, cell
separate from the yolk and use the yolk for its
nourishment. This process is known as
superficial cleavage (cleavage over the yolk
surface). The first day of A. atlas embryo may
reach the formation of blastoderm comparing
with B. mori that reached blastoderm
formation in 12 hours after oviposition
(Tazima 1978).
According to Tazima (1978), a dense
embryonic primordium will be performed as
soon as the blastoderm completed. The cells
inside the presumptive embryonic primordium
area increase in thickness and become
concentrated. This area in the blastoderm
extends broadly on the ventral to ventrolateral
side of egg and known as germ band. The
second day observation of A. atlas embryo
may has the complete germ band formation
compare with B. mori that completed germ
band formation right after blastoderm was
established in 20 hours after oviposition
(Tazima 1978).
After germ band was establish, it was
grow to transform three germ layers.
Gastrulation is the process when the germ
band enlarges and begins to lengthen and fold
into a sausage shape. The one layer of cells
that is located on the outside forms the
ectoderm and another layer of cells on the
inside form the mesoderm.
Ectoderm cells grow and differentiate to
form the epidermis, the brain and nervous
system, and most of the insect's respiratory
(tracheal) system. In addition, the ectoderm
invaginates (folds inward) at the front and rear
of the embryo's body to create front and rear
portions of the digestive system (foregut and
hindgut). Mesoderm cells differentiate to form
other internal structures such as muscles,
glands, heart, blood, fat body, and
reproductive organs. The midgut develops
from a third germ layer (the endoderm) that
arises near the fore and hindgut invaginations
and eventually fuses with them to complete
the alimentary canal (Sander et al 1985).
Embryo of B. mori begins the gastrulation in
25 hours after oviposition (Tazima 1978).
Electron microscopic scanning of the
developing Manduca sexta shows the
gastrulation and differentiation begins at 33
hours after oviposition (Richard et al 1988).
Embryo of A. atlas may begins the
gastrulation stage in the 2nd day
After completing the gastrulation and
differentiation of three germ layers, the
embryo moves to the surface (peripheral) of
egg, this movement is a part of blastokinesis.
Tazima (1978) reported that B. mori takes
this stage in two days (4th and 5th day), in A.
atlas it may be occurred in the 3rd or 4th day.
6
Organogenesis stages.
Organogenesis in insect embryo was
including differentiation of body segments
and growth of embryo. The fifth day embryo
of A. atlas has reached the organogenesis
stage, and the organogenesis continued to the
7th day. According to Sander et al (1985),
organogenesis could be started during late
germ band stage and it takes several days or
even weeks depends on specific mechanism
and characters of each insect egg. The fifth
day embryo of A. atlas showed the complete
segmentation body, but the formation of
appendages and differentiation of cephal
section was not complete yet. The embryo has
3 thoracic segments and 8 abdominal
segments. The embryo of B. mori reached the
complete segmentation body in 6th day
(Tazima 1978).
In the sixth day, the embryo of A. atlas
reached the stage in which all appendages are
formed, the thoracic legs are completely
formed and so did the prolegs (abdominal
legs). Three pairs of thoracic legs grow in
three thoracic segments, while the prolegs are
found in the 3rd, 4th, 5th , 6th , and last segment
of abdominal segments. The section of cephal
has also performed well. The cephal was
consisted of 3 segments that performed
maxilla, mandible, and labium (Figure 6). In
this stage, the pigmentation at the cephal was
beginning, while B.mori embryo begins
pigmentation of the cephal in 8th day (Tazima
1978).
The seventh day old embryo has the same
morphological structure with the larva, which
make this stage of embryo is known as
prelarva embryo or mature embryo. Besides
pigments are formed in entire body (including
epithelium and seta), the embryo also grow
bigger than the 6th day. After reaching this
stage the embryo is ready to hatch.
hatch or hatch through the hatching line
(Sander et al. 1985). Different from B. mori
that hatched in 9 or 10 days after oviposition
(Tazima 1978).
Temperature effect on embryogenesis
Insect embryogenesis was affected by
many factors, such as: temperature, humidity,
genetic, and oxygen consumption (Sander et
al. 1985).
As poichilotherm organism,
temperature has a big effect to insect embryo
development. The exposure of cold
temperature
could
caused
abnormal
development even death to the embryo
(Yamashita & Hasegawa 1985). But some
insect in temperate region has ability to
survive from the winter by suppressing the
metabolism through diapauses. The univoltine
B. mori was the common model to study the
diapause in Lepidotera (Umeya 1950). While
the polyvoltine one was the common model of
artificial hibernation. The embryogenesis of
polyvoltine B. mori can be postponed in a
month by refrigerating it in 5oC (Katsumata
1964).
A. atlas was polyvoltine and used to life in
tropical region. The cold temperature
exposure on 7 days old A. atlas embryo
caused the unsuccessful hatching. No embryo
of A. atlas could hatch after being stored in 10
O
C for several times (Appendix 2). In
polyvoltine B. mori, the egg that would be
preserved should reach the mature stage
(ready to hatch) that was indicated by the
appearance of dark blue dot in the eggshell
(Katsumata 1964).
This unsuccessful hatching might be
caused by the lack oxygen demand or
dehydration because the embryo was wrinkle
(Appendix 3). Although some embryo was
preserved in Sucrose 0.5 M and NaCl 0.89%
medium, it did not prevent the embryo from
dehydration.
CONCLUSION
Figure 6 Cephal section of A. atlas embryo.
l: labium; max: maxilla; man: mandible
Hatching
A. atlas larva hatched in 8 days after
oviposition by cracking the eggshell. Some
insect embryo is used to crack the egg shell to
The embryonic development of Attacus
atlas occurred in 7 days. The morphological
observation showed that the embryogenesis
could be divided as pre-organogenesis and
organogenesis stage. The 1st to 4th day old
embryo could not be observed by the stereo
microscope and it may be occurred the
cleavage to gastrulation stage, while the 5th to
7th day old has reached the organogenesis
stage. The exposure of cold temperature
caused unsuccessful hatching in A. atlas.
7
SUGGESTION
To observe the details of embryonic stage,
observations are recommended to be done in
every 3 to 6 hours. The serial slicing specimen
with histological staining is recommended to
observe
the
complete
process
of
embryogenesis. The using of SEM (Scanning
Electron Microscope) is recommended to
study the embryo movement.
REFERENCES
Atmosoedarjo, Katsubrata J, Kaomini M,
Saleb W, Moerdoko W. 2000. Sutera
Alam Indonesia. Jakarta: Yayasan Sarana
Wana Jaya.
[BKPPMD] Badan Koordinasi Promosi dan
Penanaman Modal Daerah. 2006.
Pengembangan
Sutera
Alam.
http//:www.westjava.com [Apr 6th 2009].
Chapman RF. 1971. The Insects, Structure
and Function 2nd Ed. New York:
Elsevier Publishing comp, Inc.
Kalshoven LGE. 1981. The Pets of Crops in
Indonesia. Laan PA van der.,
penerjemah; Jakarta: Ichtiar BarutoVan
Hoeve. Terjemahan dari : De Plagen van
de Cultuurgewassen in Indonesie.
Katsumata F. 1964. Petunjuk Sederhana bagi
Pemeliharaan Ulat Sutera. Bogor:
Balitbang Kehutanan.
Kobayashi Y. 1997. Embryogenesis of the
fairy moth, Nemophora albiantennella
issiki (Lepidoptera, Adelidae), with
special emphasis on its phylogenetic
implications. J Insect Morphol Embryol
27:157to166:
abstract
[terhubung
berkala]. http://www.sciencedirect.com.
[March 12th 2009].
Nurmalitasari GRAj, Kuroda F. 2002.
Indonesia Progress in The Development
of Wild Silkmoths. [terhubung berkala].
www.jogja.com/images/WildsilkEnglish
02.pdf. [March 20th 2009].
Oppenheimer SB. 1944. An Introduction to
Embryonic development. Massachusetts:
Allyn & Bacon,Inc.
Peigler RS. 1989. A revision of the
IndotoAustralian
genus
Attacus.
California:The Lepidoptera research
Foundation, Inc.
Richard CD, Stanley DC, Walter GG. 1988. A
scanning electron microscope study of
the developing embryo of Manduca
sexta (L.) (Lepidoptera : Sphingidae). Int
J Insect Morphol Embryol 17: 231to242:
abstract
[terhubung
berkala].
http://www.sciencedirect.com. [March
12th 2009].
Sander K, Goutzeit HO, Jäckel H. 1985.
Insect Embryogenesis: Morphology,
Physiology, Genetical and Molecular
Aspects. In: Kerkut GA, Gilbert LI,
editors.
Comprehensive
Insect
Physiology,
Biochemistry
and
Pharmacology. Vol. I. Embryogenesis
and Reproduction. Oxford: Pergamon
Press. 407–434.
Tazima Y. 1978. The silkworm; an important
laboratory tool. Tokyo: Kodansha LTD.
Triplehorn CA, Johnson NF. 2005. Borror and
De Long’s Introduction to the study of
Insects. 7th Ed. California: Thomson
Learning, Inc.
Umeya Y. 1950. Studies on embryonic
hibernation and diapause in insects. Bul
Sericul Exper Station Tokyo 26:1–9.
Yamashita O, Hasegawa K. 1985. Embryonic
Diapause. In: Kerkut GA, Gilbert LI,
editors. Comprehensive InsectPhysiology,
Biochemistry and Pharmacology. Vol. I.
Embryogenesis
and
Reproduction.
Oxford: Pergamon Press. 407–434.
8
APPENDIX
95
Appendix 1 Temperature and humidity in breeding room
Date
120209
130209
140209
150209
160209
170209
180209
190209
200209
210209
220209
230209
240209
250209
260209
270209
280209
Mean
Morning (08.00)
T (°C)
Rh
27
80
26
78
28
65
28
75
27
80
26
79
26
81
28
77
29
68
28
84
26
75
26
81
26
85
27
78
26
85
27
75
27
85
26.94±0.97 78.29±5.62
Afternoon (16.00)
T (°C)
Rh
27
70
28
70
28
65
27
70
27
82
28
72
27
80
29
75
28
70
27
70
27
71
27
70
26
73
29
62
30
60
31
65
27
80
27.82±1.29 70.88±6.04
EMBRYONIC DEVELOPMENT OF Attacus atlas L.
(LEPIDOPTERA: SATURNIIDAE)
YUSTINA YUSUF
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATIC AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2009
ABSTRACT
YUSTINA YUSUF. Embryonic Development of Attacus atlas L. (Lepidoptera; Saturniidae).
Supervised by DEDY DURYADI SOLIHIN and ARIEF BOEDIONO.
Attacus atlas was one of wild silkworm species that has been developed in Indonesia as an
alternative silk source to domestic silkworm Bombyx mori. A. atlas was spread in tropical and
subtropical rain forest and can be found almost in entire Indonesian islands. It has complete
metamorphosis stage, including egg (embryo), larvae, pupa, and imago (adult). Embryogenesis
was an important stage to know the physiology characteristic of insect such as diapauses. The
research aimed to know the embryogenesis of A. atlas. The observation of embryonic development
was done in two ways, observation within the eggshell and without the eggshell. The
embryogenesis in A. atlas was done in seven days. Morphological observation on A. atlas embryo
can be divided as pre-organogenesis and organogenesis stage. Pre-organogenesis stage occurred in
first to fourth day after oviposition. Organogenesis stage can be observed in fifth to seventh day
after oviposition. In the fifth day, body segmentation was completed. In the sixth day, cephal
section and patterning of appendages was completed. Besides, the pigmentation of cephal also
occurred in sixth day. In the seventh day, the pigmentation of entire body was completed and
embryo was ready to hatch. In the eight day after oviposition embryo was hatching by cracking the
eggshell.
Key words: Attacus atlas, embryogenesis, development.
ABSTRAK
YUSTINA YUSUF. Perkembangan Embrio Attacus atlas L. (Lepidoptera; Saturniidae).
Dibimbing oleh DEDY DURYADI SOLIHIN and ARIEF BOEDIONO.
Attacus atlas adalah salah satu species ulat sutera liar yang dikembangkan di Indonesia
sebagai alternatif penghasil sutera dari ulat sutera domestik Bombyx mori. A. atlas tersebar di
daerah tropis dan dapat ditemukan di seluruh wilayah Indonesia. A. atlas memiliki metamorfosis
sempurna mencakup fase telur, larva, pupa, dan imago. Perkembangan embrio adalah tahapan
penting untuk mengetahui karakteristik fisiologi serangga, seperti diapause. Penelitian ini
bertujuan untuk mengetahui perkembangan embrio A. atlas. Pengamatan perkembangan embrio
dilakukan dengan dua cara, yaitu pengamatan embrio dalam cangkang telur dan tanpa cangkang
telur. Tahapan perkembangan embrio pada A. atlas terjadi selama 7 hari. Pengamatan terhadap
perkembangan morfologi embrio A. atlas hanya dapat dibagi menjadi tahap, pre-organogenesis
dan organogenesis. Tahap pre-organogenesis terjadi pada 1 sampai 4 hari setelah oviposisi dan
tahap organogenesis dapat diamati pada hari ke-5 sampai 7. Pembentukan segmentasi tubuh telah
selesai pada hari ke-5 setelah oviposisi. Perkembangan embelan dan segmentasi kepala telah
selesai pada hari ke-6 setelah oviposisi, pigmentasi pada kepala juga terjadi di hari yang sama.
Pigmentasi keseluruhan tubuh terjadi pada hari ke-7 setelah oviposisi. Larva menetas pada hari ke8 setelah oviposisi dengan memecah kulit telur.
Kata kunci: Attacus atlas, perkembangan embrio, perkembangan
1
INTRODUCTION
Background
The sericulture of silkworm in Indonesia
has begun since 10th century (Atmosoedarjo et
al. 2000). So far, the cocoons of domestic
silkworm Bombyx mori have been used as the
source of silk fiber. According to BKPPMD
(Bureau of Local Region Promotion and
Infestation Coordination) (2006), almost 70%
of raw silk material (cocoons) has been
imported from China and India. Meanwhile
Indonesia has many species of wild silkworm
that can produce silk fiber. Therefore, the
Yogyakarta royal family initiated the wild silk
moth development project in Indonesia in
1994. The project aimed to introduce new
material of wild silk as an alternative to the
domestic silk. The wild silk worms in
Indonesia, on which the project focused, are
Attacus atlas and Cricula trifenestrata
(Nurmalitasari & Kuroda 2002).
Based on its taxonomy A. atlas is
classified into kingdom Animalia, phylum
Arthropoda, subphylum Atelocherata, class
Hexapoda (insecta), order Lepidoptera,
superfamily
Bombycoidea,
family
Saturniidae, subfamily Saturniinae, genus
Attacus, species A. atlas L. (Triplehorn &
Johnson 2005). It is known as giant silk moth
or “si rama-rama” and it is considered to be
the largest moth in Asia with the wingspans
about 250 mm. A. atlas is primarily found in
tropical forest and spread around South East
Asia, Southern China, and India. It can be
found almost in entire Indonesian islands
(Peigler 1989).
A. atlas has a perfect metamorphosis stage
that included egg (embryo), larval, pupae, and
adult stage (Figure 1). According to its annual
breeding, A. atlas is polyvoltine, which means
that it has more than two generation in a year
while B. mori has two characteristic
polyvoltine and bivoltine. Bivoltine here
means that it has only two generation in a
year. Therefore, A. atlas can be found all over
year (Chapman 1971). The larva of A. atlas is
polyphage that means it has more than one
host plant, such as cinchona, cinnamon, tea,
sour soup, and avocado (Kalshoven 1981).
The adult female lay about 200 to 250 eggs
that oviposited in 3 to 5 days in stage and will
hatch 8 days after oviposition. A. atlas eggs
are oval in shape, 0.008 gram in weight, 2 mm
in length, and 1.5 mm in width (Peigler 1989).
a
b
c
d
Figure 1 The developmental stage of A. atlas. Egg (embryo)
(a); larva (b); Pupae (c); imago (adult) (d).
Insect embryogenesis is an interesting
field of study. The embryonic development of
insect occurs in the egg directly after
fertilization
ended.
The
stage
of
embryogenesis in each insect can be different,
depends on temperature of environment and
species characteristics of development
(Yamashita & Hasegawa 1985). For example,
B. mori (Bombycidae) has 17 developmental
stage in 9 days (Tazima 1978) while another
Lepidoptera
Nemophora
albiantennella
(Adelidae) has 12 developmental stage in 7
days (Kobayashi 1997).
Observation of insect embryogenesis is
important in order to know the characteristic
of each embryonic stage. One of the important
physiological characteristic that is used to be
studied at the embryonic stage is diapauses.
Diapause is a condition in which animal’s cell
growth and development are reversibly
stopped or slowed (Yamashita & Hasegawa
1985). In sericulture this condition was very
useful. Because, by stopping the development
the egg hatching also was postponed. These
diapauses egg could be used as egg stock for
further sericulture program. Although much of
insect embryology is still a mystery, there are
remarkable progresses in some insect like B.
mori.
Nevertheless,
the
embryonic
development of A. atlas has not been observed
yet.
2
Objective
The research aims to know the embryonic
development of Attacus atlas in order to know
the possibility of artificial hibernation and
accurate time to postpone hatching.
MATERIALS AND METHODS
Materials and Equipments
Materials that were used for this research
were eggs A. atlas, NaOH 5M, glycerol 70%,
lacto phenol, and alcohol 70%. Equipments
that were used for the research were tweezers,
ependorf
tube,
serum
bottle,
stereomicroscope, digital camera Nikon
COOLPIX S210, petridish, and pipette.
Time and Place
This research was conducted in April 2008
to March 2009 in Laboratory of Molecular
Biology Research Center for Bioresources and
Biotechnology (PPSHB) and Laboratory of
Embryology Faculty of Veterinary Medicine,
Bogor Agricultural University (IPB).
Methods
Moth copulation and egg collection
A couple of A. atlas moth was isolated
from the population and separated into
different cage to have breeding. After
copulation ended, eggs from each couples was
collected in petridish. Egg collection was done
everyday at 11 to 12 am. From 13 couples that
has been breed, 4 couples produced the
highest amount of egg. The fertile egg from
these 4 couples then was used for observation
of embryogenesis and treatment of
temperature effect on embryogenesis.
Observation of embryonic development
The embryonic development of A. atlas
was observed in 7 days. The observation was
done everyday (every 24 hours) from 1 day
old to 7 day old after oviposition. It was done
in two ways, observation within egg shell and
observation without eggshell.
Observation of embryonic development
within the eggshell. Ten eggs by the age of 1
to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 70 eggs.
Those eggs then were immersed in NaOH 5 M
overnight and moved into glycerol 70% to be
stored. The egg then was observed by using
stereomicroscope.
Observation of embryonic development
without the eggshell. Five eggs by the age of
5 to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 15 eggs.
Those eggs were opened by cracking the
eggshell. The embryo then was taken and
moved into alcohol 70%. After that, the
embryo was moved into lacto phenol
overnight to be cleared and stored in glycerol
70%. The embryo then observed by using
stereomicroscope.
Temperature effect on embryogenesis
The egg was preserved in 5oC (in
refrigerator) to know temperature effect on
embryogenesis. The egg prese
rvation was done in three treatment,
preservation without medium, preservation in
sucrose 0.5 M, and preservation in NaCl
0.89%. Ten eggs were used for each
treatment. Preservation was done in 1, 2, 3,
and 4 weeks. Total eggs that have been used
are 120 eggs. After preservation, eggs then
were placed in the breeding room.
RESULT
A. atlas got copulation overnight (Figure 2).
The copulation begun at night, about 10 pm to
2 am and it ended at 8 am to 11 am (Table 1).
Female A. atlas produced about 326 to 462
eggs in five days (Table 2). The period of
embryonic stage (egg) was about 7 days in
room temperature (26 to 31oC) (Appendix 1).
Table 1 Copulation time of A. atlas
Copulation time
Couple
Begin
Ended
1
09.57 pm
08.44 am
2
01.07 am
09.38 am
3
01.07 am
10.55 am
4
01.15 am
10.13 am
Table 2 Total number of egg produced by A. atlas couple
Egg produced by couple A. atlas
Couple
Total
number
of egg
1
Day
1
119
Day
2
92
Day
3
91
Day
4
33
Day
5
7
2
171
63
39
23
30
326
3
47
153
70
39
38
347
4
124
214
64
35
25
462
Mean
115.3
130.5
66
32.5
25
369.3
342
2
Objective
The research aims to know the embryonic
development of Attacus atlas in order to know
the possibility of artificial hibernation and
accurate time to postpone hatching.
MATERIALS AND METHODS
Materials and Equipments
Materials that were used for this research
were eggs A. atlas, NaOH 5M, glycerol 70%,
lacto phenol, and alcohol 70%. Equipments
that were used for the research were tweezers,
ependorf
tube,
serum
bottle,
stereomicroscope, digital camera Nikon
COOLPIX S210, petridish, and pipette.
Time and Place
This research was conducted in April 2008
to March 2009 in Laboratory of Molecular
Biology Research Center for Bioresources and
Biotechnology (PPSHB) and Laboratory of
Embryology Faculty of Veterinary Medicine,
Bogor Agricultural University (IPB).
Methods
Moth copulation and egg collection
A couple of A. atlas moth was isolated
from the population and separated into
different cage to have breeding. After
copulation ended, eggs from each couples was
collected in petridish. Egg collection was done
everyday at 11 to 12 am. From 13 couples that
has been breed, 4 couples produced the
highest amount of egg. The fertile egg from
these 4 couples then was used for observation
of embryogenesis and treatment of
temperature effect on embryogenesis.
Observation of embryonic development
The embryonic development of A. atlas
was observed in 7 days. The observation was
done everyday (every 24 hours) from 1 day
old to 7 day old after oviposition. It was done
in two ways, observation within egg shell and
observation without eggshell.
Observation of embryonic development
within the eggshell. Ten eggs by the age of 1
to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 70 eggs.
Those eggs then were immersed in NaOH 5 M
overnight and moved into glycerol 70% to be
stored. The egg then was observed by using
stereomicroscope.
Observation of embryonic development
without the eggshell. Five eggs by the age of
5 to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 15 eggs.
Those eggs were opened by cracking the
eggshell. The embryo then was taken and
moved into alcohol 70%. After that, the
embryo was moved into lacto phenol
overnight to be cleared and stored in glycerol
70%. The embryo then observed by using
stereomicroscope.
Temperature effect on embryogenesis
The egg was preserved in 5oC (in
refrigerator) to know temperature effect on
embryogenesis. The egg prese
rvation was done in three treatment,
preservation without medium, preservation in
sucrose 0.5 M, and preservation in NaCl
0.89%. Ten eggs were used for each
treatment. Preservation was done in 1, 2, 3,
and 4 weeks. Total eggs that have been used
are 120 eggs. After preservation, eggs then
were placed in the breeding room.
RESULT
A. atlas got copulation overnight (Figure 2).
The copulation begun at night, about 10 pm to
2 am and it ended at 8 am to 11 am (Table 1).
Female A. atlas produced about 326 to 462
eggs in five days (Table 2). The period of
embryonic stage (egg) was about 7 days in
room temperature (26 to 31oC) (Appendix 1).
Table 1 Copulation time of A. atlas
Copulation time
Couple
Begin
Ended
1
09.57 pm
08.44 am
2
01.07 am
09.38 am
3
01.07 am
10.55 am
4
01.15 am
10.13 am
Table 2 Total number of egg produced by A. atlas couple
Egg produced by couple A. atlas
Couple
Total
number
of egg
1
Day
1
119
Day
2
92
Day
3
91
Day
4
33
Day
5
7
2
171
63
39
23
30
326
3
47
153
70
39
38
347
4
124
214
64
35
25
462
Mean
115.3
130.5
66
32.5
25
369.3
342
2
Objective
The research aims to know the embryonic
development of Attacus atlas in order to know
the possibility of artificial hibernation and
accurate time to postpone hatching.
MATERIALS AND METHODS
Materials and Equipments
Materials that were used for this research
were eggs A. atlas, NaOH 5M, glycerol 70%,
lacto phenol, and alcohol 70%. Equipments
that were used for the research were tweezers,
ependorf
tube,
serum
bottle,
stereomicroscope, digital camera Nikon
COOLPIX S210, petridish, and pipette.
Time and Place
This research was conducted in April 2008
to March 2009 in Laboratory of Molecular
Biology Research Center for Bioresources and
Biotechnology (PPSHB) and Laboratory of
Embryology Faculty of Veterinary Medicine,
Bogor Agricultural University (IPB).
Methods
Moth copulation and egg collection
A couple of A. atlas moth was isolated
from the population and separated into
different cage to have breeding. After
copulation ended, eggs from each couples was
collected in petridish. Egg collection was done
everyday at 11 to 12 am. From 13 couples that
has been breed, 4 couples produced the
highest amount of egg. The fertile egg from
these 4 couples then was used for observation
of embryogenesis and treatment of
temperature effect on embryogenesis.
Observation of embryonic development
The embryonic development of A. atlas
was observed in 7 days. The observation was
done everyday (every 24 hours) from 1 day
old to 7 day old after oviposition. It was done
in two ways, observation within egg shell and
observation without eggshell.
Observation of embryonic development
within the eggshell. Ten eggs by the age of 1
to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 70 eggs.
Those eggs then were immersed in NaOH 5 M
overnight and moved into glycerol 70% to be
stored. The egg then was observed by using
stereomicroscope.
Observation of embryonic development
without the eggshell. Five eggs by the age of
5 to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 15 eggs.
Those eggs were opened by cracking the
eggshell. The embryo then was taken and
moved into al
(LEPIDOPTERA: SATURNIIDAE)
YUSTINA YUSUF
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATIC AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2009
ABSTRACT
YUSTINA YUSUF. Embryonic Development of Attacus atlas L. (Lepidoptera; Saturniidae).
Supervised by DEDY DURYADI SOLIHIN and ARIEF BOEDIONO.
Attacus atlas was one of wild silkworm species that has been developed in Indonesia as an
alternative silk source to domestic silkworm Bombyx mori. A. atlas was spread in tropical and
subtropical rain forest and can be found almost in entire Indonesian islands. It has complete
metamorphosis stage, including egg (embryo), larvae, pupa, and imago (adult). Embryogenesis
was an important stage to know the physiology characteristic of insect such as diapauses. The
research aimed to know the embryogenesis of A. atlas. The observation of embryonic development
was done in two ways, observation within the eggshell and without the eggshell. The
embryogenesis in A. atlas was done in seven days. Morphological observation on A. atlas embryo
can be divided as pre-organogenesis and organogenesis stage. Pre-organogenesis stage occurred in
first to fourth day after oviposition. Organogenesis stage can be observed in fifth to seventh day
after oviposition. In the fifth day, body segmentation was completed. In the sixth day, cephal
section and patterning of appendages was completed. Besides, the pigmentation of cephal also
occurred in sixth day. In the seventh day, the pigmentation of entire body was completed and
embryo was ready to hatch. In the eight day after oviposition embryo was hatching by cracking the
eggshell.
Key words: Attacus atlas, embryogenesis, development.
ABSTRAK
YUSTINA YUSUF. Perkembangan Embrio Attacus atlas L. (Lepidoptera; Saturniidae).
Dibimbing oleh DEDY DURYADI SOLIHIN and ARIEF BOEDIONO.
Attacus atlas adalah salah satu species ulat sutera liar yang dikembangkan di Indonesia
sebagai alternatif penghasil sutera dari ulat sutera domestik Bombyx mori. A. atlas tersebar di
daerah tropis dan dapat ditemukan di seluruh wilayah Indonesia. A. atlas memiliki metamorfosis
sempurna mencakup fase telur, larva, pupa, dan imago. Perkembangan embrio adalah tahapan
penting untuk mengetahui karakteristik fisiologi serangga, seperti diapause. Penelitian ini
bertujuan untuk mengetahui perkembangan embrio A. atlas. Pengamatan perkembangan embrio
dilakukan dengan dua cara, yaitu pengamatan embrio dalam cangkang telur dan tanpa cangkang
telur. Tahapan perkembangan embrio pada A. atlas terjadi selama 7 hari. Pengamatan terhadap
perkembangan morfologi embrio A. atlas hanya dapat dibagi menjadi tahap, pre-organogenesis
dan organogenesis. Tahap pre-organogenesis terjadi pada 1 sampai 4 hari setelah oviposisi dan
tahap organogenesis dapat diamati pada hari ke-5 sampai 7. Pembentukan segmentasi tubuh telah
selesai pada hari ke-5 setelah oviposisi. Perkembangan embelan dan segmentasi kepala telah
selesai pada hari ke-6 setelah oviposisi, pigmentasi pada kepala juga terjadi di hari yang sama.
Pigmentasi keseluruhan tubuh terjadi pada hari ke-7 setelah oviposisi. Larva menetas pada hari ke8 setelah oviposisi dengan memecah kulit telur.
Kata kunci: Attacus atlas, perkembangan embrio, perkembangan
EMBRYONIC DEVELOPMENT OF Attacus atlas L.
(LEPIDOPTERA: SATURNIIDAE)
YUSTINA YUSUF
Skripsi
In partial fulfillment of the requirement for the Sarjana of Science
In Department of Biology
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATIC AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2009
Title : Embryonic Development of Attacus atlas L. (Lepidoptera: Saturniidae)
Name : Yustina Yusuf
NRP : G34104072
Approved by,
Supervisor I
Supervisor II
Dr. Ir. Dedy Duryadi Solihin, DEA
NIP 19561102 198403 1 003
Prof. Dr. drh. Arief Boediono
NIP 19640305 198803 1 002
Endoresed by,
Dean of Faculty of Mathematic and Natural Sciences
Bogor Agricultural University
Dr. drh. Hasim, DEA
NIP 19610328 198601 1 002
Graduation Date:
Acknowledgments
All praise and gratitude to Allah SWT the Almighty for His bless and mercy to all human
kind so that this minithesis could be written.
I would like to send the acknowledgment to Dr. Ir. Dedy Duryadi Solihin, DEA and Prof.
Dr. drh. Arief Boediono as their advices, knowledge, and supports during the research. Thanks
also to Dr. Anja Meryandini for the help and suggestion in my skripsi. Deepest thanks to Mama,
Papa, Teteh, and Bibi for their support, love, and sacrifices. Deepest appreciation to Mrs. Hara and
Goodwill Committee, Australia-New Zealand Association, PT. Hutchincon, and dr. Charter for the
trust, motivation, and financially support during my last two years.
Thanks to Ibu Endang Sri Ratna, Pak Heri, Pak Thomas, Mbak Tini, all staff of Department
Biology for the help and suggestion. Special thanks to Disti and family, Ifa, Iday, Achid, Bu Septi,
Mbak Andri, Kiki, Eti, Desi, Lia, Indri, Dyna, Riana, Pampam, Mas Andik, all Friends in Biology
41 and Bio-moleculer Laboratory for the support, cheerfulness, and friendship that they given to
me this far.
Hopefully this skripsi could be useful.
Bogor, Juni 2009
Yustina Yusuf
Curriculum Vitae
Author was born in Sukabumi, August 21st 1986 as the last child of Muhammad Yusuf
Wahyudin and Titi Kurniati.
Author was graduated from MA Husnul Khotimah Kuningan in 2004 and accepted in
Department of Biology, Faculty of Mathematic and Natural Science, Bogor Agricultural
University (IPB) through Seleksi Penerimaan Mahasiswa Baru (SPMB) in the same year.
During the college, author assisted the practical class for Biology in 2007 to 2009, Plant
Anatomy in 2007 and 2008, General Botany in 2008, Environmental Science in 2008, and Micro
technique in 2008. Author also was the active member of Himabio for 2005 to 2007, Department
of Science BEM MIPA 2006-2007, and Public Relation for Pesta Sains Nasional 2006.
Author did the field work entitled Processing and Use of Slaughtery Waste in PT. Sumber
Prima Anugerah Abadi at 2008. Author was the recipient of PPA scholarship in 2005-2006 and
Goodwill International Leadership Scholarship Program for 2007 to 2009.
List of Contents
Pages
List of Tables .......................................................................................................................... viii
List of Figures ........................................................................................................................ viii
INTRODUCTION ...................................................................................................................... 1
Background ..................................................................................................................... 1
Objective ......................................................................................................................... 2
MATERIALS AND EQUIPMENTS
Time and Place ................................................................................................................
Materials .........................................................................................................................
Equipments .....................................................................................................................
Methods ...........................................................................................................................
Moth copulation and egg selection ............................................................................
Observation of embryonic development .....................................................................
Observation of embryonic development within the eggshell ..................................
Observation of embryonic development without the eggshell ................................
Temperature effect on embryogenesis .......................................................................
1
2
2
2
2
2
2
2
2
RESULT
Pre-organogenesis stages ................................................................................................. 3
Organogenesis stages ....................................................................................................... 3
Hatching ........................................................................................................................... 3
Temperature effect on embryogenesis .............................................................................. 3
DISCUSSION
Pre-organogenesis stages .................................................................................................
Organogenesis stages .......................................................................................................
Hatching ..........................................................................................................................
Temperature effect on embryogenesis ..............................................................................
5
6
6
6
CONCLUSION .......................................................................................................................... 6
SUGGESTION ........................................................................................................................... 7
REFERENCES ........................................................................................................................... 7
APPENDICES............................................................................................................................ 8
List of Tables
Pages
1 Copulation time of A. atlas .................................................................................................... 2
2 Total number of egg produced by A. atlas couple.................................................................... 2
3 Percentage of egg hatching .................................................................................................... 5
4 Seven days old egg preserved in 10oC for 1 week ................................................................. 5
5 Seven days old egg preserved in 10oC for 2 weeks ................................................................. 5
6 Seven days old egg preserved in 10oC for 3 weeks ................................................................. 5
7 Seven days old egg preserved in 10oC for 4 weeks ................................................................. 5
List of Figures
Pages
1 The developmental stage of A. atlas ...................................................................................... 1
2 A. atlas having copulation ..................................................................................................... 4
3 The embryonic development of A. atlas (1 to 4 day old) within the eggshell after being
immersed by NaOH 5 M overnight ...................................................................................... 4
4 The embryonic development of A. atlas (5 to 7 day old) ........................................................ 4
5 The first day larva (instar 1) .................................................................................................. 5
6 Cephal section of A. atlas embryo ......................................................................................... 6
1
INTRODUCTION
Background
The sericulture of silkworm in Indonesia
has begun since 10th century (Atmosoedarjo et
al. 2000). So far, the cocoons of domestic
silkworm Bombyx mori have been used as the
source of silk fiber. According to BKPPMD
(Bureau of Local Region Promotion and
Infestation Coordination) (2006), almost 70%
of raw silk material (cocoons) has been
imported from China and India. Meanwhile
Indonesia has many species of wild silkworm
that can produce silk fiber. Therefore, the
Yogyakarta royal family initiated the wild silk
moth development project in Indonesia in
1994. The project aimed to introduce new
material of wild silk as an alternative to the
domestic silk. The wild silk worms in
Indonesia, on which the project focused, are
Attacus atlas and Cricula trifenestrata
(Nurmalitasari & Kuroda 2002).
Based on its taxonomy A. atlas is
classified into kingdom Animalia, phylum
Arthropoda, subphylum Atelocherata, class
Hexapoda (insecta), order Lepidoptera,
superfamily
Bombycoidea,
family
Saturniidae, subfamily Saturniinae, genus
Attacus, species A. atlas L. (Triplehorn &
Johnson 2005). It is known as giant silk moth
or “si rama-rama” and it is considered to be
the largest moth in Asia with the wingspans
about 250 mm. A. atlas is primarily found in
tropical forest and spread around South East
Asia, Southern China, and India. It can be
found almost in entire Indonesian islands
(Peigler 1989).
A. atlas has a perfect metamorphosis stage
that included egg (embryo), larval, pupae, and
adult stage (Figure 1). According to its annual
breeding, A. atlas is polyvoltine, which means
that it has more than two generation in a year
while B. mori has two characteristic
polyvoltine and bivoltine. Bivoltine here
means that it has only two generation in a
year. Therefore, A. atlas can be found all over
year (Chapman 1971). The larva of A. atlas is
polyphage that means it has more than one
host plant, such as cinchona, cinnamon, tea,
sour soup, and avocado (Kalshoven 1981).
The adult female lay about 200 to 250 eggs
that oviposited in 3 to 5 days in stage and will
hatch 8 days after oviposition. A. atlas eggs
are oval in shape, 0.008 gram in weight, 2 mm
in length, and 1.5 mm in width (Peigler 1989).
a
b
c
d
Figure 1 The developmental stage of A. atlas. Egg (embryo)
(a); larva (b); Pupae (c); imago (adult) (d).
Insect embryogenesis is an interesting
field of study. The embryonic development of
insect occurs in the egg directly after
fertilization
ended.
The
stage
of
embryogenesis in each insect can be different,
depends on temperature of environment and
species characteristics of development
(Yamashita & Hasegawa 1985). For example,
B. mori (Bombycidae) has 17 developmental
stage in 9 days (Tazima 1978) while another
Lepidoptera
Nemophora
albiantennella
(Adelidae) has 12 developmental stage in 7
days (Kobayashi 1997).
Observation of insect embryogenesis is
important in order to know the characteristic
of each embryonic stage. One of the important
physiological characteristic that is used to be
studied at the embryonic stage is diapauses.
Diapause is a condition in which animal’s cell
growth and development are reversibly
stopped or slowed (Yamashita & Hasegawa
1985). In sericulture this condition was very
useful. Because, by stopping the development
the egg hatching also was postponed. These
diapauses egg could be used as egg stock for
further sericulture program. Although much of
insect embryology is still a mystery, there are
remarkable progresses in some insect like B.
mori.
Nevertheless,
the
embryonic
development of A. atlas has not been observed
yet.
2
Objective
The research aims to know the embryonic
development of Attacus atlas in order to know
the possibility of artificial hibernation and
accurate time to postpone hatching.
MATERIALS AND METHODS
Materials and Equipments
Materials that were used for this research
were eggs A. atlas, NaOH 5M, glycerol 70%,
lacto phenol, and alcohol 70%. Equipments
that were used for the research were tweezers,
ependorf
tube,
serum
bottle,
stereomicroscope, digital camera Nikon
COOLPIX S210, petridish, and pipette.
Time and Place
This research was conducted in April 2008
to March 2009 in Laboratory of Molecular
Biology Research Center for Bioresources and
Biotechnology (PPSHB) and Laboratory of
Embryology Faculty of Veterinary Medicine,
Bogor Agricultural University (IPB).
Methods
Moth copulation and egg collection
A couple of A. atlas moth was isolated
from the population and separated into
different cage to have breeding. After
copulation ended, eggs from each couples was
collected in petridish. Egg collection was done
everyday at 11 to 12 am. From 13 couples that
has been breed, 4 couples produced the
highest amount of egg. The fertile egg from
these 4 couples then was used for observation
of embryogenesis and treatment of
temperature effect on embryogenesis.
Observation of embryonic development
The embryonic development of A. atlas
was observed in 7 days. The observation was
done everyday (every 24 hours) from 1 day
old to 7 day old after oviposition. It was done
in two ways, observation within egg shell and
observation without eggshell.
Observation of embryonic development
within the eggshell. Ten eggs by the age of 1
to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 70 eggs.
Those eggs then were immersed in NaOH 5 M
overnight and moved into glycerol 70% to be
stored. The egg then was observed by using
stereomicroscope.
Observation of embryonic development
without the eggshell. Five eggs by the age of
5 to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 15 eggs.
Those eggs were opened by cracking the
eggshell. The embryo then was taken and
moved into alcohol 70%. After that, the
embryo was moved into lacto phenol
overnight to be cleared and stored in glycerol
70%. The embryo then observed by using
stereomicroscope.
Temperature effect on embryogenesis
The egg was preserved in 5oC (in
refrigerator) to know temperature effect on
embryogenesis. The egg prese
rvation was done in three treatment,
preservation without medium, preservation in
sucrose 0.5 M, and preservation in NaCl
0.89%. Ten eggs were used for each
treatment. Preservation was done in 1, 2, 3,
and 4 weeks. Total eggs that have been used
are 120 eggs. After preservation, eggs then
were placed in the breeding room.
RESULT
A. atlas got copulation overnight (Figure 2).
The copulation begun at night, about 10 pm to
2 am and it ended at 8 am to 11 am (Table 1).
Female A. atlas produced about 326 to 462
eggs in five days (Table 2). The period of
embryonic stage (egg) was about 7 days in
room temperature (26 to 31oC) (Appendix 1).
Table 1 Copulation time of A. atlas
Copulation time
Couple
Begin
Ended
1
09.57 pm
08.44 am
2
01.07 am
09.38 am
3
01.07 am
10.55 am
4
01.15 am
10.13 am
Table 2 Total number of egg produced by A. atlas couple
Egg produced by couple A. atlas
Couple
Total
number
of egg
1
Day
1
119
Day
2
92
Day
3
91
Day
4
33
Day
5
7
2
171
63
39
23
30
326
3
47
153
70
39
38
347
4
124
214
64
35
25
462
Mean
115.3
130.5
66
32.5
25
369.3
342
3
Eggs that were used for observation of
embryogenesis were selected from: day 1 of
1st couple (119), day 1 of 2nd couple (171),
day 2 of 3rd couple (153), and day 2 of 4th
couple (214). Meanwhile, eggs that were used
for the treatment of temperature effect on
embryogenesis were selected from: day 2 of
couple1 (91), day 2 of couple 2 (63), day 1 of
couple 3 (47), and day 1 of couple 4 (124).
Pre-organogenesis stages.
The 1st day egg after oviposition of A.
atlas showed the large number of yolk
fulfilling the entire egg. The 2nd, 3rd, and 4th
day eggs also showed the same thing. In these
days, the embryo could not be seen because it
was covered by a large amount of yolk
(Figure 3).
Organogenesis stages.
In the 5th day, embryo within eggshell that
was immersed by NaOH 0.5M showed that
the embryo was migrated to the peripheral
surface of egg (surrounding the yolk). The 5th
day embryo was 3.5 to 4 mm in length. It has
reached the organogenesis stage and
performed 11 segments. In the 6th day old
embryo within eggshell, it can be seen that the
amount of yolk was decreasing while the body
size of embryo increasing. The 6th day embryo
reached 4.5 to 5.5 mm in length and it showed
the complete patterning of segmentation and
appendages.
Table 4 Seven day old A. atlas egg preserved in 10oC
for 1week
The 7 th day old embryo within the eggshell
showed the smaller amount of yolk than
before. 7th day old embryo without eggshell
showed that it has 5.5 to 6 mm length. After 7
days from oviposition, the formation of
embryo is completed, as shown in Figure 4.
Hatching
The larva hatched in 8 to 10 days after
oviposition (Table 3). It has light green color
with seta in entire body. The body length was
about 6 to 7 mm, it has 3 thoracic legs and 5
prolegs (abdominal legs) (Figure 5). A. atlas
hatched by cracking the eggshell.
Table 3 Percentage of egg hatching
% Hatching
Couple
Day 8
Day 9
Day 10
1
97%
0
2%
2
95%
2%
1%
3
90%
3%
0
4
100%
0
0
Mean
96%
1%
1%
Temperature effect on embryogenesis
The cold temperature treatment caused the
unsuccessful hatching to A. atlas embryo. The
control eggs for 1 week preservation hatched
97 %, for 2 weeks preservation hatched 93%,
for 3 weeks preservation hatched 89%, while
for 4 weeks preservation hatched 100% (Table
4 to 7).
Table 6 Seven day old A. atlas egg preserved in 10oC
for 3 weeks
Preservation time
0 week
(control)
1 week
C
S
Preservation time
Treatment
0
97%
N
0 week
(control)
C
0
S
0
N
2 weeks
0
93%
0
0
C : Cold treatment without medium
S : in medium sucrose 0.5 M
N : in medium NaCl 0.89%
Table 5 Seven day old A. atlas egg preserved in 10oC
for 2 weeks
Table 7 Seven day old A. atlas egg preserved in 10oC
for 4 weeks
Preservation time
Treatment
0 week
(control)
C
S
N
89%
3 weeks
Preservation time
Treatment
0
C
0
S
0
N
0 week
(control)
4 weeks
100%
0
0
0
4
Figure 2 A. atlas having copulation. Female
A. atlas (♀) was hanging on the cage,
while the male (♂) was hanging on
female’s body.
a
Figure 3 The embryonic development of A. atlas.
(1 to 4 day old) within the eggshell after
being immersed by NaOH 5 M
overnight. mcp: micropyle.
b
c
d
e
f
Figure 4 The embryonic development of A. atlas (5 to 7 day old). 5th day embryo within eggshell (a); 5th day
embryo without eggshell (b); 6th day embryo within eggshell (c); 6th day embryo without eggshell (d);
7th day embryo within eggshell (e); 7th day embryo without eggshell (f); ant: anterior; cep: cephal; post:
posterior; prl: prolegs; trl: thoracic leg; T1-T3: thoracic segments; A1-A8: abdominal segments
5
Figure 5 The first day larva (instar 1). ant: anterior;
cep: cephal; post: posterior; prl: prolegs;
trl: thoracic legs.
DISCUSSION
Insect embryogenesis occurs inside the
egg directly after the fertilization. It is divided
into four main processes, cleavage (formation
of blastoderm), gastrulation, differentiation,
and growth (Tazima 1978; Sander et al 1985).
In the stereomicroscopic observation of A.
atlas embryo, the only embryogenic stages
that can be seen are pre-organogenesis stage
and organogenesis stage.
Pre-organogenesis stages.
The 1st to 4th day old embryo of A. atlas
didn’t show a specific difference that could
marked it into one of embryo developmental
stage. The large amount of yolk covered the
zygote, so that the stereomicroscopic
observation could not be performed. The 1st to
4th day old embryo only can be categorized as
the pre-organogenesis stage. Oppenheimer
(1944)
mentioned
that
insect
has
centrolecithal egg with a large amount of
yolk. In the eggs with highly concentrated
regions of yolk, cleavage occurs only in the
non-yolky cytoplasm. The cleavage begins
with mitotic division of zygote nuclei (without
cytokinesis) in yolk that occurred in several
cycle. This process performs hundred nuclei
that often called as energids. The energids
then migrated from the yolk to the peripheral
surface of the egg and continued the mitotic
division. This division performs one-cell-thick
layer of cells surrounding the yolk in the
peripheral surface that is called blastoderm,
while the central yolky part enclosed by
blastoderm is known as yolk sac or yolk
system (Tazima 1978). In this time, cell
separate from the yolk and use the yolk for its
nourishment. This process is known as
superficial cleavage (cleavage over the yolk
surface). The first day of A. atlas embryo may
reach the formation of blastoderm comparing
with B. mori that reached blastoderm
formation in 12 hours after oviposition
(Tazima 1978).
According to Tazima (1978), a dense
embryonic primordium will be performed as
soon as the blastoderm completed. The cells
inside the presumptive embryonic primordium
area increase in thickness and become
concentrated. This area in the blastoderm
extends broadly on the ventral to ventrolateral
side of egg and known as germ band. The
second day observation of A. atlas embryo
may has the complete germ band formation
compare with B. mori that completed germ
band formation right after blastoderm was
established in 20 hours after oviposition
(Tazima 1978).
After germ band was establish, it was
grow to transform three germ layers.
Gastrulation is the process when the germ
band enlarges and begins to lengthen and fold
into a sausage shape. The one layer of cells
that is located on the outside forms the
ectoderm and another layer of cells on the
inside form the mesoderm.
Ectoderm cells grow and differentiate to
form the epidermis, the brain and nervous
system, and most of the insect's respiratory
(tracheal) system. In addition, the ectoderm
invaginates (folds inward) at the front and rear
of the embryo's body to create front and rear
portions of the digestive system (foregut and
hindgut). Mesoderm cells differentiate to form
other internal structures such as muscles,
glands, heart, blood, fat body, and
reproductive organs. The midgut develops
from a third germ layer (the endoderm) that
arises near the fore and hindgut invaginations
and eventually fuses with them to complete
the alimentary canal (Sander et al 1985).
Embryo of B. mori begins the gastrulation in
25 hours after oviposition (Tazima 1978).
Electron microscopic scanning of the
developing Manduca sexta shows the
gastrulation and differentiation begins at 33
hours after oviposition (Richard et al 1988).
Embryo of A. atlas may begins the
gastrulation stage in the 2nd day
After completing the gastrulation and
differentiation of three germ layers, the
embryo moves to the surface (peripheral) of
egg, this movement is a part of blastokinesis.
Tazima (1978) reported that B. mori takes
this stage in two days (4th and 5th day), in A.
atlas it may be occurred in the 3rd or 4th day.
6
Organogenesis stages.
Organogenesis in insect embryo was
including differentiation of body segments
and growth of embryo. The fifth day embryo
of A. atlas has reached the organogenesis
stage, and the organogenesis continued to the
7th day. According to Sander et al (1985),
organogenesis could be started during late
germ band stage and it takes several days or
even weeks depends on specific mechanism
and characters of each insect egg. The fifth
day embryo of A. atlas showed the complete
segmentation body, but the formation of
appendages and differentiation of cephal
section was not complete yet. The embryo has
3 thoracic segments and 8 abdominal
segments. The embryo of B. mori reached the
complete segmentation body in 6th day
(Tazima 1978).
In the sixth day, the embryo of A. atlas
reached the stage in which all appendages are
formed, the thoracic legs are completely
formed and so did the prolegs (abdominal
legs). Three pairs of thoracic legs grow in
three thoracic segments, while the prolegs are
found in the 3rd, 4th, 5th , 6th , and last segment
of abdominal segments. The section of cephal
has also performed well. The cephal was
consisted of 3 segments that performed
maxilla, mandible, and labium (Figure 6). In
this stage, the pigmentation at the cephal was
beginning, while B.mori embryo begins
pigmentation of the cephal in 8th day (Tazima
1978).
The seventh day old embryo has the same
morphological structure with the larva, which
make this stage of embryo is known as
prelarva embryo or mature embryo. Besides
pigments are formed in entire body (including
epithelium and seta), the embryo also grow
bigger than the 6th day. After reaching this
stage the embryo is ready to hatch.
hatch or hatch through the hatching line
(Sander et al. 1985). Different from B. mori
that hatched in 9 or 10 days after oviposition
(Tazima 1978).
Temperature effect on embryogenesis
Insect embryogenesis was affected by
many factors, such as: temperature, humidity,
genetic, and oxygen consumption (Sander et
al. 1985).
As poichilotherm organism,
temperature has a big effect to insect embryo
development. The exposure of cold
temperature
could
caused
abnormal
development even death to the embryo
(Yamashita & Hasegawa 1985). But some
insect in temperate region has ability to
survive from the winter by suppressing the
metabolism through diapauses. The univoltine
B. mori was the common model to study the
diapause in Lepidotera (Umeya 1950). While
the polyvoltine one was the common model of
artificial hibernation. The embryogenesis of
polyvoltine B. mori can be postponed in a
month by refrigerating it in 5oC (Katsumata
1964).
A. atlas was polyvoltine and used to life in
tropical region. The cold temperature
exposure on 7 days old A. atlas embryo
caused the unsuccessful hatching. No embryo
of A. atlas could hatch after being stored in 10
O
C for several times (Appendix 2). In
polyvoltine B. mori, the egg that would be
preserved should reach the mature stage
(ready to hatch) that was indicated by the
appearance of dark blue dot in the eggshell
(Katsumata 1964).
This unsuccessful hatching might be
caused by the lack oxygen demand or
dehydration because the embryo was wrinkle
(Appendix 3). Although some embryo was
preserved in Sucrose 0.5 M and NaCl 0.89%
medium, it did not prevent the embryo from
dehydration.
CONCLUSION
Figure 6 Cephal section of A. atlas embryo.
l: labium; max: maxilla; man: mandible
Hatching
A. atlas larva hatched in 8 days after
oviposition by cracking the eggshell. Some
insect embryo is used to crack the egg shell to
The embryonic development of Attacus
atlas occurred in 7 days. The morphological
observation showed that the embryogenesis
could be divided as pre-organogenesis and
organogenesis stage. The 1st to 4th day old
embryo could not be observed by the stereo
microscope and it may be occurred the
cleavage to gastrulation stage, while the 5th to
7th day old has reached the organogenesis
stage. The exposure of cold temperature
caused unsuccessful hatching in A. atlas.
7
SUGGESTION
To observe the details of embryonic stage,
observations are recommended to be done in
every 3 to 6 hours. The serial slicing specimen
with histological staining is recommended to
observe
the
complete
process
of
embryogenesis. The using of SEM (Scanning
Electron Microscope) is recommended to
study the embryo movement.
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Umeya Y. 1950. Studies on embryonic
hibernation and diapause in insects. Bul
Sericul Exper Station Tokyo 26:1–9.
Yamashita O, Hasegawa K. 1985. Embryonic
Diapause. In: Kerkut GA, Gilbert LI,
editors. Comprehensive InsectPhysiology,
Biochemistry and Pharmacology. Vol. I.
Embryogenesis
and
Reproduction.
Oxford: Pergamon Press. 407–434.
8
APPENDIX
95
Appendix 1 Temperature and humidity in breeding room
Date
120209
130209
140209
150209
160209
170209
180209
190209
200209
210209
220209
230209
240209
250209
260209
270209
280209
Mean
Morning (08.00)
T (°C)
Rh
27
80
26
78
28
65
28
75
27
80
26
79
26
81
28
77
29
68
28
84
26
75
26
81
26
85
27
78
26
85
27
75
27
85
26.94±0.97 78.29±5.62
Afternoon (16.00)
T (°C)
Rh
27
70
28
70
28
65
27
70
27
82
28
72
27
80
29
75
28
70
27
70
27
71
27
70
26
73
29
62
30
60
31
65
27
80
27.82±1.29 70.88±6.04
EMBRYONIC DEVELOPMENT OF Attacus atlas L.
(LEPIDOPTERA: SATURNIIDAE)
YUSTINA YUSUF
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATIC AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2009
ABSTRACT
YUSTINA YUSUF. Embryonic Development of Attacus atlas L. (Lepidoptera; Saturniidae).
Supervised by DEDY DURYADI SOLIHIN and ARIEF BOEDIONO.
Attacus atlas was one of wild silkworm species that has been developed in Indonesia as an
alternative silk source to domestic silkworm Bombyx mori. A. atlas was spread in tropical and
subtropical rain forest and can be found almost in entire Indonesian islands. It has complete
metamorphosis stage, including egg (embryo), larvae, pupa, and imago (adult). Embryogenesis
was an important stage to know the physiology characteristic of insect such as diapauses. The
research aimed to know the embryogenesis of A. atlas. The observation of embryonic development
was done in two ways, observation within the eggshell and without the eggshell. The
embryogenesis in A. atlas was done in seven days. Morphological observation on A. atlas embryo
can be divided as pre-organogenesis and organogenesis stage. Pre-organogenesis stage occurred in
first to fourth day after oviposition. Organogenesis stage can be observed in fifth to seventh day
after oviposition. In the fifth day, body segmentation was completed. In the sixth day, cephal
section and patterning of appendages was completed. Besides, the pigmentation of cephal also
occurred in sixth day. In the seventh day, the pigmentation of entire body was completed and
embryo was ready to hatch. In the eight day after oviposition embryo was hatching by cracking the
eggshell.
Key words: Attacus atlas, embryogenesis, development.
ABSTRAK
YUSTINA YUSUF. Perkembangan Embrio Attacus atlas L. (Lepidoptera; Saturniidae).
Dibimbing oleh DEDY DURYADI SOLIHIN and ARIEF BOEDIONO.
Attacus atlas adalah salah satu species ulat sutera liar yang dikembangkan di Indonesia
sebagai alternatif penghasil sutera dari ulat sutera domestik Bombyx mori. A. atlas tersebar di
daerah tropis dan dapat ditemukan di seluruh wilayah Indonesia. A. atlas memiliki metamorfosis
sempurna mencakup fase telur, larva, pupa, dan imago. Perkembangan embrio adalah tahapan
penting untuk mengetahui karakteristik fisiologi serangga, seperti diapause. Penelitian ini
bertujuan untuk mengetahui perkembangan embrio A. atlas. Pengamatan perkembangan embrio
dilakukan dengan dua cara, yaitu pengamatan embrio dalam cangkang telur dan tanpa cangkang
telur. Tahapan perkembangan embrio pada A. atlas terjadi selama 7 hari. Pengamatan terhadap
perkembangan morfologi embrio A. atlas hanya dapat dibagi menjadi tahap, pre-organogenesis
dan organogenesis. Tahap pre-organogenesis terjadi pada 1 sampai 4 hari setelah oviposisi dan
tahap organogenesis dapat diamati pada hari ke-5 sampai 7. Pembentukan segmentasi tubuh telah
selesai pada hari ke-5 setelah oviposisi. Perkembangan embelan dan segmentasi kepala telah
selesai pada hari ke-6 setelah oviposisi, pigmentasi pada kepala juga terjadi di hari yang sama.
Pigmentasi keseluruhan tubuh terjadi pada hari ke-7 setelah oviposisi. Larva menetas pada hari ke8 setelah oviposisi dengan memecah kulit telur.
Kata kunci: Attacus atlas, perkembangan embrio, perkembangan
1
INTRODUCTION
Background
The sericulture of silkworm in Indonesia
has begun since 10th century (Atmosoedarjo et
al. 2000). So far, the cocoons of domestic
silkworm Bombyx mori have been used as the
source of silk fiber. According to BKPPMD
(Bureau of Local Region Promotion and
Infestation Coordination) (2006), almost 70%
of raw silk material (cocoons) has been
imported from China and India. Meanwhile
Indonesia has many species of wild silkworm
that can produce silk fiber. Therefore, the
Yogyakarta royal family initiated the wild silk
moth development project in Indonesia in
1994. The project aimed to introduce new
material of wild silk as an alternative to the
domestic silk. The wild silk worms in
Indonesia, on which the project focused, are
Attacus atlas and Cricula trifenestrata
(Nurmalitasari & Kuroda 2002).
Based on its taxonomy A. atlas is
classified into kingdom Animalia, phylum
Arthropoda, subphylum Atelocherata, class
Hexapoda (insecta), order Lepidoptera,
superfamily
Bombycoidea,
family
Saturniidae, subfamily Saturniinae, genus
Attacus, species A. atlas L. (Triplehorn &
Johnson 2005). It is known as giant silk moth
or “si rama-rama” and it is considered to be
the largest moth in Asia with the wingspans
about 250 mm. A. atlas is primarily found in
tropical forest and spread around South East
Asia, Southern China, and India. It can be
found almost in entire Indonesian islands
(Peigler 1989).
A. atlas has a perfect metamorphosis stage
that included egg (embryo), larval, pupae, and
adult stage (Figure 1). According to its annual
breeding, A. atlas is polyvoltine, which means
that it has more than two generation in a year
while B. mori has two characteristic
polyvoltine and bivoltine. Bivoltine here
means that it has only two generation in a
year. Therefore, A. atlas can be found all over
year (Chapman 1971). The larva of A. atlas is
polyphage that means it has more than one
host plant, such as cinchona, cinnamon, tea,
sour soup, and avocado (Kalshoven 1981).
The adult female lay about 200 to 250 eggs
that oviposited in 3 to 5 days in stage and will
hatch 8 days after oviposition. A. atlas eggs
are oval in shape, 0.008 gram in weight, 2 mm
in length, and 1.5 mm in width (Peigler 1989).
a
b
c
d
Figure 1 The developmental stage of A. atlas. Egg (embryo)
(a); larva (b); Pupae (c); imago (adult) (d).
Insect embryogenesis is an interesting
field of study. The embryonic development of
insect occurs in the egg directly after
fertilization
ended.
The
stage
of
embryogenesis in each insect can be different,
depends on temperature of environment and
species characteristics of development
(Yamashita & Hasegawa 1985). For example,
B. mori (Bombycidae) has 17 developmental
stage in 9 days (Tazima 1978) while another
Lepidoptera
Nemophora
albiantennella
(Adelidae) has 12 developmental stage in 7
days (Kobayashi 1997).
Observation of insect embryogenesis is
important in order to know the characteristic
of each embryonic stage. One of the important
physiological characteristic that is used to be
studied at the embryonic stage is diapauses.
Diapause is a condition in which animal’s cell
growth and development are reversibly
stopped or slowed (Yamashita & Hasegawa
1985). In sericulture this condition was very
useful. Because, by stopping the development
the egg hatching also was postponed. These
diapauses egg could be used as egg stock for
further sericulture program. Although much of
insect embryology is still a mystery, there are
remarkable progresses in some insect like B.
mori.
Nevertheless,
the
embryonic
development of A. atlas has not been observed
yet.
2
Objective
The research aims to know the embryonic
development of Attacus atlas in order to know
the possibility of artificial hibernation and
accurate time to postpone hatching.
MATERIALS AND METHODS
Materials and Equipments
Materials that were used for this research
were eggs A. atlas, NaOH 5M, glycerol 70%,
lacto phenol, and alcohol 70%. Equipments
that were used for the research were tweezers,
ependorf
tube,
serum
bottle,
stereomicroscope, digital camera Nikon
COOLPIX S210, petridish, and pipette.
Time and Place
This research was conducted in April 2008
to March 2009 in Laboratory of Molecular
Biology Research Center for Bioresources and
Biotechnology (PPSHB) and Laboratory of
Embryology Faculty of Veterinary Medicine,
Bogor Agricultural University (IPB).
Methods
Moth copulation and egg collection
A couple of A. atlas moth was isolated
from the population and separated into
different cage to have breeding. After
copulation ended, eggs from each couples was
collected in petridish. Egg collection was done
everyday at 11 to 12 am. From 13 couples that
has been breed, 4 couples produced the
highest amount of egg. The fertile egg from
these 4 couples then was used for observation
of embryogenesis and treatment of
temperature effect on embryogenesis.
Observation of embryonic development
The embryonic development of A. atlas
was observed in 7 days. The observation was
done everyday (every 24 hours) from 1 day
old to 7 day old after oviposition. It was done
in two ways, observation within egg shell and
observation without eggshell.
Observation of embryonic development
within the eggshell. Ten eggs by the age of 1
to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 70 eggs.
Those eggs then were immersed in NaOH 5 M
overnight and moved into glycerol 70% to be
stored. The egg then was observed by using
stereomicroscope.
Observation of embryonic development
without the eggshell. Five eggs by the age of
5 to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 15 eggs.
Those eggs were opened by cracking the
eggshell. The embryo then was taken and
moved into alcohol 70%. After that, the
embryo was moved into lacto phenol
overnight to be cleared and stored in glycerol
70%. The embryo then observed by using
stereomicroscope.
Temperature effect on embryogenesis
The egg was preserved in 5oC (in
refrigerator) to know temperature effect on
embryogenesis. The egg prese
rvation was done in three treatment,
preservation without medium, preservation in
sucrose 0.5 M, and preservation in NaCl
0.89%. Ten eggs were used for each
treatment. Preservation was done in 1, 2, 3,
and 4 weeks. Total eggs that have been used
are 120 eggs. After preservation, eggs then
were placed in the breeding room.
RESULT
A. atlas got copulation overnight (Figure 2).
The copulation begun at night, about 10 pm to
2 am and it ended at 8 am to 11 am (Table 1).
Female A. atlas produced about 326 to 462
eggs in five days (Table 2). The period of
embryonic stage (egg) was about 7 days in
room temperature (26 to 31oC) (Appendix 1).
Table 1 Copulation time of A. atlas
Copulation time
Couple
Begin
Ended
1
09.57 pm
08.44 am
2
01.07 am
09.38 am
3
01.07 am
10.55 am
4
01.15 am
10.13 am
Table 2 Total number of egg produced by A. atlas couple
Egg produced by couple A. atlas
Couple
Total
number
of egg
1
Day
1
119
Day
2
92
Day
3
91
Day
4
33
Day
5
7
2
171
63
39
23
30
326
3
47
153
70
39
38
347
4
124
214
64
35
25
462
Mean
115.3
130.5
66
32.5
25
369.3
342
2
Objective
The research aims to know the embryonic
development of Attacus atlas in order to know
the possibility of artificial hibernation and
accurate time to postpone hatching.
MATERIALS AND METHODS
Materials and Equipments
Materials that were used for this research
were eggs A. atlas, NaOH 5M, glycerol 70%,
lacto phenol, and alcohol 70%. Equipments
that were used for the research were tweezers,
ependorf
tube,
serum
bottle,
stereomicroscope, digital camera Nikon
COOLPIX S210, petridish, and pipette.
Time and Place
This research was conducted in April 2008
to March 2009 in Laboratory of Molecular
Biology Research Center for Bioresources and
Biotechnology (PPSHB) and Laboratory of
Embryology Faculty of Veterinary Medicine,
Bogor Agricultural University (IPB).
Methods
Moth copulation and egg collection
A couple of A. atlas moth was isolated
from the population and separated into
different cage to have breeding. After
copulation ended, eggs from each couples was
collected in petridish. Egg collection was done
everyday at 11 to 12 am. From 13 couples that
has been breed, 4 couples produced the
highest amount of egg. The fertile egg from
these 4 couples then was used for observation
of embryogenesis and treatment of
temperature effect on embryogenesis.
Observation of embryonic development
The embryonic development of A. atlas
was observed in 7 days. The observation was
done everyday (every 24 hours) from 1 day
old to 7 day old after oviposition. It was done
in two ways, observation within egg shell and
observation without eggshell.
Observation of embryonic development
within the eggshell. Ten eggs by the age of 1
to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 70 eggs.
Those eggs then were immersed in NaOH 5 M
overnight and moved into glycerol 70% to be
stored. The egg then was observed by using
stereomicroscope.
Observation of embryonic development
without the eggshell. Five eggs by the age of
5 to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 15 eggs.
Those eggs were opened by cracking the
eggshell. The embryo then was taken and
moved into alcohol 70%. After that, the
embryo was moved into lacto phenol
overnight to be cleared and stored in glycerol
70%. The embryo then observed by using
stereomicroscope.
Temperature effect on embryogenesis
The egg was preserved in 5oC (in
refrigerator) to know temperature effect on
embryogenesis. The egg prese
rvation was done in three treatment,
preservation without medium, preservation in
sucrose 0.5 M, and preservation in NaCl
0.89%. Ten eggs were used for each
treatment. Preservation was done in 1, 2, 3,
and 4 weeks. Total eggs that have been used
are 120 eggs. After preservation, eggs then
were placed in the breeding room.
RESULT
A. atlas got copulation overnight (Figure 2).
The copulation begun at night, about 10 pm to
2 am and it ended at 8 am to 11 am (Table 1).
Female A. atlas produced about 326 to 462
eggs in five days (Table 2). The period of
embryonic stage (egg) was about 7 days in
room temperature (26 to 31oC) (Appendix 1).
Table 1 Copulation time of A. atlas
Copulation time
Couple
Begin
Ended
1
09.57 pm
08.44 am
2
01.07 am
09.38 am
3
01.07 am
10.55 am
4
01.15 am
10.13 am
Table 2 Total number of egg produced by A. atlas couple
Egg produced by couple A. atlas
Couple
Total
number
of egg
1
Day
1
119
Day
2
92
Day
3
91
Day
4
33
Day
5
7
2
171
63
39
23
30
326
3
47
153
70
39
38
347
4
124
214
64
35
25
462
Mean
115.3
130.5
66
32.5
25
369.3
342
2
Objective
The research aims to know the embryonic
development of Attacus atlas in order to know
the possibility of artificial hibernation and
accurate time to postpone hatching.
MATERIALS AND METHODS
Materials and Equipments
Materials that were used for this research
were eggs A. atlas, NaOH 5M, glycerol 70%,
lacto phenol, and alcohol 70%. Equipments
that were used for the research were tweezers,
ependorf
tube,
serum
bottle,
stereomicroscope, digital camera Nikon
COOLPIX S210, petridish, and pipette.
Time and Place
This research was conducted in April 2008
to March 2009 in Laboratory of Molecular
Biology Research Center for Bioresources and
Biotechnology (PPSHB) and Laboratory of
Embryology Faculty of Veterinary Medicine,
Bogor Agricultural University (IPB).
Methods
Moth copulation and egg collection
A couple of A. atlas moth was isolated
from the population and separated into
different cage to have breeding. After
copulation ended, eggs from each couples was
collected in petridish. Egg collection was done
everyday at 11 to 12 am. From 13 couples that
has been breed, 4 couples produced the
highest amount of egg. The fertile egg from
these 4 couples then was used for observation
of embryogenesis and treatment of
temperature effect on embryogenesis.
Observation of embryonic development
The embryonic development of A. atlas
was observed in 7 days. The observation was
done everyday (every 24 hours) from 1 day
old to 7 day old after oviposition. It was done
in two ways, observation within egg shell and
observation without eggshell.
Observation of embryonic development
within the eggshell. Ten eggs by the age of 1
to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 70 eggs.
Those eggs then were immersed in NaOH 5 M
overnight and moved into glycerol 70% to be
stored. The egg then was observed by using
stereomicroscope.
Observation of embryonic development
without the eggshell. Five eggs by the age of
5 to 7 days old after oviposition were selected
from each couple. Total eggs that have been
selected from each couple were 15 eggs.
Those eggs were opened by cracking the
eggshell. The embryo then was taken and
moved into al