Longterm Estuary Assessment Group
LaFleur
Lab
Developing biomarkers of reproductive health
in fish and amphibians of the
Barataria-Terrebonne Estuary
LaFleur, Pitre, Lasseigne, and Nelson
nicholls state university
NOAA '07
Eco Impacts
of Hypoxia
The Barataria-Terrebonne Estuary is impaired
• lack of flow
• saltwater
intrusion
• land-loss
Graphic from Restore or Retreat: www.restoreorretreat.org
Having been isolated from freshwater inputs it is lacks
flushing, sheet flow, and an ecological flood pulse
NOAA '07
Eco Impacts
of Hypoxia
USACE Butte La Rose Gage 03120 ('59-'04)
The 46-year mean stage of the Atchafalaya
still reflects an ecological flood pulse
Dissolved Oxygen (mg/L)
8
6
4
2
0
4/7/06
5/27/06
7/16/06
9/4/06
10/24/06
12/13/06
2/1/07
from Estay and Fontenot (thesis)
In the Upper Barataria basin, Hypoxia can occur
But it is usually associated with Rainwater events,
rather than flood stage
Our estuary is slated for largescale hydrologic modifications:
Graphic from Restore or Retreat: www.restoreorretreat.org
one project proposes diverting 300,000 cfs water into the BTE;
NOAA '07
up to 6 sq mi / year; at a cost of $25 / cubic yard
Eco Impacts
of Hypoxia
Pipeline Slurry System
A more recently presented scenario would utilize
a sediment pipeline slurry system to build land even quicker:
NOAA '07
up to15 sq mi / year; at a cost of $4 /cubic yard
Eco Impacts
of Hypoxia
MY OBJECTIVES
In preparation for hydrologic
changes due to further
deterioration or restoration
activities in our estuary, my lab has begun a survey
•to monitor behavioral indicators of reproduction
in amphibians of the estuary
•to monitor anatomical indicators of reproduction in
amphibians of the estuary
•to monitor molecular indicators of reproduction in
amphibians of the estuary
NOAA '07
Eco Impacts
of Hypoxia
1
2
3
4
5
Site 1 Choctaw swamp; Site 2 Chacahoula swamp
Site 3 Nicholls’ Environmental Ag Facility
Site 4 Falgout Canal fw / brackish marsh
NOAA '07
Impacts
Site 5 fish only at Isle Dernieres Barrier Islands Eco
of Hypoxia
Reproductive behavior
is being surveyed in
three LAMP survey
routes:
spanning Lafourche and
Terrebonne Parishes
NOAA '07
Eco Impacts
of Hypoxia
Northern
Cricket frogs
peaked in Jan,
but are still
calling
15
10
5
0
23Jan
7Feb
6Mar
20Mar
temp
calls
20
Call Density Score/
Temperature
In 2007,
Spring
Peepers
reached peak
calling in Feb
Call Density Score/
Temperature
20
15
10
5
temp
calls
0
23-Jan
7-Feb
6-Mar
20-Mar
Confirmed Hyla avivoca
Choctaw Swamp, Lafourche Parish
Proposed Expansion of
Geographic Range to the BTES,
south of the Mississippi River
Conant and Collins.1991
liver
ovary
Selected species are collected, dissected, and
anatomical indicators are examined
NOAA '07
Eco Impacts
of Hypoxia
Species
Repro
Behavior
Preserved
Repro Anatomy
Molec.
Biomarker
Bufo valliceps
X
X
X
X
Acris gryllus
X
X
Hyla cinerea
X
X
Hyla squirella
X
X
Hyla versicolor/
chrysoscelis
X
X
Hyla avivoca*
X
X
Pseudacris crucifer
X
X
Gastrophryne carolinensis
X
X
Rana catesbeiana
X
X
X
Rana grylio
X
Rana clamitans
X
X
X
X
Rana utricularia
X
X
X
X
Amphiuma
tridactylum
Notophthalmus
viridescens
X
X
X
X
X
X
X
Amphibian Survey Matrix
Approach to
designing biomarkers for
estrogen-induction
• Injection of estradiol into males
• Isolation of liver RNA
• RT-PCR for Vtg, Chg from
homogenates using degenerate
and heterologous primers
NOAA '07
Eco Impacts
of Hypoxia
vitellogenins and
choriogenins
vitelline envelope
precursor to the
chorion
Estrogen
induced
Liver synthesis
of teleost
choriogenins and vitellogenins
follicle
cells
HETEROSYNTHETIC
ORIGIN OF
TELEOSTEAN
EGG PROTEINS
yolk of oocyte
micropyle
NOAA '07
Eco Impacts
of Hypoxia
Choriogenin Primers
Row 78 TTCAGGTTCCAGAATTCTGAC
Row 81 CATTGGTTCATATCGCTGTCT
Vitellogenin Primers
Row 8 CGATATTGACATGTTTCCAA
Row 24 TACCAGCTTGGTTTCTACCT
Choriogenin and Vitellogenin primers derived from
F. heteroclitus, the mummichog. Row 78 and Row
81 produce a 450 bp product while Row 8 and Row
24 produce a 729 bp product
NOAA '07
Eco Impacts
of Hypoxia
RT-PCR inchoriogenin
Fundulus chrysotus
F.g.
F.c.
G.a.
A.t.
RT-PCR
in
Fundulus
grandis
liver Preliminary
liver
liver
ovary
Results
in Gambu
450
bp
Chg in female
Vtg in female and male
a
b
c
d
FemIM Male
Male IM
Fem IM IMFem Fem
Choriogenin
Vitellogenin
Choriogenin
Vitellogenin
Choriogenin cDNAs
indicating
normal female
reproductive activity.
NOAA '07
Eco Impacts
of Hypoxia
(Estrogen-injected males)
vitellogenin
RT-PCR in Fundulus
chrysotus
CR in Fundulus grandis
729
bp
a
Male
IM
Fem Male
b
c
IM
a= Fundulus
IM
Fem grandis
IM liver,
Fem
b= Fundulus chrysotus liver,
Choriogenin
Vitellogenin
c= Amphiuma tridactylum liver
oriogenin Vitellogenin
NOAA '07
Eco Impacts
of Hypoxia
(Non-injected males)
vitellogenin
Preliminary Results in Gambusia affinis
729
bp
Chg in female
Vtg
a in female
b
c and male
Vtg in female
Three wild-caught males showing the
presence of female specific RNA
a= Bufo valliceps liver,
b= Amphiuma tridactylum liver
c= Gambusia affinis
NOAA '07
Eco Impacts
of Hypoxia
Fish and
Amphibians
Through collaborations between the labs of LaFleur,
Ferrara and Fontenot we have established overlapping
projects of the estuarine fauna, including
NOAA '07
monitoring of finfish, larval fish, and amphibians. Eco Impacts
of Hypoxia
Summary
LaFleur
Lab
• Documented reproductive behavior
through calls of 12 local Anuran species.
• Established herpetology collection.
• Tracking reproductive seasons by measuring GSI on 4
Anurans, 1 Salamander, 3 fish.
• Molecular biomarkers were amplified using
RT-PCR on 3 anurans, 1 Salamander, 3 fish.
• Poised to implement reproductive survey to include water
quality and expanded sampling regime.
• Proposed range extension for Hyla avivoca to include
wetlands of the BTES.
Post Katrina Directions
Amphibian and Fish reproduction will be
utilized as a longterm assessment tool for
environmental health of the estuary
Nicholls has been awarded a grant for
restoration planting by NOAA
Nicholls has signed an MOU with NRCS for
restoration plant propagation at Nicholls Farm
LaFleur, Boopathy, and Zou are committed to
longterm monitoring programs that will offer
consistency over time
NOAA '07
Eco Impacts
of Hypoxia