Fernando Galvez - WordPress.com · 2015-08-28 · Overview of Presentation. 1. ... Basic Facts of the Deepwater Horizon Oil Spill •Approximately 800 million liters of oil was released

Fernando Galvez

Dept. of Biological Sciences,

Louisiana State University

How was the biology of a marsh fish affected by the Deepwater

Horizon oil spill?

Gulf Killifish (Fundulus grandis)

• most abundant vertebrate in coastal

marshes

• easy to raise in captivity

• relatively sensitive to chemical toxicity

• transparent embryos

• extensive characterization of embryology,

physiology, biochemistry, ecology, and

population genetics

• growing genomics tool kit (genome

sequence, RNA-seq, bioinformatics,

morpholinos)

Photo: A. Whitehead

Research model

Overview of Presentation

1. What are the physiological and molecular

impacts of oil exposure at different stages of

fish development?

- fitness level consequences

2. What is the geographical and temporal extent

of oiling in the field?

3. How does evolutionary history and exposure

history affect the sensitivity of fish to DWH oil?

Experimental Questions

Basic Facts of the Deepwater Horizon Oil Spill

• Approximately 800 million liters of

oil was released during 87 days

• Oil was spread over 180,000 km2

• Approximately 6.9 million liters of

chemical dispersant was added

Exxon Valdez oil spill released

approximately 40 million liters

Maps of Shoreline Oiling Data

1,770 km of shoreline

were oiled

790 km of Louisiana

shoreline were oiled

Sample Sites for Gulf Killifish Monitoring

GT

FMABLBBFPBSL

MB

• Grande Terre (GT), Louisiana

• Bay St. Louis (BSL) and Belle Fontaine Point (BFP),

Mississippi

• Bayou La Batre (BLB), Fort Morgan (FMA) and Upper

Mobile Bay (MB), Alabama

Track marsh fish health before, during DWH

oil exposure, and during a recovery period

GT

FMABLBBFPBSL

MB

• In our sampling, GT was the only location that oil made

landfall (between June 9-14, 2010).

• Extremely high concentrations of polyaromatic

hydrocarbons in the sediment.

Experimental Design

1. Measured tissue-specific responses in field-

collected fish.

• Physiology and protein-level expression changes

• Genome-wide mRNA transcript changes

2. Performed early-life exposures to field-collected

waters and sediments.

• time-to-hatch, hatching success, gene expression.

physiology

Genome expression: Microarray

GT BSL BFP BLB MB FMA

AHR ligand regulated -4 +4

• Divergent gene

expression in livers of

GT fish

• Coincident with

location & timing of oil

• AHR ligand regulated

genes were

differentially expressed

PNAS (2012) 50: 20298-20302

-4 +4

Genome Expression Response in Livers of

Field-Collected Gulf Killifish

PAH

Hsp90AhR

AIP

Arnt

Nuclear

mRNA

Endoplasmic

Reticulum

CY

P1A

The Aryl-hydrocarbon Receptor (AhR)

-Biomarkers of Exposure

PNAS (2012) 50: 20298-20302

AhR-mediated CYP1A Protein

Expression in Gulf Killifish Gills

Dark red stain = CYP1A protein in gills from fish collected in situBlue stain = hematoxylin (nuclei)

Trip 48/28/2011

PNAS (2012) 50: 20298-20302


AHR ligand regulated -4 +4

• Divergent gene

expression in livers of

GT fish

• Coincident with

location & timing of oil

• AHR ligand regulated

genes were

differentially expressed

PNAS (2012) 50: 20298-20302

-4 +4



HYPOTHESIS:Exposure to toxic components of oil


Reproduction +4-4

• Genes turned OFF coincident with timing/location of oiling:

Zona pellucida

Choriogenin

Vitellogenin



PNAS (2012) 50: 20298-20302

Ligand (PAH)-activated AHR

Ubiquitin-ligase activity

Proteolytic degradation of

sex steroid receptors

Suppression of sex

hormone induced gene

expression

Proteolysis -4 +4




PNAS (2012) 50: 20298-20302

Ligand (PAH)-activated AHR

Ubiquitin-ligase activity

Proteolytic degradation of

sex steroid receptors

Suppression of sex

hormone induced gene

expression

Proteolysis -4 +4




PNAS (2012) 50: 20298-20302

HYPOTHESIS:Compromised reproduction

(altered estrogen signaling)

Cardiovascular+4-4


-2 +2Cardiovascular



Field Collected Gulf Killifish

PNAS (2012) 50: 20298-20302

Cardiovascular+4-4


-2 +2Cardiovascular



Field Collected Gulf Killifish

PNAS (2012) 50: 20298-20302

HYPOTHESIS:Altered cardiac

physiology

Conclusions

• Large signal of hydrocarbon exposure exists in

Louisiana marsh fish collected in situ.

• CYP1a expression is highly elevated in the gills

and livers of fish collected from oil-contaminated

sites.

• Transcriptomics data suggest potential for

reduced reproductive capacity and alterations in

cardiovascular physiology

Gulf of

Mexico

Bay

Batiste

Bay Jimmy

Barataria

Bay

Grand Terre

Island, LA

***

**

***

* ** *

**

*

Sampling Sites in Southern Louisiana

*

*

Gulf of

Mexico

Bay

Batiste

Bay Jimmy

Barataria

Bay

Grand Terre

Island, LA

***

**

***

* ** *

**

*

Sampling Sites in Southern Louisiana

Gulf of

MexicoNBS WB GT GT

To

tal

[PA

H]

µg

/g

0204060

300

350

400

2011 2010

Killifish embryo exposures to

field-collected sediments

EST (2013) 47: 5074-5082

“Clean” 12 ppt water

Sediment

EST (2013) 47: 5074-5082

Killifish embryo exposures to

field-collected sediments

Effects of PAHs on Heart Function

Sampling Locations

NBS 2011WB 2011 GT 2011 GT 2010

tPA

Hs

(ng

/g)

0

15x1

03

30x1

03

300x

103

325x

103

350x

103

NBS WB GT GT

20

11

20

10

20

10

2011

2011

2011

Sampling Locations

NBS 2011 WB 2011 GT 2011 GT 2010

Hea

rt

Ra

te (

BP

M)

0

20

40

60

80

100

120

*

*

NBS WB GT GT

P≤0.05

EST (2013) 47: 5074-5082

20

10

2011

2011

2011

Hatching and tPAHs

Sampling Locations

NBS 2011WB 2011 GT 2011 GT 2010

tPA

Hs

(ng

/g)

0

15x1

03

30x1

03

300x

103

325x

103

350x

103

Days Post Fertilization

8 10 12 14 16 18 20 22

Per

cen

tag

e H

atc

h

-20

0

20

40

60

80

100

NBS 2011

WB 2011

GT 2011

GT 2010

*

**

* *

* *

**

** * *

*****

NBS WB GT GT

2011

2010

2010

2011

2011

2011

P≤0.05

EST (2013) 47: 5074-5082

Hatch

Influence of Oiled Sediments on Fish Hatch

EST (2013) 47: 5074-5082

NBS WB GT GT

Pe

rcen

t o

f T

ota

l

0

25

50

75

100

2011 2010

Dead

Hatched

Unhatched

PAH-induced Teratogenesis

Elongated Heart and Improper Folding

Pericardial Edema

Craniofacial Deformities Hemorrhage

Conclusions

• Exposure to field-collected sediments increases

time-to-hatch and reduces the percent hatch of

killifish embryos.

• DWH oil leads to teratogenic effects and affects

cardiovascular fitness

Oceans and Human Health Centers:

Mechanisms of reproductive,

developmental, and early life stage

impacts of marine

DWH Oil Spill: NEXT?...

Andrew WhiteheadFernando Galvez Christopher Green

Health risk

Early-life development

Juvenile/adult

performance

Reproduction

Low level exposures during critical developmental stages impacts in later life


Juvenile/adult performance

Reproduction

+

Health risk


Adult exposures impacts on reproduction



Reproduction

+

Health risk



Adult & early-life exposures trans-generational impacts



Reproduction

+

Health risk




Mechanisms morphology, physiology, genome expression, genome imprinting



Reproduction

Health risk




Mechanisms morphology, physiology, genome expression, genome imprinting

Genetic variation & exposure history protect or sensitize individuals



Reproduction

1 2

3

1 TX

2 LA

3 FL

4

4 LA – recent exposure history

5

5 TX – long exposure history

Genetic diversity

Health risk

Gulf killifish with different “exposure”

histories were collected along the nGOM

1 Gang’s Bayou, TX

2 LSU Aquaculture, LA

3 St. Teresa, FL

4 Grande Terre, LA (2015) – prior DWH oil exposure

5 Vince Bayou, TX – long exposure history

Reference populations

1 2

3

45



• Each population of adult fish were exposed to control

water (i.e., parental controls; PC) or to 25% high-

energy oiled WAF for at least 40 days (i.e., parental

exposed; PE)

Brood fish collection

Parental exposures

LSU, LA

PEPC

Grand Terre, LA (2015)

PEPC

Gang’s Bayou, TX

PEPC

Vince Bayou, TX

PEPC

Fertilization




Parental exposures

LSU, LA

PEPC


PEPC

Gang’s Bayou, TX

PEPC

Vince Bayou, TX

PEPC

Fertilization

0

20

40

60

80

100

VinceBayou

Gang'sBayou

GrandeTerre

LSU-LA

Fert

iliz

ati

on

(%

)

PC

PE




Parental exposures

LSU, LA

PEPC


PEPC

Gang’s Bayou, TX

PEPC

Vince Bayou, TX

PEPC

Fertilization

0% 10% 32% 56%

Embryo DWH Oil exposures for all treatments

Conclusions

• Genetic variation & exposure history can either

protect or sensitize individuals to subsequent oil

exposure

• F1 embryos from Louisiana populations are more

sensitive to DWH oil

• F0 adults from an adapted population are

recalcitrant to DWH oil (i.e., retains high

reproductive capacity

• F1 embryos from an adapted population is more

resistant to cardiovascular teratogenesis

Acknowledgements

Whitehead lab• Jennifer Roach

• Jan Park

Galvez lab• Charles Brown

• Tiffany Simms

• Chelsea Adams

• Ben Dubansky

Green lab• Chelsea Bonner

• Zachary McDaniel

QUESTIONS?

Documents

Fernando Galvez - WordPress.com · 2015-08-28 · Overview of Presentation. 1. ... Basic Facts of the Deepwater Horizon Oil Spill •Approximately 800 million liters of oil was released