Environmental Heat Stress Induces Epigenetic Inheritance of Robustness in Artemia Model

Parisa Norouzitallab, Kartik Baruah, Gilbert Van Stappen, Karel De

Schamphelaere, Patrick Sorgeloos & Peter Bossier

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Crustacean aquaculture

Highly demanded High market value

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Finding solution

Finding solution to the problem of the disease is a primery goal

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Parental manipulation to improve phenotypes in progenies

F0 manipulation

Selection Epigenetics

Intense pressure Across generations Long period of time

Major loss of the miss-fit Labor intensive

Mild pressure One generation

Short period of time Minor loss of miss-fit

Labor intensive

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DNA molecule can record the environment

Epigenetic is the study of heritable changes in gene expression and function that cannot be explained by

changes in DNA sequence

HERITABLE REVERSIBLE

Stressed

mom

Anxious

daughter

Anxious

granddaughter

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Constraints for using shrimp as model organism

Long culture period of approximately 1 year for one generation

Labour intensive

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Selection of the model organism

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Short generation time – 3 generations in about 6 months

Genome is sequenced

Easy and cheap to maintain and manipulate

Produce large numbers of offspring

Artemia as model organism for our studies

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Parthenogenesis

NO MALE

Clonal population

Bisexual

Wild population maternal/paternal

heredity

Artemia as model organism for epigenetic studies

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F1 F2 F3 or more

Cyst (dormant egg) production – Common garden experiments

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F1 F2 F3 or more

Common garden experiments – axenic /gnotobiotic

Chemical removal of the embryo outer shells Sodium hydroxide hypochlorite solution

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Abiotic stressor and transgenerational inheritance of robustness

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No

n-l

eth

al

he

at s

ho

ck

Induction of heat shock protein 70 (HSP70)

Baruah et al. (2012). Aquaculture. 334-337:152-58

Protection against subsequent stressors abiotic - lethal heat shock biotic - pathogenic Vibrio campbellii

Background information

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Heat shock protein (HSP) 70 against abiotic stressors

Induced after exposure

to stressors

Synthesized constitutively in the cells

(heat shock cognate 70)

Role in house keeping Danger signal

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(clonal population)

Parthenogenetic Artemia

TF1 CF1

TF2 CF2

TF3 CF3

I S O

T H E R M I C

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Common garden test - verifying stress resistance phenotypes

42°C for 15 min

Or

Vibrio campbellii 107 cells/ml

Or

Zinc

20 instar II nauplii in 6 replicates

F1 F2 F3

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Thermo-tolerance test

*** ***

*** *** *** *** *** ***

0

0,2

0,4

0,6

0,8

1

0 3 6 9 12 15 18 21 24 27

Pro

po

rtio

n o

f th

e s

urv

ive

d a

nim

als

Time (h)

TF1

CF1

*** ***

*** ***

*** *** *** ***

** **

0

0,2

0,4

0,6

0,8

1

0 3 6 9 12 15 18 21 24 27

Pro

po

rtio

n o

f th

e s

urv

ive

d a

nim

als

Time (h)

TF2

CF2

*

* *

* * * *

0

0,2

0,4

0,6

0,8

1

0 3 6 9 12 15 18 21 24 27

Pro

po

rtio

n o

f th

e s

urv

ive

d a

nim

als

Time (h)

TF3

CF3

TF1 > CF1 TF2 > CF2

TF3 > CF3

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***

***

0

0,2

0,4

0,6

0,8

1

0 12 24 36 48

Pro

po

rtio

n o

f th

e s

uvi

ved

an

imal

s

Time (h)

TF1 CF1

***

***

0

0,2

0,4

0,6

0,8

1

0 12 24 36 48

Pro

po

rtio

n o

f th

e s

urv

ive

d a

nim

als

Time (h)

TF2 CF2

**

***

0

0,2

0,4

0,6

0,8

1

0 12 24 36 48

Pro

po

rtio

n o

f th

e s

uvi

ved

an

imal

s

Time (h)

TF3 CF3

TF1 > CF1 TF2 > CF2

TF3 > CF3

V. campbellii resistance

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Zn in O (P) and F3 generation

LC50 = 13.4 (6.6 - 15.7) mg/l

Heat shock (F3)

Non-heat shock (O)

Non-heat shock (F3)

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Phenotypic trait - HSP70 production

TP CP TF1 CF1 TF2 CF2 TF3 CF3

0

100

200

300

400

500

T-F0 C-F0 T-F1 C-F1 T-F2 C-F2 T-F3 C-F3

Hsp

70

(n

g/µ

g o

f p

rote

in)

Treatment

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Histone acetylation

Transcriptionally active

Transcriptionally repressed

HAT HDAC

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*

*

*

0

1

2

3

4

5

6

7

T-F0 C-F0 T-F1 C-F1 T-F2 C-F2 T-F3 C-F3

His

ton

e H

3 a

cety

lati

on

(n

g/µ

g o

f h

isto

ne

)

Treatment

Epigenetic mark - histone H3 acetylation

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TP CP TF1 CF1 TF2 CF2 TF3 CF3

0

0,3

0,6

0,9

1,2

1,5

T-F0 C-F0 T-F1 C-F1 T-F2 C-F2 T-F3 C-F3

His

ton

e H

4 t

ota

l ace

tyla

tio

n

(ng

/µg

of

his

ton

e)

Treatment

Epigenetic mark - histone H4 acetylation

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DNA methylation

Addition of methyl group to 5´ cytosine on CpG sites

Reduced chance of transcription

Silenced gene

DNA methylation

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*

*

0

0,1

0,2

0,3

0,4

0,5

T-F0 C-F0 T-F1 C-F1 T-F2 C-F2 T-F3 C-F3

%m

dc/

dG

Generations

Epigenetic mark – global DNA methylation

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Environmental heat stress on parental generation induces epigenetic inheritance of

resistance against disease, lethal heat shock and Zn

These transgenerational epigenetic inheritence of traits were associated with

production of HSP70 and sustained hyper-acetylation of histones H3 and H4

Abiotic environmental stimuli in parental generation can result in

transgenerational modification of phenotypes

Conclusions

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Future perspectives

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Thank you Acknowledgement

Promoter Prof. Dr. ir. Peter Bossier Funding BOF PhD Grant, UGent Belspo, AquaStress Project

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Cu in Wild population

LC50 = 0.18 (0.15 - 0.26) mg/l

LC50 = 0.26 (0.18 - 0.3) mg/l

Heat shock (W)

Non-heat shock (W)

axenic

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Zn in Wild population

LC50 = 10.6 (6.6 - 15.7) mg/l

LC50 = 4.6 (4.3 - 7.6) mg/l

Heat shock (W)

Non-heat shock (W)

axenic