Tilapia seeds culture in Taiwan

Genetic Engineering in Aquaculture Genome & Gene Manipulation

Dr. JK, Lu 5-2008

Tilapia seed culture in Taiwan



chromatin - the DNA/protein complex making up chromosomes heterochromatin - chromatin that tends to take up large amounts of stain, thus staining darkly. Heterochromatin tends to be densely-coiled. euchromatin - chromatin that tends to take up small amounts of stain, thus staining lightly. constitutive heterochromatin - chromatin that remains denselycoiled through out interphase, in all tissues. Often found near centromeres, telomeres and nucleolar organizing regions (NORs). facultative heterochromatin - chromatin that is either denselycoiled or loosely coiled during interphase, depending of the developmental state or tissue

Animals
Goldfish Common carp Nile tilapia
Carassius auratus

CV= haploid= pg 1.35-2.05 1.70 1.20 2.64 2.60 0.93

CN=diploid (2N)
94 100 44 74 60 48 50

Cyprinus carpio Oreochromis niloticus

Oncorhynchus keta Oncorhynchus mykiss

Chum salmon Rainbow trout Red seabream Zebrafish

Pagrus major Danio rerio

Shrimp

Penaeus sp.

2.50

2n=88-92

Chromosome of teleost & crustacean

The genome of L. vannamei consists roughly of two billion nucleotide pairs or 2/3rds the size of the human genome. In shrimp, there are about 90 chromosomes, each with about 22 million base pairs of DNA nucleotides per molecule In humans, there are 23 chromosomes each with about 130 million base pairs contained in each molecule.

Smallest teleost genome size: ~0.4pg in several pufferfishes of the Family Tetraodontidae. Largest teleost genome size: 4.4pg in Corydoras metae, the masked corydoras.

Use of Biotechnology to Enhance Fish Production

1. Induced ovulation and spermiation 2. Chromosomal manipulation 3. Controlled sex differentiation - monosex culture, sertilization 4. Endocrine regulation of fish growth & administration

Schematic diagram of individual selection for weight

Controlled sex differentiation

5. Transgenic technologies - approaches for manipulating phenotype - Genes available for construct 6. Nutrition - transgenic animal - manipulate digest enzymes /secrete into gastrointestine tract - transgenic food (atemia, rotifer, blue green algae) - enriched planktons - HND diet 7. Intensive aquaculture technology

Sex reveral & production of monosex population Sterilization

Manipulation of Genetic Sex

Purpose: increase growth rate of population control propagation speed extend growth period improve meat quality

Characteristics of sex determination in aquatic species

fish sex chromosome is lacking or primitive phenotypic sex easy to change normal function after sex reversal YY male are fertiled

Sex-specific linkage maps for linkage group 8 in Oreochromis niloticus

Tilapia genetic map

The karyotype of Oreochromis species is considered to be highly conserved, with a diploid chromosome complement of 2n = 44

Lee et al. 2003

Sex control in aquatic species

Mechanism of sex-determination in fish

(Table 2.1) 1. XY sex-determining system - Y chromosome determine sex - homogametic sex: XX (female); heterogametic XY (male) 2. WZ system ( a mirror image of XY system) - W Cs determine sex - homogametic ZZ (male); heterogametic ZW (female)

surgery (remove gonad) temperature manipulation prior to gonad differentiation sex hormones aministration(via oral, dipping,mmersion

methods)
interspecies hybridization - tilapia: female (XX), cross w/ ZZ male close 100%

male hybrids
sex reversal individual cross w/ normal individual - produce YY male (e.g. tilapia) - produce XX female (e.g. salmon)

3. Multiple sex-determining system 3.1. Multiple XY system - Homogametic (X1X1X2X2, female; - Heterogametic X1X2Y male - male Cs determine offsprings sex 3.2. Multiple WZ system - homogametic ZZ , male), - heterogametic W1W2Z , female) - female recessive Cs (W1W2) determine offsprings sex

3.3. Multiple Y Cs system - heterogametic male (XY1Y2) - homogametic female (XX) - male determine offsprings sex ; receive Y1Y2 is male 3.4. WXY system - varing from XY system - W Cs is modified from X Cs , - W can block male determining ability of Y Cs - XY, YY are males - XX, XW, WY are females - both male and female can be homogametics - i.e. either parent can determine offsprings sex

4. XO system - variant from XY system - O means no Cs - homogametic XX female - heterogametic (?) XO male - male determine the sex, receive his X Cs female, reveive no Cs (O) male 5. ZO system - variant from WZ system - heterogametic ZO female - homogametic ZZ male - female determine offsprings sex, offspring receive her Z Cs -> male, receive O no Cs become female

Sex determination
sex determination has been intensively studied in the genus Oreochromis, where both male homogamety (ZZ / WZ) and female homogamety (XX / XY) XX / XY system: O. niloticus and O. mossambicus; T. zillii, WZ (female heterogametic)/ ZZ (male) system: in O. aureus; O. karongae (2n = 38) ; O. urolepis hornorum

* in some case autosomal sex-influence genes or sex-modified gene may influence fish sex

Hybridization
- interspecies hybridization

interspecies hybridization
tilapia: female (XX) cross w/ (ZZ) male close 100% male (XZ) hybrids
O. mossambica F X O. hornorum M ---> 98-100% M O. nilotic F X O. aurea M ---> 100% M O. nilotic F X O. variablilis M ---> 100% M O. nilotic F X O. macrochir M ---> 100% M O. nilotic F X O. leucostica M ---> 100% M O. nilotic F X O. hornorum M ---> 100% M O. nigra F X O. leucostica M ---> 100% M O. nilotic F X O. aurea M ---> 100% M X Orange T. mossambica T. hornorum

100% Supermale Hybrid

White Bass Hybrid striped bass :

Striped Bass

female white bass X male striped bass.

Top to bottom: white bass, striped bass and hybrid striped bass (Bodie bass)

Sex Determination Gene(s) Isolation

Y- Cs encode testis determining gene (TDF, testis-determining factor) a DNA-binding motif Two possible genes determine sex (ovary or testis formation): - SRY (sex-determining region Y) - Z gene (autosomal-x-linked gene) Salmon & Tilapia Y-chromosomal DNA
Cytogenetical mapping of the sex-determining region (SD) of medaka.

YY (Supermale) & superfemale (WW)

YY (Supermale) & superfemale (WW) in tilapia; prevent reproduction & eliminate female (grow slower) produce 100% monosex population in salmon; eliminate precocious male (die after mature) female growth faster than male

YY male

Table 1. Key developments of GMT

Genetically Improved Farmed Tilapia (GIFT) tilapia

Growth comparisons of Genetically Male Tilapia

Schematic diagram depicting the model for largescale production of monosex male tilapia

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All male fry production

The sex of tilapia is basically governed

YY Super male tilapia

by 2 types of sex determination genes. For O. niloticus, O. mossambicus the sex gene is X & Y type (XY male; XX female) For O. aureus, the sex gene is Z & W type. The genotype (ZZ male; ZW female) Cross the female O. niloticus (XX) with male O. aureus (ZZ), the resulting F1 generation (ZX), and this ZX genotype happen to expressing a phenotype as male fish.

ALL-MALE RED FRY GROW AFTER 4 MONTHS

Producting All-Male Progeny by Crossing Sex Reversed Founders

Production of All-male Triploid Tilapia (1)

Gonadally Undifferentiated XX XY

Super YY male YY
(sex reversed)

Normal Female X XX
All XY progeny or Femalized male (XY)

Estrogen sex reversal

OR
Thermal or hydrostatic schock Polyploid; (block 2nd PB) XXY (3N)

XX

XY (phenotypic female)

Large scale production

X XX : 1: 1 XY XY : 1: 3 XY or YY : 1:1 or all-males
Thermal or hydrostatic schock

X XY
Male progey tested with normal female to identify

(2) XY* (female) X YY male

X XX
(Cull)

Sex reveral (estrogen) XY* (female) OR Progeny test YY* female

possible genotypes (XX; XY; YY)

YY
male All YY progeny

OR

YY

(Select YY male as brood fish)

X YY XX Large scale all-male production

Triploidy : (block 2nd PB) YYY male

All-Male Triploid Tilapia (Large scale production for aquaculture purpose)

Producing YY Male Tilapia (Gynogenesis Method)

Gonadally Undifferentiated

Producing YY-Male Tilapia (Gynogenesis Method)

Gonadally Undifferentiated Fry XY XX Sex Reversal Administration
(Estrogen)

XX

XY

estrogen administration sex reversal Progeny test

" XX
(Cull) Normal

"
XY (phenotypic female) (Gynogenesis) :
5 min post-fertilization 6000-8000 psi; or 41.1 for blocking 2nd PB, 3-5 mins duration

Progeny test*

" XX (Cull)

" XY (phenotypic female) Gynogenesis: UV irradiated Sperm Schock- blocking 2nd PB

XY
UV irradiated sperm

oC

Normal

XY

OR

XX
(Cull)

" YY"

su p e r m a le

XX (Cull) Progeny test* 1:1 OR

" Y Y - m a le "
X

Progeny test

all-males X (Normal female) XX

1:1
(Brood fish)

or

all-males
(Brood fish)

X " YY" XX

(Normal female)

"YY-male"

Large scale all-male production for aquaculture purpose

100 % XY Large scale all-male production for aquaculture purpose

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Methodology for production of supermale/superfemale

sex reversal / cross with normal opposite sex sex reversal / chromosomal manipulation

Micropyle of Fish Egg

Micropilar canal
Sperm

Chorion

1st polar body Cortical alveoli Cytoplasm Metaphase II

Fertilization Event
Depolar wave Sperm and eggs membrane fusion Ca+2 wave increase CA breakdown SB form; pH increase Meiosis restart Nucleus membrane disappear NM reform PB release; DNA synthesis; As form Nucleolus NO form; PN form Pronuclei fuse Chromosome form

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(A)
O o g o n iu m

Polyploidy (Chromosomal) Manipulation

P r im a r y oocyt es

Meiosis I

Meiosis & early mitosis process in fish and shellfish (Figure ) * inhibit meiosis I heterozygosity remain; heterozygosity loss when inhibit meiosis II *inhibit meiosis I can increase overall genomic heterozygosity, may see somatic growth enhancement before maturity

Se co nd ary

oocytes

Meiosis II

O o t id s

( B)
O o g o n iu m

( C)
O o g o n iu m

P r im a r y P r im a r y Shock Meiosis II oocyt es Meiosis I

oocyte

Seco nd ary oocyt es Shock O o t id s O o t id s

Retain 1st polar body Retain 2nd polar body

..

P o ly p lo id y Triploid
Sperm

In d u c t io n

in

F is h Androgenesis

Gynogenesis
(I)
UV

Chromosomal Manipulation
Triploid Gynogenesis
(I)
Sperm U V

(II)

UV

UV

Egg
EN Fertilization

Androgenesis
(II)
U V U V

Egg

Shock
2N (Meiogynes)

Shock

2 nd

PB

EN

Fertilization
2 n d P B

Shock

Shock

3 N

N 1st cleavage

3N
Shock
N

2N
(Meiogynes)
N

N
1st cleavage Shock Shock
N N N

Shock

2 N (Mitogynes)

2 N

2N
(Mitogynes)

2N

Metaphase Chromosomes of O. niloticus

1N= 22 2N=44

3N=66

4N=88

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Triploid grass carp

Advantages of polyploidy individuals

growth rate increase (?) survival rate meat qualtiy improvement sterilization

Aquatic plants provide the following benefits: - cover for young fish to hide from predators, - food for many insects that fish eat, - protection from currents and silt for fish eggs in spawning nests , and structure which sportfish use for shade and camouflage, which in turn helps anglers locate them.

Polyploidy Induction: - triploidy - tetraploidy Physical methods for generating polyploidy 1.temerature shock - heat shock - cold shock 2. hydrostatic pressure shock Chemical methods: - colchicine (antimitotic drug) - cytochalasin B

Polymerization Inhibitors
Therapeutic agents for gout and cancer

Colchicine, from autumn crocus, inhibits MT polymerization, mitosis and also white cell movement - it is a remedy for gout and an inducer of larger, healthier plants Vinblastine, vincristine also inhibit MT polymerization - anticancer agents Taxol, from yew tree bark, stimulates polymerization, stabilizes microtubules and inhibits tumor growth, (esp. breast and ovarian)

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Colchicine, an alkaloid inhibit microtubule formation (polymerization of tubulin protomers) - would not inhibit Cs duplication and separation - inhibit microtubule assemble, cause spindle disassemble (I.e. Inhibit spindle formation or damage has assembled spindle) Cytochalasin B: fungal alkaloid interfere actin aggregation, i.e. block actin polymerization by bind to the end of F-actin filament) - inhibit actin filament assemble (e.g. cytokenesis; daughter cell separate)

colchicine is a drug that can be used to artificially break down microtubules.

The structure of cytochalacin B ( a fungal metabolite)

Oyster life cycle & settlement

Planktonic larvae Free-swimming stage Crawling stage

Attachment stage Two days after attachment Ten days after attachment

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Embryonic stages of Crassostrea gigas in relation to time at water temperature of 2021 degrees Celsius.
Developmental stage 1st polar body releasing 2nd polar body releasing 1st division 2nd division Morula stage Blastula stage Rotary movement Free swimming Gastrula stage Trochophore D-shaped larva 5 hour 20 minute - 5 hour 50 minute 5 hour 30 minute - 6 hour 00 minute 6 hour 10 minute - 6 hour 30 minute 9 hour 40 minute - 10 hour 00 minute 15 hour 00 minute - 28 hour 00 minute Time required 50 minute - 1 hour 10 minute 1 hour 00 minute - 1 hour 20 minute 1 hour 20 minute - 1 hour 40 minute 2 hour 00 minute - 2 hour 20 minute 3 hour 5 minute - 3 hour 25 minute

Polyploidy of Pacific Oyster (C. gigas)

Haploid (1N)

Diploid (2N) Triploid(3N) Tetraploid(4N)

Flow cytometric histogram

Triploidy Induction in Bivalves

Diploid
Egg
EN

Triploid
Sperm Egg
EN

Triploid
Sperm Egg
EN

Sperm

1s tP B

Inhibit 1st PB releasing

2 nd PB

1s

tP B

Fertilization

Fertilization

Fertilization

2 nd

PB

Shock

Inhibit 2nd PB releasing

Shock

device for exerting pressure on eggs to prevent chromosomal reduction through the suppression of meiosis. B - experiments in the cryopreservation of bivalve gametes and larvae

2N

3N

3N

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Representative metaphases of diploid, triploid and aneuploid Pacific oysters produced from diploid triploid crosses: (a) 2n=20; (b) 2n+1=21; (c), 2n+2=22; (d), 2n+3=23; (e), 3n-2=28; (f) 3n-1=29; (g) 3n=30 chromosomes

Metaphases from polyploid and aneuploid Pacific oysters produced by chromosome manipulation. Chromosome constitutions shown are A, 21; B, 30; C, 31; D, 32; E, 33; F, 38; G, 39; H, 40; and I, 40. The normal diploid number is 20.

Polyploidy Identification
- chromosome number/ karyotyping - nucleoli silver staining - (RBC) nuclear volume measure - DNA content analysis - flow cytometry - fluorescent intensity - isotope labeling

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Application of chromosomal manipulation

1. Sex control - producing all-male or all-female population - one sex is growth faster than the other sex - population control 2. Sterilization fish - control population - increase growth rate special in reproduction season(steriled) - survive rate increase (after spawning season) - reduce precocious maturation - meat quality improved (higher glycogen content)

3. Producing homozygosity (pure strain) /clone fish - faster process

Pathenogenesis
- gynogenesis - androgenesis Gynogenesis induction - polar body gynogenesis - inhibit 2nd PB releasing - mitotic gynogenesis - inhibit 1st mitotic cleavage * mitotic gynogenesis can generate pure strain (homozygosity) in one time * PB gynogenesis to generate homozygosity requeires several crossing

gynogenesis : an all-maternal inheritance,

sperm cell does not contribute genetic materials to embryo androgenesis:an all-parantal inheritance, egg does not contribute genetic materials to embryo

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Andogenesis
Inactivate gametes (sperm or egg) Physical methods - gamma ray, x-ray, UV Chemical methods - toluidine, EMS, trypaflavine, thiazine overmatures (aging) eggs - female Cs degraded

Gynogenesis + diploidization (inhibit 1st cleavage) Purposes: - to establish pure strain (homozygosity) - genetic analysis - sex determine mechanism - environmental factors influence phenotype or heritage - gene map or recessive gene - control sex of population

Nuclear Transplant at ion in Fish

Cloning fish
Clones: genetically identical species of certain cells & organisms - monozygous twins (vertibrates) - parthenogenetic progenies (pisces, amphibia, reptilia) - nuclear transplatation - genetically homozygous individuals

Fish

egg

Fish e mbryo ( blast ula st age )

Act iv at ion e gg ( add wat er)

Embry onic disc ( b la s t o m e r e ) is o la t io n

D e c h o r io n a t io n

Blast ular

cell

se parat ion

Enucleat e d

e gg

micrope pet t e

donor

nucleus

Nucle ar T ranspla nt at ion ( insert donor nucleus int o enucleat ed cell)

Em bry o

cult ure

Cloning fish induction

- two-step gynogenesis - two-step androgenesis - nucleus transplantation - transplant embryonic & somatic cells to embryos - Embryonic stem cells

Genetic engineering in aquatic species

1. Nuclear transplantation in fish species 2. Transgenics

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