Francesc Piferrer

Seminari Departament d’Ecologia, UB. 19 de febrer de 2016

Determinació per factors ambientals de la proporció de sexes

a les poblacions: Mecanismes, contribució de l’epigenètica

i aspectes adpatatius en un context de canvi global

1.- Sex ratio and sex determination. Environmental factors

Outline

2.- Epigenetics. Mechanisms and analytical methods

3.- Application of epigenetics to research in ecology

4.- Epigenomics in natural populations

5.- Adpative aspects in a global change scenario

“Applied” aspects

– Reproductive capacity

- Fisheries

– Sex control

- Aquaculture

- Invasive species

– Monitoring

- Chemical pollution

- Climate change

“Basic” aspects– Mating competition and sexual selection

– Evolution of sex determining mechanims

– Sex allocation theory

– Speciation

Penman and Piferrer (2008). Rev. Fish. Sci. 16 (Suppl. 1): 4–34

Major sex

factors

Minor

sex

factors

Environmental

differences

monofactorial

system

polyfactorial

system

environmental

sex

determination

GSD

ESD

Sex Determination

– Genetic sex determination (GSD)

GSD ESD

– Environmental sex determination (ESD)

Environmental Sex Determination (ESD)

TSD TSD

TSD GSD

Warner and Shine (2008). Nature 451, 566-568

Temperature (TSD)

pH

Population density

Photoperiod

Hypoxia

Salinity

…

ESD

Species with TSD = Natural system in which populations exhibit a

direct unambiguous fitness response to thermal fluctuations

Sex Ratio Response to Temperature in Fish

T. min T. max. Nat. Range Temp. Nat. Range Temp. during Develop.

Temperature

Pro

po

rtio

n o

f m

ale

s

GSD

GSD

+ Temp.

Effects

TSD

RTD

Ospina-Álvarez & Piferrer (2008) PLoS One 3(7): e2837

1. Less TSD species

2. One response pattern

3. Possible effects of cl. change

The European Sea Bass Temperature and Sex Enigma

Sex ratio analysis

Fertilization

0 50 100 150 200 250 300 350 TSP

Formation of gonadal

ridges 35 dpf

Increase in PGC number

60-90 dpf

First sex-related differences in

cyp19a expression 120 dpf

Sex differentiation

First observable signs of morphological

sex differentition 150 dpf

Piferrer et al. (2005). Gen.

Comp. Endocrinol., 142: 102-110

Dicentrarchus labrax (F. Moronidae).

Polyfactorial SD system w. temp. influence

G x E interactions.

Epigenetics

“The study of phenomena and mechanisms that cause chromosome-

bound, mitotically and/or meiotically heritable changes to gene

expression or phenotype that are not dependent on changes to

DNA sequence”

Epigenetic Mechanisms

Small RNAs (sncRNA)

Long RNAs (lncRNAs) Regulatory RNA

Xist, …

Monoallelic gene expression

miRNA (21-22 nt) Translational repression

3. Non-coding RNAs

1. DNA methylation 2. Histone modifications & Histone variants

H3K9me3

repression

H3K4me3

activation

ON Me Me Me

OFF CGATTCGAACG

CGATTCGAACG

Cell membrane

Nucleus

2. Epigenetic initiators - DNA binding factors

- ncRNAs

3. Epigenetic maintainers - DNA modifying enzymes

- Histone modifying enzymmes

- Histone variants

Environment 1. Epigenators - Temperature

- Differentiation signals

- Metabolites

Signaling

(epigenator)

pathway

ncRNA

DNMT

HAT

DNA-BP

HDAC

Cytoplasm

Genotype

Short-term, transient outcomes

- transcription

- DNA replication & repair

Long-term outcomes

- chromatin conformation

- heritable markers

Phenotype

Establishment of Epigenetic Marks

Genotype + Environment = Phenotype

Epigenetic Inheritance

Cell-cell transfer (mitosis)

Organism-organism transfer (meiosis) Multi- vs. transgenerational effects

P F1 F2Multigenerational effects

Transgenerational effects

F3

P F1 F2

Multigenerational Transgenerational Shao C, et al. (2014) Genome Res. 24:604-15

Inheritance of sexual reversal

in tongue sole

(ZW/ZZ sex det. system)

F1 ZW fish exclusively derived

from the paternal pseudo-males

ZW offspring develop as pseudo-

males without temp. treatment

R. Margalef

(1919–2004)

Margalef, 1955.Temperatura, dimensiones y evolución. Publ Inst Biol Apl 19: 13–94

“Merece especial interés la coincidencia entre los carácteres resultantes de

una selección (e incluso producción directa: determinación del sexo,

poliploidía) de variaciones del genotipo y los que consisten en simples

modificaciones o somaciones no hereditarias.

Es comprensible que la observación de este paralelismo haya sido tomada

como argumento a favor del lamarckismo, aunque sin dar una explicación

racional que justifique su asimilación”.

Met

hyla

tion

(%)

Low Temp.

High Temp.

a

b

c

c

14

8 9 9

MalesFemales

100

50

0

environment DNA methylation gene expression phenotype

* *

*

cyp19a +SF1

cyp19a +Foxl2

cyp19a +SF1

+Foxl2

Ctrl cyp19aLu

cife

rase

Act

ivity

0

2

4

6

8

10

12

14

16

18

Control Methylated

*

Foxl2 Sf-1 cyp19a

Foxl2 Sf-1

cyp19a

Males > Females

An Epigenetic Switch Linking Temperature and cyp19a Expression

Temperature group

Low High

cyp19a

(R

Q)

0

2

4

6

8

10

Methylation (%)0 20 40 60 80

cyp

19a

(R

Q)

0

5

10

15

20

Females only

a

b

r2 = 0.3 P = 0.01

Navarro-Martín et al. (2011). PLoS Genet 7(12): e1002447

J. Experimental Zoology Aquaculture PLoS Genetics

Science

Time

Patent

Private company

Matsumoto et al. (2013). PLoS ONE 8(6): e63599

Red-eared slider turtle

(Trachemys scripta)

26°C cyp19a1 hyperMe 100% males

31% cyp19a1 hypoMe 100% females

Does a similar mechanism operate in reptiles with TSD?

Thermosensitive period Sex differentiation period

Thermosensitive period

Sex differentiation period

FISH

REPTILES

Individual variation in

ecologically important traits

Evolutionarily important

• Provides the raw material for natural selection

• Conditions the evolutionary trajectory of populations

• Shapes the demographic structure of populations

• Affects the dynamics of populations and communities

Ecologically important

Contribution of Epigenetics to Ecological Research

How Epigenetics Integrates Genetic and Environmental Information

After Bossdorf et al 2008. Ecol Lett.11: 106–115

Alter

ECOLOGICAL

EPIGENETICS

Regulates Epigenetic

variation

Alters

Affects

Gene

expression

Phenotypic

variation

Ecological

interactions

Natural

selection

Genetic

variation

Affects

Affects

Through Alters

ECOLOGICAL

GENETICS

Affects

Morán & Pérez-Figueroa, 2011. BMC Genetics 12:86

Circles – River Tea Triangles – River Ulla

Genetic (AFLP) Epigenetic, liver (MS-AFLP) Epigenetic, gonads (MS-AFLP)

Open – Immature Filled – Mature

Extent and Structure of Epigenetic Variation

within and among Natural Populations

VP = VG + VE + VGE

VG = VA + VD + VI VG = VA + VD + VI + VEpi

Abiotic factors: Thermal stress

Nutrients

Light

Water

What is the relative importance of epigenetic variation in

determining the outcome of abiotic and biotic interactions?

Biotic interactions: Competition

Resistance to predators

Resistance to pathogens

Herrera & Bazaga 2011. Mol Ecol. 20: 1675–1688

Effects of

herbivory on

Viola

cazorlensis

Model 4

Genotype

Phenotype

Epiphenotype

Model 3

Genotype

Epiphenotype

Phenotype

Model 2

Genotype

Phenotype

Epiphenotype

Model 1

Genotype

Epiphenotype

Phenotype

Epigenetics Has a Long Way to Go in Ecology

Topic 22/04/2013 14/02/2016 % increaseCancer 2675 4563 71 Development 2250 4252 89 Genetics 650 1127 73 Evolution 400 719 80 Animal production 60 76 27 Neurobiology 40 61 53 Ecology 30 58 93

0500

100015002000250030003500400045005000

22/04/2013

14/02/2016

Individual variation in

ecologically important traits

Evolutionarily important

• Provides the raw material for natural selection

• Conditions the evolutionary trajectory of populations

• Shapes the demographic structure of populations

• Affects the dynamics of populations and communities

Ecologically important

Individual Variation in Ecologically Important Traits

production

Production

artificial

performance

Production

2. Epigenetics and Animal Production

Growth, Product quality, Health, Welfare, Reproduction

gamete development

fertilization

embryonic development

Post-hatch/birth development. Internal External

External External

Environmental influences

(biotic and abiotic)

Fertilization

Internal

Early develop.

Internal

Effects of early environment in the sea bass

Illes Medes natural reserve

Experimental Aquarium facility

Size of genome (Mb) 675

Genes with

description 25,433

Number of CpG sites 10,325,421

% of CpGs 1.53

% Cs in CG context 7.66

Number of CpG

islands 30,391

Tine et al. (2014). Nature Comm. 5:5770.

Wild sea bass vs.

Famed sea bass (NT or HT)

DNA methylation – RRBS

Histone modification – ChIP

Gene expression – RNA-seq

Tissues

Early

environment

Genetic

variation

Epigenetic

modifications

Gene

expression Phenotype

Temperature

Pollutants

Stress

Farming

AFLP

SNPs

MSAP

RRBS

ChIP

Bis-PCR2

qPCR

RNA-seq

Fitness

Performance

Pr. quality

H-O Pörtner & Peck 2011 Encyclopedia of Reproduction.

• Changing water temperatures

• Hypoxia events

• Ocean acidification

• Shifts in hydrodynamics

• Sea level rise

Effects of Climate Change

Organismal Population Community/EcosystemMolecular/Cellular

Sex differentiation

Behavioral responses

Alterations in developmental time

Changes in productivity Changes in food-web interactions

DNA methylation Gene expression Metabolism

Decrements in aerobic performance

Larval dispersal

ncRNA H3K9MeT

H3K9DMe Polycomb

Epigenetic modification-

related genes

Sex differentiation-

related genes

LT HT

Long-lasting temperature effects on gene expression

Common Temperature

Gene expression is altered in 170 dpf sea bass treated with high

temperature (HT) during the thermosensitive period (TSP)

0 50 100 150 200 250 300 350

TSP Sex differentiation

LT HT

Age

(dpf)

HT LT

Díaz & Piferrer. 2015. BMC Genomics 16:679

Genes upregulated transgenerationally:

- Metabolic genes shifts in energy production.

- Immune- and stress-responsive genes the second generation of fish to better cope with elevated temperatures.

- Genes involved with tissue development and transcriptional regulation

- Similar suite of dierentially expressed genes among developmental and transgenerational inheritance

- Heat-shock protein genes were surprisingly unresponsive

Species

Pivotal temp.

(ºC)

Percent of sexes (:) at

pivotal temp. + 1.5ºC

Percent of sexes (: )

at pivotal temp. + 4ºC

Apistogramma spp. (n = 33 sp) 25.3 62 : 38 81 : 19

Hoplosternum littorale 18.8 56 : 44 65 : 35

Limia melanogaster 25.8 57 : 43 68 : 32

Menidia menidia 14.5 61 : 39 75 : 25

Menidia peninsulae 26.6 57 : 43 69 : 31

Odontesthes argentinensis 25.7 60 : 40 76 : 24

Odontesthes bonariensis 24.2 73 : 27 98 : 2

Poeciliopsis lucida 25.6 68 : 32 92 : 8

Pivotal temp. (mean ± S.E.M.) 23.3 ± 1.5 - -

Percent males (mean ± S.E.M.) 50.0 61.7 ± 2.1 78.0 ± 4.1

Pivotal temperature in fish species with TSD

and predicted sex ratio shifts with temperature increases

Ospina-Álvarez & Piferrer (2008) PLoS ONE 3(7): e2837

Summary/Take home messages

- In a global change scenario, epigenetics provide organisms with a source

of phenotypic plasticity that can allow adaption to a new environment

- The study of variation in natural populations can be greatly benefited

if research in ecology integrates epigenetics

- The sex ratio is a fundamental parameter in population demography

and in many cases is determined by a combination of

genetic and environmental factors

- Epigenetic mechanisms provide organisms the capability to integrate

genomic and environmental information to produce a given phenotype

Laia Ribas

Laia Navarro

Natalia Ospina

Noèlia Díaz

Dafni Anastasiadi

Alejandro Valdivieso

Susanna Pla

Silvia Joly

Gemma Fuster

Núria Sánchez

Paula Gómez

Acknowledgments

Collaborators:

Christopher Bock, Univ. of Vienna

Marco Álvarez, Univ Andrés Bello,Chile

Luciano di Croce, CRG, Barcelona

Paloma Morán, Univ. Vigo

0.285 Project

Project Epigen-Aqua

Program I-LINK

differentiation

eeppiiggeenneettiiccss

LLaammaarrcckk selection

environment

phenotypic

plasticity

heritability

hybridisation

induction Arabidopsis

speciation

Darw

in

chromatin global change

apomixis

development

mem

ory

natural populations

DNA sequence paramutation

phenotype

ecology epigenome

DNA methylation conservation

miRNA

evolution epialleles imprinting variation

inheritance

maternal effects histones

stress

interactions

transgenerational

animal production

genotype

aquaculture

cloning

tarantin

o

Waddington

non-lin

eal n

arra

tive