Department of Coastal Sciences, The University of Southern Mississippi, Department of Physiological Sciences , University of Florida

R82945801

IMPACTS OF CHRONIC AND CYCLIC HYPOXIA ON GENE EXPRESSION AND REPRODUCTION IN GRASS SHRIMP, PALAEMONETES PUGIO 

Marius Brouwer; Nancy Brown-Peterson; Thea

Brouwer; Steve Manning; Tiandao Li; Nancy Denslow

Aquatic Research Consortium (ARC)Aquatic Research Consortium (ARC)

Objectives Grass Shrimp Hypoxia Objectives Grass Shrimp Hypoxia ProjectProject

Clone hypoxia (chronic and intermittent)-Clone hypoxia (chronic and intermittent)-responsive genes using subtractive responsive genes using subtractive hybridization and use clones to construct hybridization and use clones to construct macroarraysmacroarrays

Use the gene arrays to identify hypoxia-Use the gene arrays to identify hypoxia-responsive genes in grass shrimp in controlled responsive genes in grass shrimp in controlled laboratory experimentslaboratory experiments

Identify hypoxia-responsive reproductive Identify hypoxia-responsive reproductive parameters in laboratory experiments – needed parameters in laboratory experiments – needed to scale up to the population levelto scale up to the population level

Evaluate laboratory-derived parameters in Evaluate laboratory-derived parameters in field-collected grass shrimpfield-collected grass shrimp

Composition Gene Array. Will be Expanded with Cyclic DO

Responsive Genes

mitochondrial rRNA

Protein Synthesis-Folding

Protein degradation

Lipid metabolism/Vitellogenin

ATP synthesis-Electron transport

Oxygen transport/sensing

Carbohydrate metabolism

Contraction-Cytoskeleton

Heme-Fe metabolism

Antioxidant defense

Coagulation/Immune Function

Miscellaneous **

*

*

*

2 3 4 5 6 7 8 9 10

B

C

D

E

F

G

H

I

J

K

L

Hif Hcy Hcy Hcy Hcy

pepck

actin

Hsp70c 16kDa Hsp70PPI Hsp70 L13 L5

S6 L31 L27 S2 L21 L3 EF2 L6 S20

S14 CathL Apep CathC Cprot Crus Apep

ACoA ACoA Vtg Alip Chor Vtg

CcOx cytb Asyn CcOx Asyn CcOx

Amyl

Perl gbp Chit SugT

tub 16SrRNA

trop haembp

fer cSOD

Sp 2 Sp 3 Spike 3

hist

cuti odc

odc

mt1

Hybridized with RNA from 1.5 ppm DO 4 d grass shrimp

Cell myosin

mSOD

Macroarray Membrane LayoutMacroarray Membrane Layout

Gene Expression Studies

• Chronic hypoxia - laboratory

• Cyclic Hypoxia - laboratory

• Cyclic hypoxia - field studies

D a y 3 1 . 5 p p m - F S 0 1 9

- 6

- 4

- 2

0

2

4

6

D a y 7 1 . 5 p p m - F S 0 1 9

- 6

- 4

- 2

0

2

4

6

D a y 1 4 1 . 5 p p m - F S 0 1 9

- 6

- 4

- 2

0

2

4

6

Lo

g(b

as

e2)

Hyp

ox

ic/N

orm

ox

icL

og

(ba

se2

) H

ypo

xic

/No

rmo

xic

Lo

g(b

as

e2)

Hyp

ox

ic/N

orm

ox

ic

Changes in Gene Expression in Grass Shrimp Maintained in 1.5 ppm DO for 3 Days Relative to Same Day Normoxic Controls

0.1

1

10

100

rpro

t S

20

AT

PS

ynth

ase

d

AT

PS

ynth

ase

f

HE

MO

CY

AN

IN

hem

ocy

anin

hem

ocy

anin

4

tro

po

nin

C

ferr

itin

Fo

ld C

han

ge

(No

rmo

xic/

Hyp

oxi

c)

Changes in Gene Expression in Grass Shrimp Maintained in Cyclic DO for 3 Days Relative to Normoxic Controls

0.01

0.1

1

10

100

1000

HSP 70 mMnSOD

Fo

ld C

ha

ng

e (

Hy

po

xic

/No

rmo

xic

)

ChronicHypoxiaDay 3

Cyclic HypoxiaDay 3

ChronicHypoxiaDay 7

CyclicHypoxiaDay 7

Changes in Gene Expression in Grass Shrimp in Response to 7 Days Hypoxia (1.5 ppm DO) Relative to Normoxic Controls

0

2

4

6

8

16S rRNA Ccox I Heme BP ferritin

Fold

Cha

nge

(Hyp

oxic

/Nor

mox

ic)

Changes in Gene Expression in Grass Shrimp Maintained in Cyclic DO for 7 Days Relative to Normoxic Controls

0.01

0.1

1

10

rprot S2 rprot S14 CrustapainAcylCoA DH sterolreductase

ATP Synthased

PEPcarboxykinase

Fo

ld C

ha

ng

e (

Hy

po

xic

/No

rmo

xic

)

AcylCoA dehydrogenase

PEP Carboxykinase

TCA CYCLE

Phosphoenol Pyruvate Glucose Cyclic DO

Gluconeogenesis

fatty acid oxidation

Cyclic DO

GTP

GDP + CO2

CyclicHypoxiaDay 14

Chronic HypoxiaDay 14

Changes in Gene Expression In Grass Shrimp Maintained in 1.5 ppm DO for 14 Days Relative to Normoxic Controls

0.001

0.01

0.1

1

10

16SrRNA

rprot S6 fert env Ccox III cytochrb

Ccox 1 PmAV ferritin

Fo

ld C

ha

ng

e (

Hy

po

xii

c/N

orm

ox

ic)

Changes in Gene Expression in Grass Shrimp Kept in Cyclic DO for 14 Days Relative to Normoxic Controls

0

20

40

60

80

100

Vitellogenin-1 apolipoprotein A-I Cuticle protein

Fo

ld C

ha

ng

e (

Hy

po

xic

/No

rmo

xic

)

Conclusions

Chronic hypoxia induces time and dose-dependent changes in gene expression in grass shrimp

Intermittent diurnal hypoxia induces changes in gene expression that are distinct from the chronic hypoxia signal

Conclusion - Continued Mitochondrial genome-encoded 16S

rRNA, cytochrome b and cytochrome c oxidase subunits I and III

Nuclear-encoded mitochondrial proteins (ATP synthase subunits) and

Oxygen transport proteins (Hemocyanin subunits) appear to be robust molecular indicators of chronic hypoxia (1.5 ppm DO) exposure

Conclusion- continued

Mitochondrial MnSOD is a potential indicator of short-term cyclic DO

AcylCoA dehydrogenase and PEP carboxykinase may be useful as indicators of moderate length cyclic DO exposure

Gene expression profiles of grass shrimp form normoxic and hypoxic

field sites

WeeksBayMobile BayMobile Bay

EastEast BayBay

GarconPoint

EPA-EPA-GEDGED

EscambiaEscambiaBayBay

Mobile Bay Sampling SitesMobile Bay Sampling SitesSampled 2004, 2005Sampled 2004, 2005

Pensacola Bay Sampling SitesPensacola Bay Sampling SitesSampled 2002, 2003, 2004, 2005Sampled 2002, 2003, 2004, 2005

Marsh Creek Cyclic Hypoxic Site - August 2004

0.01

0.1

1

10

16S

rR

NA

Cat

hep

sin

L

tryp

sin

Cru

stap

ain

cyto

chro

me

b

AT

P S

ynth

d

Cco

x III

Cco

x I

hae

mo

cyan

in I

HE

MO

CY

AN

IN

hem

ocy

anin

hem

ocy

anin

4

per

luci

n

PE

Pca

rbo

xyki

nas

e

gly

cog

enin

hem

e B

P

ferr

itin

Gln

rep

eat

pro

teinF

old

Ch

ang

e (M

arsh

Cre

ek/C

on

tro

ls)

Normalized to alpha tubulin

GP1 Normoxic Site - August 2004

1

10

Cysteine proteinase trypsin Vitellogenin-2

Fo

ld C

ha

ng

e (

GP

1/C

on

tro

ls) Normalized to alpha tubulin

0

2

4

6

8

10

12

14

16

18

7/31/20041300

8/1/20040215

8/1/20041515

8/2/20040415

8/2/20041715

8/3/20040615

8/3/20041915

8/4/20040815

8/4/20042115

8/5/20041015

8/5/20042315

Date and Time

Diss

olve

d Ox

ygen

(ppm

) and

Sa

linity

(ppt

)

DO mg/L

Salinity

0

5

10

15

20

25

4-Aug 5-Aug 5-Aug 6-Aug 6-Aug 7-Aug 7-Aug 8-Aug 9-Aug 9-Aug10-Aug10-Aug11-Aug

Date and Time

Disso

lved O

xyge

n (mg

/L) an

d Sa

linity

(ppt)

DO Conc mg/L

Salinity ppt

Pensacola Bay, August 2004Pensacola Bay, August 2004

Water QualityWater Quality Gene Expression Relative to Normoxic ControlsGene Expression Relative to Normoxic Controls

Gene Expression Relative to Normoxic ControlsGene Expression Relative to Normoxic Controls

Similar across years and Similar across years and sites for cyclic hypoxia!sites for cyclic hypoxia!

Marsh Pond Cyclic Hypoxic Site--September 2002

0.01

0.1

1

10

100

Vit

ello

ge

nin

-1

AT

P S

yn

th d

AT

P S

yn

th f

HIF

HE

MO

CY

AN

IN

he

mo

cy

an

in

he

mo

cy

an

in 4

PE

Pc

arb

ox

yk

ina

se

ch

itin

as

e

su

ga

r tr

an

sp

ort

er

tro

po

nin

I

OD

C a

nti

zy

me

Gln

re

pe

at

pro

tein

Fo

ld C

ha

ng

e (

Hy

po

xic

/No

rmo

xic

)

Normalized to alpha tubulin

Marsh Creek Cyclic Hypoxic Site - August 2004

0.01

0.1

1

10

16S

rRN

A

Cat

heps

in L

tryp

sin

Cru

stap

ain

cyto

chro

me

b

ATP

Syn

th d

Cco

x III

Cco

x I

haem

ocya

nin

I

HEM

OC

YAN

IN

hem

ocya

nin

hem

ocya

nin

4

perlu

cin

PEP

carb

oxyk

inas

e

glyc

ogen

in

hem

e B

P

ferr

itin

Gln

repe

atpr

otei

nFold

Cha

nge

(Mar

sh C

reek

/Con

trol

s) Normalized to alpha tubulin

Weeks Creek Cyclic Hypoxic Site - September 2004

0.01

0.1

1

10

HS

P 7

0C

HS

P 7

0

rpro

t L

31

tryp

sin

AcC

oA

BP

Vtg

2

AT

P S

ynt

d

HIF

HE

MO

CY

AN

IN

hem

ocy

anin

hem

ocy

anin

4

amyl

ase

I

amyl

ase

pre

pro

pro

tein

PE

Pca

rbo

xyki

nas

esu

gar

tran

spo

rter

Fo

ld C

han

ge

(Wee

ks C

reek

/Co

ntr

ols

) Normalized to alpha tubulin

Marsh Pond Cyclic Hypoxic Site--September 2002

0.01

0.1

1

10

100

Vit

ello

ge

nin

-1

AT

P S

yn

th d

AT

P S

yn

th f

HIF

HE

MO

CY

AN

IN

he

mo

cy

an

in

he

mo

cy

an

in 4

PE

Pc

arb

ox

yk

ina

se

ch

itin

as

e

su

ga

r tr

an

sp

ort

er

tro

po

nin

I

OD

C a

nti

zy

me

Gln

re

pe

at

pro

tein

Fo

ld C

ha

ng

e (

Hy

po

xic

/No

rmo

xic

)

Normalized to alpha tubulin

Seasonal Change/Recovery in Gene ExpressionSeasonal Change/Recovery in Gene Expression

Marsh Pond September 2002

0.01

0.1

1

10

100

Vit

ello

ge

nin

-1

AT

P S

yn

th d

AT

P S

yn

th f

HIF

HE

MO

CY

AN

IN

he

mo

cy

an

in

he

mo

cy

an

in 4

PE

Pc

arb

ox

yk

ina

se

ch

itin

as

e

su

ga

r tr

an

sp

ort

er

tro

po

nin

I

OD

C a

nti

zy

me

Gln

re

pe

at

pro

tein

Fo

ld C

ha

ng

e (

Hy

po

xic

/No

rmo

xic

)

Normalized to alpha tubulin

Marsh Pond November 2002

0.01

0.1

1

10

Cy

s p

rote

ase

try

psin

Vit

ell

og

en

in-2

Cc

ox 1

hem

ocyan

in

PE

P

carb

oxykin

as

e

cellu

lar

myo

sin

acti

n

hem

e B

P

Fo

ld C

han

ge (

Mars

h P

on

d/C

on

tro

ls)

Normalized to alpha tubulin

p=

0.0

9

Weeks Creek - September 2004

0.01

0.1

1

10

HS

P 7

0C

HS

P 7

0

rpro

t L

31

try

ps

in

Ac

Co

A B

P

Vtg

2

AT

P S

yn

t d

HIF

HE

MO

CY

AN

IN

he

mo

cy

an

in

he

mo

cy

an

in

4

am

yla

se

I

am

yla

se

pre

pro

pro

tein

PE

Pc

arb

ox

yk

ina

se

su

ga

rtr

an

sp

ort

erFo

ld C

han

ge

(Wee

ks C

reek

/Co

ntr

ols

)

Normalized to alpha tubulin

Weeks Creek - November 2004

0.01

0.1

1

10

16S

rR

NA

EF

-2

Cys

pro

teas

e

tryp

sin

fert

en

v

Vtg

2

cyto

ch

rom

eb Cco

x II

I

AT

P S

ynt f

Cco

x I

HIF

hae

mo

cyan

inI

amyl

ase

I

glu

can

BP

glu

cosi

da

se

hem

e B

P

ferr

itin

OD

Can

tizym

eG

ln r

epea

tp

rote

in

Fo

ld C

ha

ng

e (

We

ek

s C

ree

k/C

on

tro

ls)

Normalized to tubulin

Summary Field Gene Expression

Grass shrimp exposed to cyclic hypoxia in the field show distinct changes in gene expression profiles, not observed in shrimp from normoxic control sites

1. Downregulation of mitochondrial gene transcription similar to changes induced by chronic 1.5 ppm DO exposures in the lab

2. Downregulation of hemocyanin transcription in late summer, not observed in lab exposures, but indicative of anaerobic metabolism

3. Return to aerobic, oxidative metabolism in late fall

New Directions Construction of 700 gene array (with cyclic-DO sensitive genes)

has been completed (Tiandoa Li)

Cyclic DO library contains many genes that are involved in sulfur redox metabolism and (possibly) in controlling cysteine and glutathione levels – a classic response to oxidative stress

Proteomics studies on mitochondrial protein expression (protein fractionation in 2 dimensions and MS) have been initiated

Reproductive Studies: Ecologically

Important Parameters

• chronic hypoxia • cyclic DO• Field

Female Oxygen Exposure

HH NN HN

Re

lati

ve

Fe

cu

nd

ity

0

200

400

600

800

1000

1200

A

BB

2-3 ppm DO

1.5 ppm DO

P = 0.003

P = 0.027

P = 0.010

HH NN HN0

200

400

600

800

1000

1200

A

B BR

elat

ive

Fec

un

dit

y

Hypoxia Increases Relative Fecundity

Hypoxia (2.5 ppm) Increases Time Interval Between Brood Production

0

5

10

15

20

25

30

Brood 1 Brood 2

Day

s to

Eg

g P

rod

uct

ion

Hypoxia

Normoxia

Hypoxia (2.5 ppm DO) Increases Survival of Starved Larvae from Hypoxic Females

Brood 1 Brood 2

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Hypoxic Normoxic A A

BB

Larv

al S

urvi

val (

days

)

Effects of Cyclic Hypoxia (1.5 8 ppm DO) on Grass Effects of Cyclic Hypoxia (1.5 8 ppm DO) on Grass Shrimp Reproduction: Laboratory ExperimentsShrimp Reproduction: Laboratory Experiments

HH HN NN

Day

s to

Eg

g P

rod

uct

ion

0

10

20

30

HH HN NN0

5

10

15

20

25

Treatment

HH HN NN0

5

10

15

20

First Brood

Second Brood

Third Brood

A

B B

A

B

C

D

D

E

F

E,F

Interbrood Interval in Cyclic Hypoxic

HH HN NN

Rel

ati

ve

Fec

un

dit

y (#

eg

gs/

g)

0

100

200

300

400

500

600

Treatment

HH HN NN0

100

200

300

400

500

600

First Brood

Second Brood

A

A,B

B

Relative Fecundity in Cyclic Hypoxia

Population Growth RatesChronic Hypoxia

Brood Number

Brood 1 Brood 2

La

mb

da

1.0

1.2

1.4

1.6

1.8Hypoxic Normoxic

Population Growth RatesCyclic Hypoxia

Brood Number

Brood 1 Brood 2

La

mb

da

1.0

1.2

1.4

1.6

1.8HH HN NN

Cyclic DO decreasesestimated populationgrowth rate

(in an aquarium)

Conclusions

Chronic hypoxia stimulates production of more and “healthier” eggs and increases time interval between broods

Intermittent diurnal hypoxia decreases fecundity and increases time interval between broods

Hypoxia (chronic and intermittent) may have population-level impacts on grass shrimp

StationStation % Egged% Egged Ratio M:FRatio M:F

WCWC 18.2%18.2% 1:11:1

WBMWBM 44.9%44.9% 1.3:11.3:1

Weeks Bay, July 2004Weeks Bay, July 2004

StationStation % Egged% Egged Ratio M:FRatio M:F

GP1GP1 75.8%75.8% 1.1:11.1:1

MCMC 45.8%45.8% 1.5:11.5:1

MPMP 29.5%29.5% 1.25:11.25:1

Pensacola Bay, August 2004Pensacola Bay, August 2004

Reproduction of Grass Shrimp in the FieldReproduction of Grass Shrimp in the Field

SiteWC WBM

Re

lati

ve

Fe

cu

nd

ity

(#

eg

gs

/g)

0

200

400

600

800

1000

SiteGP1 MP MC

Re

lati

ve F

ec

un

dit

y (

# e

gg

s/g

)

0

200

400

600

800

1000

1200

1400

Conclusions

There are distinct differences in gene expression profiles in grass shrimp from cyclic DO marsh sites and marsh edge, normoxic open bay sites

There are distinct differences in reproductive function in grass shrimp from cyclic DO field sites and normoxic sites

The two responses may be combined to provide diagnostic and predictive tools to asses effects of hypoxia on crustacea at the individual and population level

Thanks to students and staff

and NOAA and EPA

Normalized to Median Intensity - log base 2 transformed

y = 0.9554x - 0.399

R2 = 0.9326

-4.0

-3.0

-2.0

-1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

-3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Membrane 76

Mem

bran

e 78

Variability Due to Experimental Error is Small

Comparison Mean Normalized Gene Intensities of Normoxic Control Shrimp from Two Experiments

y = 0.8866x - 0.1122

R2 = 0.7971

-10.0

-8.0

-6.0

-4.0

-2.0

0.0

2.0

4.0

6.0

-10.0 -8.0 -6.0 -4.0 -2.0 0.0 2.0 4.0 6.0

Mean FS019 (n=33)

Me

an

FS

00

1 (

n=

21

)

Variability of Gene Expression Levels Among Shrimp is Modest

Develop Microarrays with ~ 5000 Genes from Sheepshead Minnow Embryos and

Larvae Responsive to Environmental Stressors

Chronic hypoxia – stratified systemsIntermittent hypoxia – tidal creeksPolycyclic Aromatic HydrocarbonsEndocrine disruptors Toxic metalsPesticides and herbicidesEffects of hypoxia and anthropogenic stressors on

embryos/larvae development and gene expression