SILKWORM EGG SCIENCE

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◆ Overall introduce of achievement
I. The history of our laboratory KNU
- 1987 " Silkworm Genetics and Breeding"
- 2000 "Insect Genetic Resources“
- 2010 “Lab. Genetic Resources of Silkworm”
(National BioResources Project (RDA) - National Design Lab)

II. Main subject (Research and Teaching)

- Silkworm Genetics and Breeding
- Silkworm Egg Science
- Insect Functional Utilization
- Insect Molecular Biology

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(1) Several fields are combined

(2) A crop finished shortly

(3) Complicated operation

(4) Light labor

(5) Product high marketability

(6) Yong larvae can be reared cooperatively

(7) Required technics and knowledge

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- Silkworm eggs are sensible to change of temperature, causing bad influence
easily in a long distant transport. Recent years silkworm eggs has become
common as having less influence in transport if employing air transport
system.

- However, any country has to establish such principle as they produces

necessary amount of silkworm eggs in the country

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 The objective of improvement varies depending upon the period of
requirements. One of the most important aspects of improvement is to
increase productivities.

* The following can be considered as the major objectives ;

(1) Healthy and strong variety

- This refers particularly to the health of larvae and pupae. If the larvae
are healthy and strong, rearing become smooth, easier and crop is steady,
with a higher cocoon yield.

- Particularly, during rearing season, when the temperature as well as the

humidity is high and also mulberry leaves are not of desired quality, it's
desirable to rear the silkworm varieties which are strong and healthy.

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(2) Egg Production
- The variety should be such that higher normal eggs laid, the collecting
efficiency is good and the eggs have a higher rate of hatchability.

(3) Cocoon yield

- If the larvae are strong and healthy, the cocoon yield increase.
However, if the cocoon weight is higher, there is a tendency toward
weaken of health.
Therefore, a suitable balance between the cocoon yield and healthy
should be ensured.

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(4) Efficiency of silk production
- The quantity of cocoon against the unit quantity of total mulberry
should be higher and the yield of raw silk must be higher.

* Based on the General theory (Silkworm Egg Science) and

Temperate zones sericulture technics

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- The history of silk is as old as the history of mankind and so is the history of
silkworm. According to archaeological and bibliographical evidence, it is probable
that sericulture was practiced in China about 2,500 B.C. and Bombyx mandarina L.
a kind of wild silkworm were reared in North China be guessed. Repeated selection
and purification by humans for a long time has become the silkworm of today

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- Silkworm race is defined as a genetically, and as a general rule
geographically, isolated and distinct interbreeding unit of a species.

- Different climates and areas produced several region specific silkworm

races with different characteristics, due to geographical isolation for
long time.

- Silkworm races are classified by aboriginal region, named Chinese

Race, Japanese Race, European Race, Tropical Race and Korean Race.
Since the birth of genetics, extensive investigation have been carried
out with those the characteristics.
European Uni. ⇦ Chinese Uni. ⇨ Korean Uni.

⇩ ⇩
Chinese Bi. Japanese Bi.

⇩ ⇩
Tropical Poly Japanese Uni.
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- Besides the geographical classification, silkworm varieties are variously
classified according to the different in their physiological and ecological
characteristics such as voltinism, moltinism, cocoon color and shape,
larval marking and so forth.

- As mentioned above, silkworm varieties are classified into five groups

according to the original region of silkworm races.

- The major characteristics of the races are as fellows,

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 1) Classification by Regional Races
[Chinese Races]

- The silkworm larvae of Chinese races eat

mulberry leaves actively, and the progress of
larval growth is comparatively fast and
uniform.

- The larval marking of many races is plain

(picture reference) and the larvae are not
sensible to high temperature and muscardine
but sensible to high humidity.

- The cocoon shape are oval (elliptical) or

spherical in many races but some races make
spindle shape cocoon.
- The cocoon colors of this race is white,
golden yellow, flesh colored or red and pink.
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- The cocoon filament is thin and long and its reelability is good.

- Univoltine, bivoltine and polyvoltine are comprised in this races,

and a few three-moulter races are also included in this group,
though most races are four- moulters.

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[Japanese Races]

- Generally the larvae of this races are strong

and resistant to adverse environments but
larval period is comparatively long.

- The marking on the silkworm larvae are

normal in many races. (picture reference)
- Almost of this races cocoon shape is
constricted spinned shape and peanut
shape.
The cocoon color is usually white, but a few
races are green or yellow.

- These races produce more double cocoons

and slightly short and thick cocoon filament.
There are univoltine and bivoltine in this
races.
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[European Races]

- All races of this group are univoltine, and eggs are big and heavy
compared with the other races.
- The larval marking is lightly normal, and silkworm larvae eat mulberry
leaves actively. The duration of larval stage, especially 5th instar stage
is long and long larval body.

- The larvae grow fat easily but slightly difficult to rear as they are
sensible to bad environment, pebrine, muscardine and C type virus
disease.
- They spin large and long oval cocoons with little constriction, and
double cocoons are rare.
- The majority of cocoons are white or flesh colored.
- The cocoon shell weight of this race is high and cocoon filament is
long, with much sericin and good reelability.

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[Tropical Races]

- The tropical races are multivoltine, which

produce non-hibernation (non diapause) eggs
and has a white egg 3 (w-3, 10:19.6) gene.

- The silkworm larvae are tolerant of high

temperature and humidity, and the larval
duration is very short.
- This race has small and slender bodies of larvae
and produce small cocoon with spindle shape,
and green, yellow or white color.
- The cocoon are loose and flossy with light cocoon
shell weight and thin cocoon filament.
- They few double cocoons but sericin percentage
of these races is very high (about 30%), so raw
silk percentage is very low. (sericin silk , Thai Jim Thompson House Shop
traditional silk). However the cocoon filament is
thin and clean with little lousiness.
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Korean Races :

- Korean races are all univoltine and tri-molters, and larval

duration is comparatively short. The cocoon of these races are
small with low cocoon shell ratio and the cocoon color are almost
yellow, yellow green, and a few white. Short and thin cocoon silk
filament.

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 General characteristics of regional origin races

larval larval voltinism, cocoon

race silk
marking duration moltinism (shape, color..)

1, 2 vol. peanut or spinned,

Japanese normal m. longer, strong thick
teramoulter almost white

ellipse or oval,
1,2,poly,
Chineses plain m. shorter, weak white, yellow, flesh thin & long
3, 4moulter
etc

normal m. 1 vol.4 mol.

the longest, big long ellipse long & good
European eggs are big sensible to pebrine,
larva size white and flesh reelability
and heavy CPV

univolter, almost spindle, colored &

Korean various shorter thin (3 mol.)
trimolter white
flossy with
small, white, green
Tropical plain ? very short w-3, all non-diapause thin cocoon
& yellow
shell

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 2) Classification by Voltinism characteristics
- The genetic characteristics that determine the number of generation
in a year are called voltinism.
- There are univoltine, bivoltine and multivoltine (polyvoltine) in the
silkworm. The appearance of voltinism depends in part on the
environment, particularly temperature and light condition, and
genetical base.

- The voltinism of the silkworm is greatly influenced by environment

conditions during the embryonic development.
- If bivoltine eggs are incubated at 15 °C after blastokinesis of the
embyogenesis, the moths lay non-diapause eggs, whereas when the
eggs are incubated at 25°C, moths lay diapause eggs.
- However when incubation temperature is 20°C, lighting influence
the voltinism of the next generation. Lighting more than 16 hrs a
day results in univoltine while lighting less than 12 hrs a day results
in bivoltine.

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 The general characteristics are fellows,
Univoltine (one generation a year) :

- The univoltine races are suitable for the cold regions. Larval duration of
these races is long and the larval body is large in size.
- The cocoon filament is of good quality, but they are not suitable for the hot
season rearing because the larvae are not resistant to the bad
environmental condition such as high temp and humidity.

Bivoltine (two generation a year) :

- These races are suitable for the temperate zone. The duration of larval stage is
short compared to univoltine and the larvae are robust.
- The cocoon quality of these races are inferior to that of univoltine races but most
of present commercial varieties are bivoltine to which the characters of good
cocoon filament of univoltine races were introduced.
- They can be reared summer and autumn.

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Polyvoltine (more than 3 generations a year) :

- Polyvoltine (multivoltine) races are suitable for the tropical region.

- The duration of larval stage of these races is very short, and the larvae are
very robust and tolerant of high temp climate.

- The cocoons are small in size, and the percentage and the yield of raw silk is
poor.
However, the cocoon filament is thin in size and clean with little lousiness.

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 3) Classification by Moltinism characteristics
- Moltinism is one of the most important economic characters along with
voltinism.

- There are at least 5 different types of molting in the silkworm, namely di-, tri-,
tetra-, penta-, and hexamolters. The most common type in the primitive
domestic silkworm varieties and in the wild moths are trimolters, where as
almost all present-day commercial strains are tetramolters.
- Tetramolting character has been considered as the standard (normal) type in
silkworm genetics. The expression of molting character is also affected partly
by the sex-linked maturity genes (Lm).

- The dominance relations are M3 > +M > M5

- The manifestation of molting characters are also controlled by the balance of
juvenile hormone of corpus allatum and ecdysone of prothoracic gland.

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- In addition to the above, the silkworms may be classified by the size, the
shape and the color of silkworm eggs, the markings and color of newly
hatched larvae, the shape, the body color and marking of silkworm larvae,
the color and the shape of cocoons, and the morphology of pupae and
moths.

- 1912 F1 hybrid (K. Toyama), From 1914 Commercial F1 Hybrid egg

production

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- The oogenesis, spermatogenesis, and embryogenesis of the silkworm have
been well analyzed from the viewpoints of histology and genetics.

- Furthermore, silkworm eggs have a special feature, their hibernation ability,

which provides an important approach for the study of mechanisms of
inhibition during embryogenic development.
- These properties will be described briefly in this chapter.
- Methods of artificial hatching and preservation of hibernating eggs will also
be dealt with from a practical viewpoint.

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 3.1 Gametogenesis and Egg formation
3.1.1 Oogenesis
- In the larva a pair of ovaries are located separately on both sides
of the dorsal vessel under the epidermis in the 8th segment.

- By the 3rd instar the ovary becomes triangular in shape and is

distinguishable from the kidney-shaped testes. Each ovariole
gradually increase in size, taking a tubular form. It becomes
longer and resembles a curved ribbon.
- The ovarioles continue to grow with the progress of larval
growth, until they break from the capture and project into the
body cavity on the 2nd day of the pupal stage. They then show a
drastic growth in length and volume until eclosion.

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 3.1.2 Further development of the Oocyte during the Pupal stage
- One oocyte and 7 nurse cells, together with some 5,000
follicle cells aggregate into one groups arrange themselves in a
row in the ovarian tubule and oocyte egg cell lies proximal to
the nurse cell group toward the distal end.
- These cells increase in size, the egg cell initially lies close to the
nurse cell. This is connected with the nurse cells through a
cyctoplasmic passage.

- In the earlier stages the egg nucleus is spherical and is located

in the center of egg cell. When the egg grows further, the
nucleus is shifted toward the upper corner of the egg cell,
where it assumes a more or less irregular shape.
- After the nucleus has attained its final size, the egg cell
continues to grow, deposition more nutritive yolk substances.
Consequently the nucleus becomes relatively small compared
with the egg cell.

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 3.1.3 The Nurse cells and Follicle epithelium

- The nurse cells begin to act when the eggs grow. The nurse cells are
generally arranged in 2 rows like a turban and are very active in synthesis.

- The follicle epithelium begins to supply cortex substances to the egg cell.
In this way the egg cell receives various substances from both the nurse
cells, and the follicle cells in addition to the voluminous yolk substances
derived from blood proteins.
- When the egg cell is fully grown, the follicle cells secrete the chorion around
the egg and thus egg formation is completed.

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 3.1.4 Spermatogenesis

- Since spermatogenesis has been described in detail by Y. Tajima.

- Spermatogenesis includes all the successive processes, divisions and
transformation, undergone by germ cells in the formation of spermatozoa.

Approximate Duration of Each Stage of Spermatogenesis

---------------------------------------------------------------------------------------
Spermatogenic stage Duration
---------------------------------------------------------------------------------------
Primary spermatogonia 34 h
Secondary spermatogonia 5-6 days
(sum of 6 generations)
Spermatocyte
Resting stage < 1 day
Meiotic prophase I 9 days
Metaphase I – Anaphase II < 1 day
Spermatid stage 9 days
Spermatozoa (1-14 dyas)
Total spermatogenesis about 26 days
--------------------------------------------------------------------------------------

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 3.2 Fertilization and Embryogenesis

3.2.1 Silkworm egg

A; anterior pole, P ; posterior pole, V; ventral side, D; dorsal side,

mp; micropyle, c; chorion, y; yolk, en; egg nucleos, p; periplasm

- Size : long length 1.2~1.3mm, short length 1.0 mm, thickness 0.6 mm
The size of eggs is determined by the variety. Even the same varieties
vary in rearing condition, rearing time, environment, temperature.
- Generally egg size is European race > Japanese race > Chinese

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 3.2.2 Chorion (Egg shell)
- At the last stage of egg formation, chorion protein are
secreted from follicle cells to form.
- The chorion weigh is about 10% of egg weight.
- It depends on location of egg
and variety.

- The eggshell has elasticity to protect

the contents of eggs.
- There is a aeropyle for embryo breath
and micropyle for fertilization.

The Out figure of micropyle region of silkworm egg

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Out surface figure of lateral side of silkworm egg

- The aeropyle structure on cross-section of silkworm chorion

- Inner layer – trabecular layer, Middle and outer layer – lamellar layer

- Chorion thickness : European race > Japanese race > Chinese race

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The surface of egg-shell of wild silkworm, Antheraea pernyi

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Antheraea yamamai

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3.2.3 Inner structure of egg

A : anterior pole
P : posterior pole
V : ventral side (胚子形成側)
D : dorsal side (背側)
m : micropyle (mp)
C : chorion
V : vitelline membrane (em)
n : egg nucleous (제1성숙분열中期)
y : yolk
p : periplasm

- Egg shell
- vitelline membrane
- serosal membrane
- periplasm
- yolk (protein and lipid yolk)

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3.2.4 Fertilization

od; oviduct, op; ovipositor, gm; glandula mucosa,

ip; intestine postrius bc; bursa copulatrix,
rs; receptaculum, vt; vestivulum

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 M; micropyle, pb; polar body,
sn; sperm nuleus (psn),
En; egg nucleus (pen),
f n; fertilized nucleus

- Fertilization : 120 ~140min after oviposition, 25℃

- The egg nucleus just after oviposition is metaphase of the first maturation
division (meiosis). The second meiosis takes place about 60min after
oviposition and maturation of the egg nucleus is completed after the second
polar body is expelled.

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- A sperm enters into the ovum and stays in the anterior region of the egg until the
completion of the egg pro-nucleus.
- In the meantime, the tail of the sperm separates from the head ; near the top of
the egg a centrosome appears and an aster is formed.
- As soon as the maturation division is completed and the female pro-nucleus is
produced, the male and female pro-nuclei approach and finally fuse.

- During this period, we should be pay totally attention to egg handling (no shaking,
no moving and suitable temp. and humidity, 24~25 ℃, 60~70%)

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 3.2.5 Embyogenesis (Embryo development)
It is important to understand the embryonic development of silkworm
eggs, for a understanding and proper preservation of the eggs

Diagrammatic representation of embryonic development in the silkworm.

a : fertilization, 2 h after oviposition ; b : early cleavage, 8 h after ovi. ; c: late cleavage ;

d : formation of blastoderm ; e : formation of germ band, 15h after ovi. ; f : germ band, 24h after
ovi. ; g : gastrulation ; h : formation of stomodeum and proctodeum, 2 days after ovi ;
I : formation of appendages, 3 days after ovi. ; j : blastokinesis, 4days after ovi. ;
k : completion of embryo, 8-10days after ovi.

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 (1) Cleavage, blastoderm and germ band formation

- After fertilization, cleavage proceeds through a succession of synchronous

mitotic divisions of the zygote nucleus and its daughter nuclei migrate
through the yolk mass.
- The mitotic spindles of each synchronous division are randomly oriented and a
number of nuclei progressively approach the periplasm.
- The rate at which synchronous divisions proceed within the yolk mass is one
mitotic cycle per 60min (11 times synchronous mitosis).

- When the cleavage nuclei migrate to the periplasm surrounding the yolk mass
and indicate the formation of the blastoderm, a number of nuclei remain
within the yolk masses.

- Blastoderm formation occurs about 12h after oviposition.

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 Blastoderm formation (left) and
germband formation (right)

- At about 15hours after oviposition, the cells inside the presumptive

embryonic primordium area increase in thickness and become
concentrated, still increasing in number through cell divisions.

- This area in the blastoderm extends broadly on the ventral to ventrolateral

side of the egg and is called the germ band.

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 (2) Growth of the embryo

- These are followed rapidly by a surface partitioning whereby the body

segmentation and appendages are set out. The early embryonic
development -> diapause -> organogenesis -> blastokinesis

- The germ band grows by cell division and differentiation, and the band
differentiates into the three germinal layers ; ectoderm, mesoderm and
endoderm.
- These are followed rapidly by a surface partitioning whereby the body
segmentation and appendages are set out.

am; amnion membrane, c; chorion,

ect; ectoderm, em; vitelline membrane,
fc; egg cavity, mes; mesoderm,
mp; micropyle, ser; serosal membrane,
y; yolk

Diapause stage embryo and cross section figure of diapause egg

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 (3) Blastokinesis

- The silkworm embryo, like other species of Lepidoptera undergoes marked

changes in position and orientation within the egg space as development
proceeds.
- This embryonic movement is referred to as blastokinesis.

21A ; Before the blastokinesis

21C ; After the blastokinesis

- After about 10 days from the time of oviposition,

the formation of embryo is completed, as shown in figure

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The Timetable of embryogenesis of the silkworm at 25℃
Time period Remarks
0-2 h The second maturation division and syngamy occur
Progress of synchronous cleavage mitosis is followed by
2-10 h
uniform invasion of the periplasm.
12 h The syncytial blastoderm is established
The germ band is formed
20 h
(Suitable for common acid treatment for artificial hatching)
Gastrulation takes place, accompanied by elongation of the
25 h
germ band
Segmentation of the mesoderm is completed and 17 segments
35 h
can be clearly observed
The appendages at the head and thoracic region develop.
40 h
At this stage, the egg enters diapause in univoltines.
Differentiation of the appendages at the abdominal region
48 h
occurs.
Invagination of the proctodeum takes place and reaches the
2.5 days
between 18th and 19th segment.
3 days Invagination of the trachea is initiated.
Invagination of the proctodeum proceeds and reaches the 16th
3.5 days
segment.
4 days Blastokinesis has begun
5 days Blastojinesis is completed
6 days External processes formed
7 days Taenidium in spiral band is formed in the tracheal tube.
8 days Pigmentation begins at the head.
9 days Pigments formed at seta and epithelium
9.5 days Hatching
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 (4) Differentiation of ectoderm, mesoderm and endoderm

- From the ectoderm are formed the epidermis and its appendages, ocellus,
salivary gland, prothoracic gland, corpora allta, molting gland, oenocyte, silk
gland, fore-gut and hind-gut, Malpighian tube, trachea, nervous organ, and
outer reproductive organ.

- mesoderm - inner part of reproductive organ, muscles, dorsal vessel,

suboesophagial ganglion, fat body, blood cells and pericardial
glands.

- endoderm - only the mid-gut is formed

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4.1 Parental silkworm race (P1) rearing (seed cocoon production)

Production and distribution system of silkworm eggs in Korea

Department of Sericulture, Breeding of new leading varieties and multiplication of
RDA foundation silkworm eggs (p3 & p2)

Provincial Sericultural Station Production of parental silkworm egg (p1)

Rearing of parental silkworm (p1) and production of

Private Silkworm egg Producer
F1 hybrid silkworm eggs

Sericulture Farm Rearing F1 hybrid silkworm eggs for commercial use

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- The commercial sericulture (silk cocoon production) purpose and parental
silkworm rearing (seed cocoon production) purpose is different.

- The purpose of parental silkworm rearing is to produce the healthy larvae

and moth, non infected (pathogen-free) silkworm eggs.
- For that purpose, all process fit to silkworm physiological conditions.

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 4.1.1 Rearing parental race seed cocoons

- Rearing for seed cocoon is entrusted in private silkworm egg production

company or provincial station.
- Technically qualified supervisory personnel trained in seed cocoon
production and non technical staff for carrying out manual operations
must be employed.
- The chief of the seed cocoon producer must be a scientific and much
experience officer who can do ordinate the activities of the seed cocoon
producer with the station, parental race silkworm rearers and the actual
farmers for whom the seeds are intended.

- The egg production company must also have trained extension workers to
supervise and assist the seed cocoon producer and station to
synchronize their activities.

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 4.2 Egg production process
4.2.1 The development of oocyte
- After mounting of the parental silkworm, all environmental condition
must be adapted to physiological condition of pupae and oocyte
development.

- The 1 oocyte and 7 nurse cells are differentiate from the early 4th instar.
- One oocyte and 7 nurse cells, together with some 5,000 follicle cells
aggregate into one group.

- After the mid pupal stage, the egg cell goes into a late growth period,
when the chromosomes disappear and deposition of yolk substances
proceeds very rapidly.
- Finally, when the egg is nearly completed, the upper side, where it was
formerly flattened by nurse cells, also become convex as the latter cells
almost completely degenerate by the time of egg maturation.
- The egg assumes an oval shape, lying lengthwise in the ovariole connected
with adjacent eggs by bridges at both poles.

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- In the earlier stages the egg nucleus is spherical and
is located in the center of the egg cell.
- When the egg grows further, the nucleus is shifted
toward the upper corner of the egg cell, where it
assumes a more or less irregular shape.

- After the nucleus has attained its final size, the egg
cell continues to grow, depositing more nutritive
yolk substances.
- Consequently the nucleus becomes relatively small
compared with the egg cell.

bg; connected tissue, en; egg nucleus,

fc; follicle cell, nc; nurse cell
nn; nucleus of nurse cell,
oc; oocyte cell, y; yolk granule

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The process of oocyte development

(1) Early rounder stage

From end of 5th instar to pupal 1 day

(2) Flat bowl shape stage

Lower side of oviduct in 2nd day of pupae,

upper side of 5th day

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(3) Half lunar shape stage

Almost complete formation of follicle cell

(4) Degeneration of nurse cell stage

Nurse cells degenerate and oocyte connected,

influx of nutrients is recognized

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(5) Complete formation of follicle cell (6) Chorion secretion

Lower side of 7th day pupa and upper side of 10th day

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4.2.2 Seed cocoon preservation

The oxygen consumption during pupal stage

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 4.2.3 Separation of sexes
- Commercial seeds are hybrids. One of the seed cocoon race (parental race)
will be used as the male and the other as the female.
- Therefore, males and females of both the parent seed cocoons have to be
separated before crossing them. This process is called sex separation

- Theoretically sex separation can be done in

the larval and pupal stage using
morphological differences.

- Separation of the sexes in the larval stage

based on the presence of Ishiwata glands
in female and Herold gland in the male is
not only too time consuming, but can be
done during only few days after the fourth BM; black spot, C; caudal legs,
moult, it also involves handling the larvae H; Herold gland, SPR; Ishiwata gland
thus increasing the chances of injuring frontal (11 seg.), SPO; Ishiwata gland postal
(12 seg.)
them.

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- Sex separation in the pupal stage is done by one of two methods.

- In the first, the cocoons are cut open, the pupae removed and examined for
morphological differences between mal and female pupa, and sorted
accordingly.
- By manually cutting open the cocoon and examining the pupa, a single person
can separate 12,000~15,000 cocoons in 8 hrs.
- Automatic cocoon cutting machines have been developed which increase
efficiency tenfold , as 200kg (each kg 700 cocoons) can be sorted in the same
duration.
- The main advantage in manual sorting is that each pupa is examined
individually and chances of error are less. The drawback of this method is
that it involves handling the pupae and after sex separation the pupae have
to stored outside the cocoon.
- The naked pupa is very delicate and needs to be stored under optional
conditions. The naked pupae are covered with perforated paper kept under
constant temperature conditions.

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- The second method uses the differences in weight of cocoons of the two
sexes for their separation by a machine.

- Separation machine works on the principle that cocoons with above-

average weight are females and those below average, males. The error
is only 6~8%. The machine is initially adjusted to the average weight of
the batch.
- During operation, the machine sorts the cocoon into three categories,
one above the average of the batch (females), one below the average of
the batch (males) and third falling within range of the average.

- The third category of cocoons has to be sorted manually by cutting the

cocoon open as in the first method.

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4.2.4 Moth eclosion and mating
- After the completion of the pupal period,
the silkworm moths emerge by breaking
open the cocoon, called ‘eclosion’.

- The silkworm moth emerge in the early

morning in response to light. To achieve
uniformity in emergence of moths in the
lot, the cocoon (pupae) are kept in
darkness one day prior to the expected
date of emergence and suddenly exposed
on the day of emergence to bright lights
switched on early morning.
- The peak period of emergence for the day is
06:00~08:00. The figure of silkworm moth eclosion

- After collecting the emerged moths, the

lights are switched off and the remaining
pupae once again kept darkness until next
morning.

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- The major problem in egg production (moth mating) is synchronization of
emergence of two parental moths to be crossed for producing the seed.

- In order to achieve this matter, planning is necessary right from the

brushing of the parental races.
- Eggs for raising seed cocoons are so distributed to farmers that the
difference in rearing period and pupation period of the races is fully taken
into account.

- Even then, simultaneous emergence is not always achieved. Under these

circumstances, the cocoons having advanced pupae of emerged moths are
refrigerated at 5~7°C for 3~7 days depending upon the stage refrigerated.

- Chilling female moths before coupling is avoid as far as possible.

- The maximum duration for which the different stages can be safely stored
and the temperature at which they are to be stored are given, male pupae
and moth 7 °C for 7 days, female pupae 5°C for 2~3 days.

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 - The possible duration for which storage is needed can be inferred by
cutting the cocoon of the late emerging race and judging the age of the
pupa from its coloration.
- If the color of the compound eyes of pupa has changed from brown to
black, the pupa has completed half its pupal duration, if its antennae are
black it is likely to emerge in 2~3 days, and if the pupal body is soft and
black, it may emerge the next day.
- The refrigeration schedule may be planned accordingly.

- After emerged moth, male and female, become to self mating.

- The coupling room is maintained at a temperature of 23~25°C and a
photoperiod of 17hrs. Generally mating is allowed in for 3 hrs, after which
the males are separated to be used for a second mating and the females
taken to the oviposition room.

- Mating duration is reduced to not less than 2 hrs when temperature is lower
than optimum or prolonged to 4 hrs but not more than 6 hrs when it is higher.
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- The decoupled male moths are preserved at 5~7°C in total darkness for a
maximum period of 2 days and used for a second or even a third mating.

- Preservation of males is essential because they belong to a rare delicate

race while the female is of the local hardy race, the mortality rate is
greater among males than females.

External genitalia of silk moth (left; male moth, right; female moth)

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 4.2.5 Oviposition

- Mated female moths are transferred to the

oviposition room immediately, not later than 10
minutes after decoupling.
- The method of seed preparation followed decides
the method of transfer of females.
- If the cellular method is used (P2, P3 level), they
are transferred single to special paper or cellular. Egg oviposition tool for P2, P3 level

Egg oviposition tool for P1 level

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4.3 Egg handling and preservation
4.3.1. Preservation of eggs produced in spring :
(1) Preservation in summer after egg laid

Preservation of silkworm eggs deposited in June.

1, Oviposition; 2, inn warm regions, a decrease of the temperature
may be necessary; 3, exposure to low temperature; 4, cold storage;
5, intermediate care; 6, removal from cold storage for incubation.

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- In about a week, the embryo enters a state of diapause.
- The egg color is lightly yellow when first laid and after 36-48 hours
it gradually changes into reddish brown and becomes darker.

- On the 4-5th day, the egg acquires its inherent color of the variety.
- During this period, respiration is brisk because of the embryonic
development.
- Care should be taken not to shock, crush or rub the eggs. So the eggs
should be kept in well ventilated clean rooms at a constant temp. of
24～25℃ and a relative humidity of 75%.

- High temperature in summer is a requisite for stabilizing the diapause

of the hibernation eggs.
- The proper temperature for completion diapause is 25℃.
- Temperature of 30℃ is harmful to the eggs, and higher than 30℃ is the
more harmful it will be.

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- Lower below 20℃ may disturb the diapause completion, making the
hibernating eggs unable to withstand cold storage, and disturbs
uniformity in embryo development.
- For the purpose of safe production of the eggs, it is advisable to
preserve the eggs around 50-60 days at 23-25℃ in summer.
- Chilling days needed to obtain more than 80% hatchability from the
eggs preserved at 25℃ for 10 to 210 days.
- The optimum humidity for the preservation in summer is 75-80%.
- If it is too dry, the eggs will be lost too much water and too wet, mold
are apt to grow on the eggs.

(2) Preservation in autumn and winter

- In September through October (during autumn), the eggs are still in
diapause , so a slight variation in temperature does no harm, but in Nov.
through Dec.(early winter), the eggs enter the pre-termination of
diapause, and high temperature should be avoided.
- In order to secure simultaneous hatching, it is necessary to expose the
eggs to a low temperature of 7.5 to 5℃ or lower for more than 50-60
days, as shown in step 3 in figure.
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- At the end of Jan. or beginning of Feb., diapause will be almost
terminated at 5 to 7.5℃. At this time the embryos increase in length
and the head lobe is wide open. If kept at 5℃ for a long time at this
stage, the embryos that have terminated diapause become unviable.

- Therefore, within about 60 days they must be transferred to 2.5℃, as

shown in step 4 in figure. Thereafter, the eggs can safely be stored for
40-60 days at 2.5℃
- Intemediate care : to ensure survival, eggs are allowed to develop one
step by conditioning at 15℃ for a few days (step 5).
- Thus the embryos reach the stage of the 'the longest embryo', just one
day before appearance of the neutral furrow. Then, the eggs are kept at
2.5℃ until the time of incubation.
- In sub-tropical or tropical areas, the natural atmospheric temperature is too
high to break the diapause even in winter.
- It is, therefore, necessary to transfer the eggs into cold storage earlier to
render the eggs to go through an artificial hibernation for the purpose of
awakening the embryo from diapause

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 4.3.2. Preservation of eggs produced in autumn :
- Eggs produced in autumn for the rearing in the next spring are called
autumn eggs and they are to be hatched in the fellowing spring.

- As the duration of preservation of the eggs is shorter, the nutrient in

the eggs are less consumed and it is easy to maintain the quality of the
eggs.

- The time from egg production to incubation is 6 months from Oct. to

Apr.-Mar. next year, and the time for low temperature preservation
needed to terminate the diapause takes 2-3 months.

- During this period, the eggs can be treated as follows ;

(1) Preserved at 25℃ within 20 days after egg laying.
(2) Lower the temperature step by step in following 10 days. Kept at
20℃ for the first days, and at 15℃ for the following 5 days.
(3) After being preserved in this way for a month, the eggs are kept in
cold storage for the hibernation

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 4.4 Artificial hatching
The silkworm eggs can be converted to nondiapausing by treatment with hot
hydrochloric acid just or shortly after the onset of diapause.

4.4.1. Common acid treatment :

- The time suitable for this treatment is a about 20h after oviposition, when
eggs have been preserved at 25℃.
- However, the development of the embryos varies depending on the
preservation temperature after oviposition, and at a constant
temperature it varies with strain : bivoltine develop fast, while univoltines
develop rather slowly.

* Optimum time for common acid treatment for bivoltine eggs

Preservation (Temperature, ℃) Time after

Oviposition (hr)
21 25 - 35
24 18 - 26
27 16 -23

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(If there is no equipment to control the temperature, next table can be
consulted for the optimum time for the acid-treatment :

- Hydrochloric acid of specific gravity 1.075 (15℃), or about 15%

concentration is heated to 46℃ before use. The egg are then dipped into
the hot solution.
- The standard duration of dipping is 5 min, but varies depending on the
strain and age of the egg :
(Japanese - 5～6min, Chinese - 4～5min, European - 6～7min)

* Unheated hydrochloric acid treatment :

Time after oviposition Treatment time by HCl solution temp.
at 25℃ 24℃ 27℃ 29℃
10 hrs 50-70 (min) 40-60 (min ) 40 (min)
15 55-75 55-70 40-50
20 55-80 55-80 40-50
25 60-90 60-80 40-50

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 4.4.2. Acid treatment after cold storage :
- For the postponement of hatching by more than 40 days, acid treatment
is performed after cold storage of the eggs.
- According to the duration needed to cold storage, the best time for cold
storage of eggs varies from 35～50 hours after oviposition at 25℃.

treatment short-term cold storage long-term cold storage

egg age 35-40 h after 40-50 h after
for chilling oviposition oviposition

20-30 days, 5℃ 40-60 days, 5℃

duation
2.5℃ after 40 ds chilling
30-50 days 50-75 days,
days from oviposition
or 90-100 days
to begining of rearing

HCl solution, 1.100(20%) at 15℃ 1.100(20%) at 15℃

temperature 47.8 (48)℃ 47.8 (48)℃
(The eggs are transferred from 25℃ to 5℃ via an intermediate temperature of 15℃ for about 6 h. )

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- High humidity of 75-80% is necessary. After taking eggs from cold place,
they can be kept at room temperature for 3-6 hours before acid
treatment.

- Treatment time : for European and Japanese univoltines, 6 to 7 min;

for Chinese uni. and bivoltines and Japanese bivoltines, 5 to 6 min;
hybrids follows the standard for the mother races.
- Immediately after the treatment the eggs should be washed
thoroughly in running water at 20℃ about, to wash off any traces of
the hydrochloric acid.

- The washing should be done until a sour taste completely disappears

from the egg-card. After deacidizing, a rapid drying is recommended.

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 4.5 Incubation
- Incubation is a process where the eggs kept in a state with ideal
temperature, humidity and light, air, etc, for the purpose of hatching.

- The objective is to make the eggs hatch uniformly on a day marked

for brushing. Further, if the incubation is correctly done, the rate of
hatching and the health of the young larvae will be ensured.

- This will improve the cocoon yield and the quality of cocoon fiber.

4.5.1 Condition of incubation

(1) Sterilization
- The incubation chamber or room and all implements are properly
sterilized before starting the process of incubation.
- Care should be taken to avoid the production of toxic gas which
might be released by the paint used on the inner side of the
incubation chamber.

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 (2) Temperature
- The temperature during incubation greatly influences the development
of the embryo.

- The ideal temperature is 23~25 ℃.

- When embryo which are at varied degrees of development are
incubated at a higher temperature, the difference in the development
is widened further.
- On the other hand, if these embryos are kept at a low temperature, the
difference in the development of the embryo, on the whole, is
lessened.

- In side the incubation chamber or room, there are variations in the

temperature, depending upon the location of the eggs.
- Therefore, it is necessary to make the eggs up and down, so that all the
eggs exposed to the same temperature conditions.

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 (3) Humidity
- It will be desirable to have 75~85% humidity during incubation.
- During the last phase of incubation, if the condition in the chamber is
dry, the number of dead eggs increase. This will decrease the rate of
hatching and subsequently, the final crop production.

(4) Light
- Light has influence on the voltinism of silkworms.
- This factor is utilized in preparing moths which will not produce non-
diapause eggs.
- For the diapause egg laying, it requires light for more than 16 hours per
day. Also, during incubation light rays influence the development of
embryo and can accelerate or suppress hatching.
- From the beginning of the incubation to the pigmentation stage,
development progresses better when the chamber is the bright rather
than dark conditions.

- On the other hand, during the period between the pigmentation and the
end of incubation, development progresses better under dark conditions
rather than under bright condition.

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- At the time of hatching, dark conditions tend to suppress hatching.

- Generally, the eggs are transferred to a dark place when pigmentation

begins. On the day of hatching, when these eggs are exposed to
bright condition in the early morning, hatching occurs almost
simultaneously.

Pigmentation egg

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 4.5.2 Regulation during incubation
- When there is a sudden change of plan relating to the date of brushing,
the embryo attain stage No.29 (bluish egg stage – completed stage),
they can be kept for a week at 5℃.

- When the young larvae (newly hatched larvae) need to be controlled,

they can kept at 10℃ for 3 days or 7.5℃ for few more days.

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SPRING

79
SUMMER

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 AUTUMN

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 WINTER

THANK YOU!
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